1 00:00:05,320 --> 00:00:08,520 >> TODAY'S SESSION OF THE 2 00:00:08,520 --> 00:00:09,480 WEDNESDAY AFTERNOON LECTURE 3 00:00:09,480 --> 00:00:10,920 SERIES HERE AT THE NIH FOR THOSE 4 00:00:10,920 --> 00:00:13,520 WHO DON'T KNOW ME, I'M HEAD OF 5 00:00:13,520 --> 00:00:14,720 THE SECTION OF STRUCTURAL AND 6 00:00:14,720 --> 00:00:16,400 CHEMICAL BIOLOGY OF MEMBRANE 7 00:00:16,400 --> 00:00:18,120 PROTEINS IN THE INTRAMURAL 8 00:00:18,120 --> 00:00:20,240 PROGRAM OF NICHD. 9 00:00:20,240 --> 00:00:22,360 TODAY'S TALK IS HOSTED BY THE 10 00:00:22,360 --> 00:00:22,920 STRUCTURAL BIOLOGY INTEREST 11 00:00:22,920 --> 00:00:25,760 GROUP OF WHICH I'M ONE OF THE 12 00:00:25,760 --> 00:00:27,040 COORDINATORS, AND IT'S A REAL 13 00:00:27,040 --> 00:00:28,520 PRIVILEGE AND HONOR FOR ME TO 14 00:00:28,520 --> 00:00:30,080 INTRODUCE TODAY'S SPEAKER, 15 00:00:30,080 --> 00:00:31,640 PROFESSOR JOHN KURIYAN, WHO 16 00:00:31,640 --> 00:00:34,360 COMES TO US FROM THE UNIVERSITY 17 00:00:34,360 --> 00:00:36,520 OF CALIFORNIA AT BERKLEY, 18 00:00:36,520 --> 00:00:37,360 CHANCELLOR'S PROFESSOR IN THE 19 00:00:37,360 --> 00:00:38,360 DEPARTMENTS OF MOLECULAR AND 20 00:00:38,360 --> 00:00:39,920 CELL BIOLOGY AND THE DEPARTMENTS 21 00:00:39,920 --> 00:00:42,120 OF CHEMISTRY AS WELL AS A HOWARD 22 00:00:42,120 --> 00:00:44,960 HUGHES MEDICAL INSTITUTE 23 00:00:44,960 --> 00:00:45,280 INVESTIGATOR. 24 00:00:45,280 --> 00:00:47,960 JOHN WAS BORN AND RAISED IN 25 00:00:47,960 --> 00:00:50,760 INDIA AND RECEIVED HIS 26 00:00:50,760 --> 00:00:51,880 UNDERGRADUATE DEGREE IN 27 00:00:51,880 --> 00:00:53,680 CHEMISTRY FROM PENNSYLVANIA, 28 00:00:53,680 --> 00:00:55,480 THEAND 29 00:00:55,480 --> 00:00:57,760 THEN HE WENT TO THE DEPARTMENT 30 00:00:57,760 --> 00:00:58,920 OF CHEMISTRY AT HARVARD WHERE HE 31 00:00:58,920 --> 00:01:00,800 GOT HIS PH.D. IN PHYSICAL 32 00:01:00,800 --> 00:01:02,240 CHEMISTRY FOLLOWED BY A VERY 33 00:01:02,240 --> 00:01:03,160 SHORT POSTDOCTORAL POSITION 34 00:01:03,160 --> 00:01:08,360 WHERE HE WAS . 35 00:01:08,360 --> 00:01:10,640 JOHN'S THESIS RESEARCH WAS 36 00:01:10,640 --> 00:01:11,240 MOSTLY COMPUTATIONAL CHEMISTRY 37 00:01:11,240 --> 00:01:15,760 FOR THOSE OF YOU WHO ARE SEEN AS 38 00:01:15,760 --> 00:01:17,160 USERS, YOU REMEMBER THE 39 00:01:17,160 --> 00:01:23,400 REFINEMENT OF R FACTOR. 40 00:01:23,400 --> 00:01:26,480 SO WHEN HE STARTED HIS LAB AT 41 00:01:26,480 --> 00:01:27,560 THE ROCKEFELLER UNIVERSITY AND 42 00:01:27,560 --> 00:01:29,200 MADE THE SWITCH TO EXPERIMENTAL 43 00:01:29,200 --> 00:01:31,040 STRUCTURAL BIOLOGY, IT WAS A 44 00:01:31,040 --> 00:01:33,680 DRASTIC AND BOLD MOVE, AND TO 45 00:01:33,680 --> 00:01:35,640 JOHN'S KRE IT, 46 00:01:35,640 --> 00:01:39,440 JOHN'S CREDIT, HE DID NOT ONLY 47 00:01:39,440 --> 00:01:40,480 SURVIVE THE TRANSITION AS WELL 48 00:01:40,480 --> 00:01:42,320 AS ANYONE COULD IMAGINE, HIS 49 00:01:42,320 --> 00:01:45,160 RESEARCH HAS THRIVED ALONG TWO 50 00:01:45,160 --> 00:01:47,800 VERY DISTINCT DIRECTIONS, SIGNAL 51 00:01:47,800 --> 00:01:49,200 TRANSDUCTION BY PROTEIN KINASES. 52 00:01:49,200 --> 00:01:51,760 JOHN ROSE THROUGH THE RANKS AT 53 00:01:51,760 --> 00:01:54,680 ROCKEFELLER AND BECAME THE 54 00:01:54,680 --> 00:01:55,880 HAGGERTY PROFESSOR. 55 00:01:55,880 --> 00:01:58,040 IN 2001, JOHN MOVED TO UC 56 00:01:58,040 --> 00:02:00,120 BERKLEY, WHERE HE HAS REMAINED 57 00:02:00,120 --> 00:02:01,800 SINCE THEN. 58 00:02:01,800 --> 00:02:03,240 JOHN'S HONORS AND ACCOLADES HAVE 59 00:02:03,240 --> 00:02:04,480 BEEN MANY. 60 00:02:04,480 --> 00:02:06,760 HE'S A MEMBER OF BOTH THE 61 00:02:06,760 --> 00:02:07,880 NATIONAL ACADEMY OF SCIENCES AND 62 00:02:07,880 --> 00:02:08,240 MEDICINE. 63 00:02:08,240 --> 00:02:10,080 HE HAS RECEIVED MANY AWARDS 64 00:02:10,080 --> 00:02:12,160 INCLUDING THE PRESTIGIOUS STEIN 65 00:02:12,160 --> 00:02:15,200 AND MOORE AWARD FROM THE PROTEIN 66 00:02:15,200 --> 00:02:16,960 SOCIETY, THE MARK AWARD FROM THE 67 00:02:16,960 --> 00:02:17,640 AMERICAN SOCIETY OF BIOCHEMISTRY 68 00:02:17,640 --> 00:02:20,480 AND MOLECULAR BIOLOGY, AND THE 69 00:02:20,480 --> 00:02:23,680 RICHARD LANCEBURY AWARD FROM THE 70 00:02:23,680 --> 00:02:24,880 NATIONAL ACADEMY OF SCIENCES AND 71 00:02:24,880 --> 00:02:26,320 MANY OTHERS I DON'T HAVE TIME TO 72 00:02:26,320 --> 00:02:27,360 NAME HERE. 73 00:02:27,360 --> 00:02:28,040 HOWEVER ANOTHER SIGNIFICANT MARK 74 00:02:28,040 --> 00:02:30,760 OF AN ACADEMIC SCIENTIST IS THE 75 00:02:30,760 --> 00:02:32,360 SCIENTIST THAT THEY TRAIN AND 76 00:02:32,360 --> 00:02:34,560 JOHN HAS TRAINED AN OUTSTANDING 77 00:02:34,560 --> 00:02:35,680 GROUP OF SCIENTISTS WHO HAVE 78 00:02:35,680 --> 00:02:37,200 GONE ON TO BECOME LEADERS IN 79 00:02:37,200 --> 00:02:38,480 THEIR OWN RITE. 80 00:02:38,480 --> 00:02:40,480 FOR THOSE WATCHING REMOTELY, WE 81 00:02:40,480 --> 00:02:41,320 ENCOURAGE YOU, YOU CAN 82 00:02:41,320 --> 00:02:43,440 PARTICIPATE IN THE Q & A AFTER 83 00:02:43,440 --> 00:02:43,960 TODAY'S PRESENTATION. 84 00:02:43,960 --> 00:02:46,280 YOU CAN DO SO BY CLICKING ON THE 85 00:02:46,280 --> 00:02:48,840 BUTTON JUST BELOW YOUR VIDEOCAST 86 00:02:48,840 --> 00:02:51,440 WINDOW THAT SAYS "SEND LIVE 87 00:02:51,440 --> 00:02:51,680 FEEDBACK." 88 00:02:51,680 --> 00:02:53,320 CLICK ON THAT, TYPE IN YOUR NAME 89 00:02:53,320 --> 00:02:55,680 AND QUESTION, AND WE WILL RELAY 90 00:02:55,680 --> 00:02:59,640 THE QUESTION TO DR. KURIYAN AT 91 00:02:59,640 --> 00:03:00,680 THE END OF THE LECTURE. 92 00:03:00,680 --> 00:03:02,080 YOU CAN ALSO SUBMIT A QUESTION 93 00:03:02,080 --> 00:03:03,160 AT ANY TIME AND DON'T HAVE TO 94 00:03:03,160 --> 00:03:03,760 WAIT UNTIL THE END. 95 00:03:03,760 --> 00:03:05,400 FOR THOSE OF YOU IN THE ROOM 96 00:03:05,400 --> 00:03:07,280 TODAY, PLEASE USE THE MICS ON 97 00:03:07,280 --> 00:03:07,680 EITHER SIDE. 98 00:03:07,680 --> 00:03:09,760 ALSO WE ARE OFFERING CONTINUING 99 00:03:09,760 --> 00:03:12,320 MEDICAL EDUCATION CREDITS FOR 100 00:03:12,320 --> 00:03:12,880 THIS PRESENTATION. 101 00:03:12,880 --> 00:03:19,640 THE CME CODE FOR TO THE IS 102 00:03:19,640 --> 00:03:21,320 37946. 103 00:03:21,320 --> 00:03:24,040 THE TITLE OF JOHN'S TALK IS DEEP 104 00:03:24,040 --> 00:03:26,080 MUTAGENESIS ANALYSIS OF 105 00:03:26,080 --> 00:03:29,680 EVOLVABILITY IN A DNA POLYMERASE 106 00:03:29,680 --> 00:03:31,520 AAA PLUS CLAMP LOADER SYSTEM. 107 00:03:31,520 --> 00:03:32,600 JOHN, THANKS FOR ACCEPTING OUR 108 00:03:32,600 --> 00:03:32,880 INVITATION. 109 00:03:32,880 --> 00:03:37,120 [APPLAUSE] 110 00:03:37,120 --> 00:03:40,560 >> THANK YOU VERY MUCH FOR THE 111 00:03:40,560 --> 00:03:43,600 PRIVILEGE OF SPEAKING TO YOU 112 00:03:43,600 --> 00:03:45,800 THIS AFTERNOON. 113 00:03:45,800 --> 00:03:46,400 IN THIS HYBRID 114 00:03:46,400 --> 00:03:47,240 FORMAT. 115 00:03:47,240 --> 00:03:50,680 IT'S NICE TO SEE PEOPLE ALIVE 116 00:03:50,680 --> 00:03:52,480 AND WELL IN THE AUDIENCE BECAUSE 117 00:03:52,480 --> 00:03:54,000 IF YOU CAN MAKE EYE CONTACT WITH 118 00:03:54,000 --> 00:03:57,000 ONE OR TWO, IT LETS YOU KNOW 119 00:03:57,000 --> 00:03:59,560 THAT YOU'RE NOT SPEAKING INTO 120 00:03:59,560 --> 00:04:03,960 THE VOID. 121 00:04:03,960 --> 00:04:07,560 AS WE WOULD TEACH UNDERGRADUATES 122 00:04:07,560 --> 00:04:08,480 OVER THE PANDEMIC. 123 00:04:08,480 --> 00:04:10,480 I'M GOING TO TALK TO YOU TODAY 124 00:04:10,480 --> 00:04:12,520 ABOUT SOME WORK WE'VE BEEN DOING 125 00:04:12,520 --> 00:04:13,800 TO UNDERSTAND THE ABILITY OF A 126 00:04:13,800 --> 00:04:16,760 MOLECULAR MACHINE KNOWN AS THE 127 00:04:16,760 --> 00:04:19,320 DNA POLYMERASE CLAMP LOADER TO 128 00:04:19,320 --> 00:04:21,560 TOLERATE ADDITIONAL INSULTS 129 00:04:21,560 --> 00:04:24,080 WHILE IT'S FUNCTIONING IN THE 130 00:04:24,080 --> 00:04:29,000 DNA REPLICATION SYSTEM. 131 00:04:29,000 --> 00:04:35,840 SO WHILE DNA PO POLYMERASE SYSTEMS 132 00:04:35,840 --> 00:04:36,840 ACHIEVE GREAT SPEED THROUGH 133 00:04:36,840 --> 00:04:38,720 ATTACHMENT TO A SLIDING CLAMP, 134 00:04:38,720 --> 00:04:40,240 AND THE SLIDING CLAMP IS A 135 00:04:40,240 --> 00:04:41,800 CIRCULAR PROTEIN THAT GOES 136 00:04:41,800 --> 00:04:44,960 AROUND DNA, AND BY ATTACHING THE 137 00:04:44,960 --> 00:04:46,720 POLYMERASE TO THE SIDING CLAMP 138 00:04:46,720 --> 00:04:49,400 THE SYSTEM CAN ACHIEVE VERY HIGH 139 00:04:49,400 --> 00:04:51,600 SPEED WITHOUT LETTING GO OF DNA. 140 00:04:51,600 --> 00:04:53,760 SO THIS IS A PRINCIPLE THAT 141 00:04:53,760 --> 00:04:54,680 EMERGED, THE STRUCTURAL BASIS 142 00:04:54,680 --> 00:04:56,440 FOR THIS EMERGED FROM STUDIES 143 00:04:56,440 --> 00:04:58,400 DONE OVER THE YEARS BY MY LAB 144 00:04:58,400 --> 00:05:00,480 AND THE LAB OF MICHAEL 145 00:05:00,480 --> 00:05:02,160 O'DONNELL. 146 00:05:02,160 --> 00:05:03,800 AND THIS SIMPLIFIED DIAGRAM 147 00:05:03,800 --> 00:05:07,800 ESSENTIALLY SHOWS YOU HOW THE 148 00:05:07,800 --> 00:05:08,920 DNA POLYMERASE CLAMP LOADER 149 00:05:08,920 --> 00:05:10,560 SYSTEM WORKS TO LOAD SLIDING 150 00:05:10,560 --> 00:05:13,400 CLAMPS ON TO DNA, AND IT DOES 151 00:05:13,400 --> 00:05:14,240 THIS REPEATEDLY FOR EVERY 152 00:05:14,240 --> 00:05:16,440 FRAGMENT THAT'S GENERATED. 153 00:05:16,440 --> 00:05:18,400 SO THE DNA POLYMERASE SLIDING 154 00:05:18,400 --> 00:05:20,760 CLAMP SHOWN HERE IS A COMPLEX OF 155 00:05:20,760 --> 00:05:23,040 FIVE DIFFERENT PROTEINS LABELED 156 00:05:23,040 --> 00:05:30,640 A, B, C, D, E, AND THEY ARE ATP 157 00:05:30,640 --> 00:05:33,160 ACES OF THE TRIPLE A FAMILY AND 158 00:05:33,160 --> 00:05:34,760 THE ACTION OF THE MOLECULAR 159 00:05:34,760 --> 00:05:37,760 MACHINE IS BASED ON THE ABILITY 160 00:05:37,760 --> 00:05:38,960 TO HYDROIZE ATP. 161 00:05:38,960 --> 00:05:40,240 I'M GOING TO BE TELLING YOU 162 00:05:40,240 --> 00:05:42,120 TODAY ABOUT ONE SPECIFIC CLAMP 163 00:05:42,120 --> 00:05:46,160 LOADER CALLED THE T4 PHAGE CLAMP 164 00:05:46,160 --> 00:05:47,440 LOADER SYSTEM, AND IN THAT 165 00:05:47,440 --> 00:05:50,800 PARTICULAR ONE, THERE ARE TWO 166 00:05:50,800 --> 00:05:52,600 GENES THAT ENCODE THE FIVE 167 00:05:52,600 --> 00:05:54,440 PROTEIN COMPLEX, ONE GENE 168 00:05:54,440 --> 00:05:57,200 ENCODES THE PROTEIN COLORED 169 00:05:57,200 --> 00:06:00,680 GREEN HERE, WHICH IS ACTUALLY A 170 00:06:00,680 --> 00:06:02,320 DEGENERATE AAA PLUS PROTEIN SO 171 00:06:02,320 --> 00:06:03,600 IT DOESN'T LOOK LIKE THE OTHERS 172 00:06:03,600 --> 00:06:04,680 BUT IT ORIGINATED LONG AGO FROM 173 00:06:04,680 --> 00:06:07,880 THE OTHERS. 174 00:06:07,880 --> 00:06:10,080 AND THE OTHER GENE ENCODES FOUR 175 00:06:10,080 --> 00:06:12,920 OF THE ATP ACES WHICH ARE 176 00:06:12,920 --> 00:06:15,080 LABELED A, B, C, D AND E IN THIS 177 00:06:15,080 --> 00:06:21,400 COMPLEX SO TOGETHER THEY FORM AN 178 00:06:21,400 --> 00:06:25,560 ASSEMBLY THAT DOES NOT HYDROLIZ, 179 00:06:25,560 --> 00:06:28,040 ATP UNTIL IT RECOGNIZES THE 180 00:06:28,040 --> 00:06:30,000 SLIDING CLAMP AND IT RECOGNIZES 181 00:06:30,000 --> 00:06:31,360 A PRIMARY JUNCTION SHOWN 182 00:06:31,360 --> 00:06:31,800 SCHEMATICALLY HERE. 183 00:06:31,800 --> 00:06:33,720 WHEN THOSE TWO THINGS ARE 184 00:06:33,720 --> 00:06:35,760 RECOGNIZED, THE ASSEMBLY 185 00:06:35,760 --> 00:06:37,240 HYDROLYZES ATP WHICH RELEASES 186 00:06:37,240 --> 00:06:38,480 THE CLAMP LOADER FROM THE 187 00:06:38,480 --> 00:06:39,840 SLIDING CLAMP AND DNA, LEAVING 188 00:06:39,840 --> 00:06:42,000 THE SLIDING CLAMP ON DNA. 189 00:06:42,000 --> 00:06:44,960 SO THAT'S THE ESSENTIAL FUNCTION 190 00:06:44,960 --> 00:06:54,000 OF A CLAMP LOADER. 191 00:06:54,000 --> 00:06:55,960 THE STRUCTURE EV THESE THAT TOLD 192 00:06:55,960 --> 00:06:57,560 MANY OF THE ESSENTIAL ASPECTS OF 193 00:06:57,560 --> 00:06:58,920 HOW THESE THINGS PULL TOGETHER 194 00:06:58,920 --> 00:07:00,000 AND HOW THEY WORK BUT TODAY I 195 00:07:00,000 --> 00:07:02,000 WANT TO FOCUS TWO QUESTIONS THAT 196 00:07:02,000 --> 00:07:03,280 CAME OUT OF THINGS THAT WE WERE 197 00:07:03,280 --> 00:07:04,480 WONDERING ABOUT. 198 00:07:04,480 --> 00:07:05,560 THE FIRST QUELL IS WE KNOW THAT 199 00:07:05,560 --> 00:07:09,000 THE DIVERSITY OF AAA+ PROTEINS, 200 00:07:09,000 --> 00:07:15,280 CLAMP LOADERS SPECIFICALLY, 201 00:07:15,280 --> 00:07:17,200 AAA -- IS ENORMOUS BOTH IN 202 00:07:17,200 --> 00:07:19,200 SEQUENCE AND STRUCTURAL DETAILS. 203 00:07:19,200 --> 00:07:21,720 LIKE ALL BIOLOGISTS WHO STUDY 204 00:07:21,720 --> 00:07:23,560 MOLECULAR SYSTEMS AND EVEN KNOW 205 00:07:23,560 --> 00:07:24,600 CROWSCOPIC SYSTEMS WE WONDER HOW 206 00:07:24,600 --> 00:07:26,920 DOES NATURE COME UP WITH THIS 207 00:07:26,920 --> 00:07:29,080 DIVERSITY AND STRUCTURE, WHEN 208 00:07:29,080 --> 00:07:29,640 MAINTAINING AN ESSENTIAL 209 00:07:29,640 --> 00:07:30,200 MECHANISM? 210 00:07:30,200 --> 00:07:31,720 ONE WAY TO ASK THAT IS TO ASK 211 00:07:31,720 --> 00:07:33,880 THE QUESTION, WHAT'S THE 212 00:07:33,880 --> 00:07:36,320 CAPACITY OF THE CLAMP LOADER 213 00:07:36,320 --> 00:07:38,440 SYSTEM TO ACCEPT CHANGES TO ITS 214 00:07:38,440 --> 00:07:40,800 SEQUENCE WHILE IT ACTUALLY 215 00:07:40,800 --> 00:07:42,200 PERFORMING A CRITICAL TASK. 216 00:07:42,200 --> 00:07:43,960 IN THIS CASE, DNA REPLICATION. 217 00:07:43,960 --> 00:07:46,240 SO WHAT I'M GOING TO TELL YOU IS 218 00:07:46,240 --> 00:07:49,760 ABOUT A SYSTEM DEVELOPED IN MY 219 00:07:49,760 --> 00:07:53,560 LAB WHICH ALLOWS US TO PROBE THE 220 00:07:53,560 --> 00:07:56,200 MUTATIONAL TOLERANCE OF THE 221 00:07:56,200 --> 00:07:58,080 SYSTEM WHILE IT'S ACTUALLY 222 00:07:58,080 --> 00:07:59,400 REPLICATING DNA. 223 00:07:59,400 --> 00:08:00,600 AND THE SECOND QUESTION WE'D 224 00:08:00,600 --> 00:08:02,840 LIKE TO ASK AND ANSWER IS WHAT 225 00:08:02,840 --> 00:08:04,480 DOES THE MUTATIONAL RESPONSE OF 226 00:08:04,480 --> 00:08:07,240 THE SYSTEM TELL US ABOUT THE 227 00:08:07,240 --> 00:08:07,520 MECHANISM? 228 00:08:07,520 --> 00:08:09,320 NOW WE KNOW A LOT ABOUT THE 229 00:08:09,320 --> 00:08:10,600 MECHANISM FROM THE STRUCTURAL 230 00:08:10,600 --> 00:08:11,800 WORK AND THE BIOCHEMISTRY THAT 231 00:08:11,800 --> 00:08:14,280 WE'VE DONE, BUT AS YOU'LL SEE, 232 00:08:14,280 --> 00:08:17,840 THE ANALYSIS OF THE MUTATIONAL 233 00:08:17,840 --> 00:08:19,400 RESPONSE DID TEACH US SOME 234 00:08:19,400 --> 00:08:20,480 COMPLETELY UNEXPECTED THINGS 235 00:08:20,480 --> 00:08:21,440 ABOUT THE SYSTEM. 236 00:08:21,440 --> 00:08:22,760 SO THAT'S WHAT THIS LECTURE IS 237 00:08:22,760 --> 00:08:26,280 GOING TO BE ABOUT. 238 00:08:26,280 --> 00:08:27,640 SO THE WAY THE EXPERIMENTS WERE 239 00:08:27,640 --> 00:08:29,400 CARRIED OUT WAS TO USE AN 240 00:08:29,400 --> 00:08:33,360 EXPERIMENTAL PLATFORM DEVELOPED 241 00:08:33,360 --> 00:08:35,320 WHICH IS EXTREMELY ROBUST IN 242 00:08:35,320 --> 00:08:36,760 TERMS OF BEING ABLE TO TELL US 243 00:08:36,760 --> 00:08:39,000 THE EFFECT ON FITNESS OF ANY 244 00:08:39,000 --> 00:08:40,080 MUTATION MADE IN THE CLAMP 245 00:08:40,080 --> 00:08:41,200 LOADER SYSTEM. 246 00:08:41,200 --> 00:08:42,600 SO FIRST I'D LIKE TO JUST 247 00:08:42,600 --> 00:08:45,040 EXPLAIN WHAT THIS PLATFORM IS 248 00:08:45,040 --> 00:08:49,000 USING THIS DIAGRAM, THEN I'D 249 00:08:49,000 --> 00:08:51,240 LIKE TO SHOW YOU SOME DATA WHICH 250 00:08:51,240 --> 00:08:52,840 CONVINCED US THAT THE SYSTEM IS 251 00:08:52,840 --> 00:08:54,320 ACTUALLY REALLY ROBUST IN TERMS 252 00:08:54,320 --> 00:08:57,200 OF TELLING US THE RELATIONSHIP 253 00:08:57,200 --> 00:08:58,320 BETWEEN SEQUENCE AND THE 254 00:08:58,320 --> 00:08:59,840 FUNCTION OF THE PROTEIN. 255 00:08:59,840 --> 00:09:01,800 SO AS I MENTIONED, THE SYSTEM 256 00:09:01,800 --> 00:09:05,600 WE'RE USING IS A MODEL SYSTEM IS 257 00:09:05,600 --> 00:09:06,240 T4 BACTERIOPHAGE WHICH IS 258 00:09:06,240 --> 00:09:08,280 SPECIAL BECAUSE T4 BACTERIOPHAGE 259 00:09:08,280 --> 00:09:10,200 BRINGS WITH IT ITS OWN DNA 260 00:09:10,200 --> 00:09:13,600 REPLICATION PROTEINS WHETHER 261 00:09:13,600 --> 00:09:14,920 IT -- E. COLI. 262 00:09:14,920 --> 00:09:16,360 SO IT BRINGS A SLIDING CLAMP, 263 00:09:16,360 --> 00:09:19,800 CLAMP LOADER, DNA LIGASE, 264 00:09:19,800 --> 00:09:20,720 STRANDING DNA BINDING PROTEIN, 265 00:09:20,720 --> 00:09:23,040 ALL OF WHICH IT USES INSTEAD OF 266 00:09:23,040 --> 00:09:25,280 USING THE E. COLI PROTEINS. 267 00:09:25,280 --> 00:09:27,240 IN ADDITION, THE -- AND THIS IS 268 00:09:27,240 --> 00:09:29,440 NOT REALLY IMPORTANT FOR THE 269 00:09:29,440 --> 00:09:30,640 WORK I'M GOING TO TELL YOU ABOUT 270 00:09:30,640 --> 00:09:32,960 TODAY BUT IN ADDITION, THESE 271 00:09:32,960 --> 00:09:34,480 PROTEINS THAT THE T4 SYSTEM 272 00:09:34,480 --> 00:09:35,800 BRINGS WITH IT ARE MORE SIMILAR 273 00:09:35,800 --> 00:09:38,200 TO THE EUKARYOTIC REPLICATION 274 00:09:38,200 --> 00:09:39,360 PROTEINS, THEY'RE ACTUALLY A 275 00:09:39,360 --> 00:09:41,200 MUCH BETTER MODEL FOR EUKARYOTIC 276 00:09:41,200 --> 00:09:43,800 REPLICATION PROTEINS THAN IS THE 277 00:09:43,800 --> 00:09:44,400 BACK 278 00:09:44,400 --> 00:09:47,520 BACTERIAL SYSTEM. 279 00:09:47,520 --> 00:09:51,880 SO WHAT SUBU DID WAS -- THE 280 00:09:51,880 --> 00:09:53,520 SPECIFIC REPLICATION PROTEINS OF 281 00:09:53,520 --> 00:09:54,880 INTEREST COULD BE ANY OF THE 282 00:09:54,880 --> 00:09:55,560 REPLICATION PROTEINS BUT TODAY 283 00:09:55,560 --> 00:09:56,720 I'M GOING TO TALK ABOUT THE 284 00:09:56,720 --> 00:09:58,600 SLIDING CLAMP AND THE CLAMP 285 00:09:58,600 --> 00:10:01,680 LOADER, ARE DELETED FROM THE 286 00:10:01,680 --> 00:10:03,680 T4 BACTERIOPHAGE SO THOSE SHOWN 287 00:10:03,680 --> 00:10:04,920 IN GREY DON'T HAVE THE 288 00:10:04,920 --> 00:10:08,200 REPLICATION PROTEINS, THE 289 00:10:08,200 --> 00:10:09,200 SLIDING CLAMP, THE CLAMP LOADER. 290 00:10:09,200 --> 00:10:11,920 SO THEY CAN INFECT E. COLI BUT 291 00:10:11,920 --> 00:10:14,400 THEY'RE UNABLE TO REPLICATE. 292 00:10:14,400 --> 00:10:17,240 HOWEVER, IF THE E. COLI CONTAIN 293 00:10:17,240 --> 00:10:18,040 PLASMIDS, SHOAP HERE AS THESE 294 00:10:18,040 --> 00:10:20,160 COLORED CIRCLES THAT CONTAIN 295 00:10:20,160 --> 00:10:21,600 CODING REGIONS FOR THESE 296 00:10:21,600 --> 00:10:22,760 REPLICATION PROTEINS THEN THE 297 00:10:22,760 --> 00:10:23,760 E. COLI WILL MAKE THE PROTEINS 298 00:10:23,760 --> 00:10:27,000 AND THOSE PROTEINS CAN BE USED 299 00:10:27,000 --> 00:10:30,760 BY THE T4 TO REPLICATION. 300 00:10:30,760 --> 00:10:32,680 SO IF THE E. COLI CONTAINS A 301 00:10:32,680 --> 00:10:36,720 LIBRARY THAT IS A VARIETY OF 302 00:10:36,720 --> 00:10:38,920 VARIANTS OF THESE REPLICATION 303 00:10:38,920 --> 00:10:40,400 PROTEINS THEN ONES THAT WORK 304 00:10:40,400 --> 00:10:42,600 BETTER WILL PRODUCE MORE PHAGE 305 00:10:42,600 --> 00:10:43,560 THAN ONES THAT DON'T WORK SO 306 00:10:43,560 --> 00:10:44,240 WELL. 307 00:10:44,240 --> 00:10:45,080 THERE'S ONE MORE THING THAT HAD 308 00:10:45,080 --> 00:10:47,600 TO BE DONE IN ORDER TO SET UP A 309 00:10:47,600 --> 00:10:49,720 SYSTEM WHERE WE COULD USE DEEP 310 00:10:49,720 --> 00:10:51,040 SEQUENCING TO ACTUALLY COUNT THE 311 00:10:51,040 --> 00:10:57,720 SUCCESS OF ANY VARIANT. 312 00:10:57,720 --> 00:11:01,240 THAT WAS CRISPR/CAS 12 WORKING 313 00:11:01,240 --> 00:11:01,840 IN REVERSE. 314 00:11:01,840 --> 00:11:03,400 YOU KNOW THAT NORMALLY THE 315 00:11:03,400 --> 00:11:07,360 CRISPR IS PULLING OUT DNA FROM 316 00:11:07,360 --> 00:11:09,240 THE BACK BACTERIOPHAGE AND TESTING 317 00:11:09,240 --> 00:11:10,800 IT, THAT'S THE CLASSICAL 318 00:11:10,800 --> 00:11:13,400 MECHANISM OF PROTECTION THAT THE 319 00:11:13,400 --> 00:11:16,440 CRISPR SYSTEM EMPLOYS, BUT HERE 320 00:11:16,440 --> 00:11:18,120 THE CRISPR IS USED TO PUT THE 321 00:11:18,120 --> 00:11:19,640 GENES -- SO WHEN A PHAGE INFECTS 322 00:11:19,640 --> 00:11:24,040 A PARTICULAR E. COLI, THE CRISPR 323 00:11:24,040 --> 00:11:27,200 SYSTEM WILL PUT THESE GENES BACK 324 00:11:27,200 --> 00:11:28,640 INTO THE PHAGE THROUGH CRISPR 325 00:11:28,640 --> 00:11:29,000 RECOMBINATION. 326 00:11:29,000 --> 00:11:33,360 SO IF THESE GENES ARE ACTUALLY 327 00:11:33,360 --> 00:11:35,800 SUCCESSFUL AT PRODUCING 328 00:11:35,800 --> 00:11:38,560 BACTERIOPHAGE, THEN THE 329 00:11:38,560 --> 00:11:40,280 BACTERIOPHAGE WILL LISE THE E 330 00:11:40,280 --> 00:11:43,360 OWE 331 00:11:43,360 --> 00:11:44,400 E. COLI, THE MORE OF THAT 332 00:11:44,400 --> 00:11:45,360 VARIANT WILL BE PRODUCED IN 333 00:11:45,360 --> 00:11:46,520 SOLUTION. 334 00:11:46,520 --> 00:11:48,400 SO THEN THE EXPERIMENT IS 335 00:11:48,400 --> 00:11:50,440 CONCEPTUALLY VERY SIMPLE. 336 00:11:50,440 --> 00:11:56,360 YOU USE ILLUMINA DEEP SEQUENCING 337 00:11:56,360 --> 00:12:00,200 OR ANY DEEP SEQUENCING METHOD TO 338 00:12:00,200 --> 00:12:01,200 SEQUENCE THE LIBRARY THAT WENT 339 00:12:01,200 --> 00:12:02,400 INTO THE EXPERIMENT THOUSANDS OF 340 00:12:02,400 --> 00:12:04,160 DIFFERENT VARIANTS IN THE POT, 341 00:12:04,160 --> 00:12:06,000 AND THEN YOU CAN BASICALLY 342 00:12:06,000 --> 00:12:07,600 NORMALIZE THE DATA TO TELL YOU 343 00:12:07,600 --> 00:12:08,960 WHICH VARIANTS WORK BETTER THAN 344 00:12:08,960 --> 00:12:09,480 THE OTHERS. 345 00:12:09,480 --> 00:12:13,200 AND YOU CAN ACTUALLY THEN TAKE 346 00:12:13,200 --> 00:12:15,440 THESE PHAGE, IF YOU WISH, AND 347 00:12:15,440 --> 00:12:17,440 USE THEM TO REINFECT E. COLI AND 348 00:12:17,440 --> 00:12:19,000 SET UP A COMPETITION, IN THAT 349 00:12:19,000 --> 00:12:21,680 CASE YOU'D BE DOING A NATURAL 350 00:12:21,680 --> 00:12:24,160 EVOLUTION VARIATION OF THIS 351 00:12:24,160 --> 00:12:24,440 EXPERIMENT. 352 00:12:24,440 --> 00:12:26,640 SO THE ESSENTIAL IDEA BEHIND 353 00:12:26,640 --> 00:12:32,280 THIS PLATFORM WAS PUBLISHED IN 354 00:12:32,280 --> 00:12:34,400 2021 TOWARDS -- I FORGET WHEN 355 00:12:34,400 --> 00:12:35,400 EXACTLY IT WAS PUBLISHED BUT 356 00:12:35,400 --> 00:12:36,960 IT'S IN THE LITERATURE SO YOU 357 00:12:36,960 --> 00:12:38,000 CAN READ ABOUT IF YOU'RE 358 00:12:38,000 --> 00:12:39,200 INTERESTED, BUT IT REALLY DOES 359 00:12:39,200 --> 00:12:44,320 PROVIDE US WITH A VERY ACCURATE 360 00:12:44,320 --> 00:12:46,360 MEASUREMENT OF THE FITNESS OF A 361 00:12:46,360 --> 00:12:46,600 VARIANT. 362 00:12:46,600 --> 00:12:47,720 ACCURATE IN TERMS OF WHAT WE 363 00:12:47,720 --> 00:12:49,440 KNOW ABOUT CHEMISTRY AND 364 00:12:49,440 --> 00:12:52,120 BIOCHEMISTRY AND ACCURATE IN 365 00:12:52,120 --> 00:12:53,720 TERMS OF REPRODUCIBILITY AND 366 00:12:53,720 --> 00:12:56,800 BIOCHEMICAL VALIDATION. 367 00:12:56,800 --> 00:12:59,800 SO TO GIVE YOU A SENSE OF THE 368 00:12:59,800 --> 00:13:01,080 ROBUSTNESS OF THE PLATFORM BUT 369 00:13:01,080 --> 00:13:02,920 ALSO TO EXPLAIN THE IMPORTANCE 370 00:13:02,920 --> 00:13:05,080 OF A MOTIF IN THE CLAMP LOADER 371 00:13:05,080 --> 00:13:06,720 THAT WILL BECOME CENTRAL TO SOME 372 00:13:06,720 --> 00:13:08,040 OF THE EXPERIMENTS I'M GOING TO 373 00:13:08,040 --> 00:13:10,120 END THIS LECTURE WITH, I WANT TO 374 00:13:10,120 --> 00:13:13,200 INTRODUCE YOU FIRST TO A MOTIF 375 00:13:13,200 --> 00:13:17,680 IN THESE ATPASES CALLED THE DEXD 376 00:13:17,680 --> 00:13:18,520 MOTIF. 377 00:13:18,520 --> 00:13:21,680 NOW ANYBODY WHO'S FAMILIAR WITH 378 00:13:21,680 --> 00:13:23,400 RNA HELICASES WILL KNOW ABOUT 379 00:13:23,400 --> 00:13:26,000 THE DEAD BOX HELI DID THE CASES. 380 00:13:26,000 --> 00:13:27,480 THEY WERE DISCOVERED MAYBE IN 381 00:13:27,480 --> 00:13:28,320 THE LATE EIGHTIES. 382 00:13:28,320 --> 00:13:32,400 THEY'RE A GROUP OF RNA 383 00:13:32,400 --> 00:13:33,680 HELICASES, VERY IMPORTANT AT 384 00:13:33,680 --> 00:13:35,400 DIFFERENT ASPECTS OF BIOLOGY 385 00:13:35,400 --> 00:13:37,720 THAT HAVE AT THE HEART ATA 386 00:13:37,720 --> 00:13:40,600 PROTEIN A KNOW DEEF DEAD, HENCE, 387 00:13:40,600 --> 00:13:41,720 DEAD BOX. 388 00:13:41,720 --> 00:13:45,280 THIS PARTICULAR ATPASE, THE 389 00:13:45,280 --> 00:13:47,040 T4 BACTERIOPHAGE CLAMP LOADER, 390 00:13:47,040 --> 00:13:51,840 HAS DE15 LAL NEEN SO I WRITE 391 00:13:51,840 --> 00:13:54,640 THAT AS XD. 392 00:13:54,640 --> 00:13:55,800 INSTEAD OF ALANINE. 393 00:13:55,800 --> 00:13:58,400 IN THE STRUCTURAL DIAGRAM, WHAT 394 00:13:58,400 --> 00:14:02,240 YOU SEE IS WHERE THE DEAD BOX IS 395 00:14:02,240 --> 00:14:02,800 LOCATED. 396 00:14:02,800 --> 00:14:04,920 THE D AND THE E OF THE DEAD BOX, 397 00:14:04,920 --> 00:14:07,400 ALL COLORED RED HERE, THE D AND 398 00:14:07,400 --> 00:14:09,920 THE E ALSO CALLED A WALKER A 399 00:14:09,920 --> 00:14:11,080 MOTIF BECAUSE JOHN WALKER 400 00:14:11,080 --> 00:14:11,680 IDENTIFIED THE IMPORTANCE OF 401 00:14:11,680 --> 00:14:14,600 THIS ON THE F1 ATPASE SYSTEM 402 00:14:14,600 --> 00:14:16,040 THAT MAKES ATP. 403 00:14:16,040 --> 00:14:20,280 SO THESE RESIDUES ARE ANCIENT 404 00:14:20,280 --> 00:14:20,840 EVOLUTIONARILY, AND THEY'RE 405 00:14:20,840 --> 00:14:21,600 CRUCIALLY IMPORTANT. 406 00:14:21,600 --> 00:14:23,600 THEY'RE IN THE MACHINE THAT 407 00:14:23,600 --> 00:14:26,480 MAKES ATP, THEY START OFF ALL 408 00:14:26,480 --> 00:14:27,760 KINDS OF PROCESSING AFTER 409 00:14:27,760 --> 00:14:28,080 TRANSCRIPTION. 410 00:14:28,080 --> 00:14:32,120 WHAT THE D AND THE E DO IS THAT 411 00:14:32,120 --> 00:14:33,600 THEY COORDINATE A MINE BELOW THE 412 00:14:33,600 --> 00:14:35,320 ATP SHOWN HERE IN COLORS, AND 413 00:14:35,320 --> 00:14:36,440 ACTIVATE A WATER MOLECULE WHICH 414 00:14:36,440 --> 00:14:39,200 IS NOT SHOWN HERE. 415 00:14:39,200 --> 00:14:42,840 SO THEY'RE CRUCIAL REGS RESIDUES AND 416 00:14:42,840 --> 00:14:45,680 ALWAYS FOUND IN THIS FAMILY OF 417 00:14:45,680 --> 00:14:46,840 ATPASES. 418 00:14:46,840 --> 00:14:48,800 THE THIRD ACIDIC RESIDUE IS NOT 419 00:14:48,800 --> 00:14:49,920 A CATALYTIC RESIDUE. 420 00:14:49,920 --> 00:14:51,840 WHAT YOU SEE DOING HERE IS IT'S 421 00:14:51,840 --> 00:14:53,440 COORDINATING AN ARGININE, WHICH 422 00:14:53,440 --> 00:14:54,720 YOU SEE IN YELLOW AND BLUE. 423 00:14:54,720 --> 00:14:57,160 THAT ARGININE, LABELED ARGININE 424 00:14:57,160 --> 00:14:59,400 122, IS PRESENTED BY NEIGHBORING 425 00:14:59,400 --> 00:15:00,040 SUBUNIT, NOT THIS ONE. 426 00:15:00,040 --> 00:15:03,200 SO IT'S IMPORTANT FOR 427 00:15:03,200 --> 00:15:03,680 INTERFACIAL COUPLING. 428 00:15:03,680 --> 00:15:04,920 SO THAT'S AN INTRODUCTION TO THE 429 00:15:04,920 --> 00:15:05,840 DEAD BOX MOTIF. 430 00:15:05,840 --> 00:15:07,280 AND WHAT I WANT TO SHOW YOU HERE 431 00:15:07,280 --> 00:15:09,240 ARE A FEW THINGS. 432 00:15:09,240 --> 00:15:10,600 FIRST, LET'S LOOK AT THE LOWER 433 00:15:10,600 --> 00:15:11,760 LEFT. 434 00:15:11,760 --> 00:15:15,720 IN THE LOWER LEFT IS A SEQUENCE 435 00:15:15,720 --> 00:15:15,920 LOGO. 436 00:15:15,920 --> 00:15:18,040 AND THE WAY THE SEQUENCE LOGO IS 437 00:15:18,040 --> 00:15:21,280 GENERATED BY SUBU WAS TO ALIGN 438 00:15:21,280 --> 00:15:23,480 1,000 PHAGE GENOMES, THESE ARE 439 00:15:23,480 --> 00:15:26,840 ALL BACTERIOPHAGE GENOMES OF 440 00:15:26,840 --> 00:15:27,440 WHICH T4 IS ONE. 441 00:15:27,440 --> 00:15:29,120 IT SHOWS YOU THE CONSERVATION OF 442 00:15:29,120 --> 00:15:30,920 RESIDUES ACROSS THIS SEGMENT OF 443 00:15:30,920 --> 00:15:31,240 1,000 PHAGES. 444 00:15:31,240 --> 00:15:32,600 SO IT'S ACTUALLY A PRETTY 445 00:15:32,600 --> 00:15:33,160 RESTRICTED LIBRARY. 446 00:15:33,160 --> 00:15:34,800 IT ISN'T LOOKING AT THE BROADER 447 00:15:34,800 --> 00:15:36,720 FAMILY OF ATPASES. 448 00:15:36,720 --> 00:15:39,520 AND YOU'LL SEE THE DEAD BOX 449 00:15:39,520 --> 00:15:42,480 MOTIF SPELLED OUT. 450 00:15:42,480 --> 00:15:44,080 D, E, A, D. 451 00:15:44,080 --> 00:15:47,400 THE FIRST THING TO NOTE IS THE 452 00:15:47,400 --> 00:15:48,880 T4 BACTERIOPHAGE IS 453 00:15:48,880 --> 00:15:49,200 IDIOSYNCRATIC. 454 00:15:49,200 --> 00:15:50,320 IT DOESN'T HAVE THE A, IT HAS AN 455 00:15:50,320 --> 00:15:51,000 F. 456 00:15:51,000 --> 00:15:52,680 NOW, LET'S MOVE OVER TO THE HEAT 457 00:15:52,680 --> 00:15:54,280 MAP IN THE MIDDLE HERE. 458 00:15:54,280 --> 00:15:55,400 AND WHAT THIS HEAT MAP IS 459 00:15:55,400 --> 00:15:58,120 SHOWING YOU IS A SEQUENCE THAT 460 00:15:58,120 --> 00:16:00,400 CORRESPONDS TO THE DEAD BOX 461 00:16:00,400 --> 00:16:04,640 REGION DEFDRSGLAE AS IS WRITTEN 462 00:16:04,640 --> 00:16:06,640 ALONG THE HORIZONTAL AXIS. 463 00:16:06,640 --> 00:16:09,240 AND AT EACH POSITION WITH THE 464 00:16:09,240 --> 00:16:10,080 SATURATION MUTAGENESIS 465 00:16:10,080 --> 00:16:11,440 EXPERIMENT USING THE 466 00:16:11,440 --> 00:16:14,200 BACTERIOPHAGE PLATFORM SUBU 467 00:16:14,200 --> 00:16:16,360 DEVELOPED DOES IS TO REPLACE 468 00:16:16,360 --> 00:16:19,400 THAT RESIDUE WITH ONE OF THE 20 469 00:16:19,400 --> 00:16:23,680 AMINO ACIDS OR STOP CODE STAR. 470 00:16:23,680 --> 00:16:26,560 FOR EACH ONE OF THOSE 471 00:16:26,560 --> 00:16:27,840 SUBSTITUTIONS, THE EFFECT ON 472 00:16:27,840 --> 00:16:34,960 PHAGE HELP RICATION IS IS MEASURED AND SH OWN ON A 473 00:16:34,960 --> 00:16:35,440 LOG 10 SCALE. 474 00:16:35,440 --> 00:16:36,440 WHAT DARK BLUE TELLS YOU IS THAT 475 00:16:36,440 --> 00:16:39,400 THE MUTATION LEADS TO LOSS OF 476 00:16:39,400 --> 00:16:39,800 FUNCTION. 477 00:16:39,800 --> 00:16:41,720 AND IF IT'S REALLY DARK BLUE, IT 478 00:16:41,720 --> 00:16:44,200 LIKE AT LEAST 100 FOLD DOWN. 479 00:16:44,200 --> 00:16:45,400 SO WHEN WE LOOK AT THIS, WE 480 00:16:45,400 --> 00:16:46,520 START TO SEE THAT THINGS START 481 00:16:46,520 --> 00:16:49,320 TO MAKE CHEMICAL SENSE. 482 00:16:49,320 --> 00:16:51,480 THE D AND THE E, THE FIRST TWO 483 00:16:51,480 --> 00:16:52,840 RESIDUES, CANNOT BE SUBSTITUTED 484 00:16:52,840 --> 00:16:55,240 EVEN BY ANOTHER ACIDIC RESIDUE 485 00:16:55,240 --> 00:16:57,000 WITHOUT TOTAL LOSS OF FUNCTION 486 00:16:57,000 --> 00:17:01,160 AS MEASURED BY PHAGE PRODUCTION. 487 00:17:01,160 --> 00:17:03,800 THAT MAKES SENSE BECAUSE IF 488 00:17:03,800 --> 00:17:04,560 YOU'RE COORDINATING MAGNESIUM 489 00:17:04,560 --> 00:17:06,000 AND ACTIVATING A WATER MOLECULE, 490 00:17:06,000 --> 00:17:08,680 IF YOU SHIFT THE POSITION OF THE 491 00:17:08,680 --> 00:17:09,680 CARBOXYL GROUP BY EVEN A 492 00:17:09,680 --> 00:17:11,520 FRACTION OF AN ANGSTROM, YOU 493 00:17:11,520 --> 00:17:12,000 LOSE ACTIVITY. 494 00:17:12,000 --> 00:17:13,960 AND THE EXPERIMENT, THE DEEP 495 00:17:13,960 --> 00:17:15,000 MUTAGENESIS EXPERIMENT IS 496 00:17:15,000 --> 00:17:17,120 REFLECTING THAT CHEMICAL TRUTH. 497 00:17:17,120 --> 00:17:19,640 IF YOU MOVE OVER AND LOOK AT THE 498 00:17:19,640 --> 00:17:22,440 FOURTH POSITION, WHICH IS 499 00:17:22,440 --> 00:17:23,640 ASPARTATE, YOU'LL SEE THAT'S 500 00:17:23,640 --> 00:17:25,720 MORE -- SLIGHTLY MORE TOLERANT, 501 00:17:25,720 --> 00:17:28,200 THE ASPARTATE CAN BE REPLACED BY 502 00:17:28,200 --> 00:17:29,720 GLUTAMATE IN THE T4 SYSTEM. 503 00:17:29,720 --> 00:17:31,600 AND AS I MENTIONED EARLIER, THAT 504 00:17:31,600 --> 00:17:33,440 ASPARTATE IS NOT A CATALYTIC 505 00:17:33,440 --> 00:17:35,120 RESIDUE, IT'S NOT INVOLVED IN 506 00:17:35,120 --> 00:17:36,120 POSITIONING ANYTHING FOR 507 00:17:36,120 --> 00:17:38,840 CATALYSIS OR INTERACTING WITH AN 508 00:17:38,840 --> 00:17:40,040 ARGININE FOR INTERFACIAL 509 00:17:40,040 --> 00:17:40,400 STABILIZATION. 510 00:17:40,400 --> 00:17:41,280 WE'LL COME BACK TO THAT AND LOOK 511 00:17:41,280 --> 00:17:43,560 AT IT IN MORE DETAIL LATER, BUT 512 00:17:43,560 --> 00:17:45,480 YOU CAN UNDERSTAND THAT MAYBE 513 00:17:45,480 --> 00:17:46,400 ASPEN GLUE WOULD STILL WORK. 514 00:17:46,400 --> 00:17:48,800 AND IN FACT, IF YOU GO DOWN AND 515 00:17:48,800 --> 00:17:52,320 LOOK OVER HERE, CYSTINE ALSO 516 00:17:52,320 --> 00:17:52,680 WORKS. 517 00:17:52,680 --> 00:17:56,880 AND WHEN YOU LOOK AT THIS LITTLE 518 00:17:56,880 --> 00:17:59,000 BIT OF THE HEAT MAP SHOWING 519 00:17:59,000 --> 00:18:00,480 MUTATIONAL RESPONSE, YOU START 520 00:18:00,480 --> 00:18:01,760 TO LEARN THINGS THAT ARE VERY 521 00:18:01,760 --> 00:18:02,040 INTERESTING. 522 00:18:02,040 --> 00:18:03,360 I'M NOT GOING TO BELABOR THIS 523 00:18:03,360 --> 00:18:05,120 MUCH, I JUST WANT TO TELL YOU 524 00:18:05,120 --> 00:18:06,960 WHY THESE EXPERIMENTS -- SHOW 525 00:18:06,960 --> 00:18:08,240 YOU WHY THESE EXPERIMENTS ARE SO 526 00:18:08,240 --> 00:18:09,600 INTERESTING TO US. 527 00:18:09,600 --> 00:18:10,800 SO FIRST OF ALL, THESE ARE DEAD 528 00:18:10,800 --> 00:18:12,160 BOX PROTEINS. 529 00:18:12,160 --> 00:18:17,120 BUT T4 DOESN'T HAVE A, IT HAS 530 00:18:17,120 --> 00:18:18,200 PHENYL ALANINE IN THE THIRD 531 00:18:18,200 --> 00:18:18,560 POSITION. 532 00:18:18,560 --> 00:18:20,880 WHAT THIS EXPERIMENT HERE TELLS 533 00:18:20,880 --> 00:18:22,120 US, IF YOU DRAW YOUR EYE TO THE 534 00:18:22,120 --> 00:18:24,160 PURPLE BOX, WHAT IT TELLS US IS 535 00:18:24,160 --> 00:18:28,080 THAT IF YOU ACTUALLY PUT A INTO 536 00:18:28,080 --> 00:18:30,760 THE T4 CLAMP LOADER, YOU GET 537 00:18:30,760 --> 00:18:31,200 LOSS OF FUNCTION. 538 00:18:31,200 --> 00:18:33,840 SO AGAIN, IT'S TELLING US THAT 539 00:18:33,840 --> 00:18:36,400 THIS PARTICULAR PROTEIN IS 540 00:18:36,400 --> 00:18:38,000 IDIOSYNCRATIC IN ITS SEQUENCE IT 541 00:18:38,000 --> 00:18:38,680 USES. 542 00:18:38,680 --> 00:18:39,800 IT DOESN'T AT CERTAIN POSITIONS 543 00:18:39,800 --> 00:18:44,640 MATCH THE CONSENSUS. 544 00:18:44,640 --> 00:18:46,240 MOST INTERESTINGLY IF YOU MOVE 545 00:18:46,240 --> 00:18:49,480 OVER AND LOOK AT THIS ARGININE 546 00:18:49,480 --> 00:18:51,720 111, YOU'LL SEE THIS ARGININE 547 00:18:51,720 --> 00:18:52,920 RESIDUE CANNOT BE REPLACED BY 548 00:18:52,920 --> 00:18:54,120 ANYTHING, NOT EVEN LYSINE. 549 00:18:54,120 --> 00:18:55,760 THE ONLY THING THAT WORKS IS 550 00:18:55,760 --> 00:18:56,360 ARGININE. 551 00:18:56,360 --> 00:18:59,960 AND THIS ARGININE IS FORMING AN 552 00:18:59,960 --> 00:19:01,760 ION PAIR WITH A PHOSPHATE 553 00:19:01,760 --> 00:19:02,920 BACKBONE OF DNA AND IT'S FORMING 554 00:19:02,920 --> 00:19:05,400 THE CLASSIC ARGININE PHOSPHATE 555 00:19:05,400 --> 00:19:08,600 BY DENTATE HYDROGEN BONDING 556 00:19:08,600 --> 00:19:09,840 SYSTEM THAT WE SEE IN OUR 557 00:19:09,840 --> 00:19:11,080 CRYSTAL STRUCTURE. 558 00:19:11,080 --> 00:19:12,400 YOU CAN'T REPLACE IT WITH 559 00:19:12,400 --> 00:19:13,480 ANYTHING, NOT EVEN LYSINE, 560 00:19:13,480 --> 00:19:15,120 BECAUSE LYSINE WOULDN'T DO THAT. 561 00:19:15,120 --> 00:19:17,520 BUT IF YOU LOOK AT THE SEQUENCE 562 00:19:17,520 --> 00:19:19,760 LOGO WHICH TELLS YOU WHAT THE 563 00:19:19,760 --> 00:19:20,840 POPULATION OF PHAGE CLAMP 564 00:19:20,840 --> 00:19:24,240 LOADERS IS DOING, ARGININE ISN'T 565 00:19:24,240 --> 00:19:25,520 EVEN SEEN IN THE LOGO AT THIS 566 00:19:25,520 --> 00:19:28,240 POSITION. 567 00:19:28,240 --> 00:19:31,560 SO WHAT THAT SAYS IS THAT THE 568 00:19:31,560 --> 00:19:33,120 IDIOSYNCRASIES ARE BECOMING 569 00:19:33,120 --> 00:19:35,960 EVIDENCE NOW. 570 00:19:35,960 --> 00:19:37,320 ARGININE IS CRITICAL FOR 571 00:19:37,320 --> 00:19:38,880 T4 CLAMP LOADER, MOST OF THE 572 00:19:38,880 --> 00:19:40,720 PHAGE CLAMP LOADERS DON'T USE 573 00:19:40,720 --> 00:19:41,320 ARGININE. 574 00:19:41,320 --> 00:19:42,120 THEY'RE DOING SOMETHING ELSE. 575 00:19:42,120 --> 00:19:44,080 THIS IS WHY THE DEEP MUTAGENESIS 576 00:19:44,080 --> 00:19:48,640 EXPERIMENT IS ACTUALLY GIVING US 577 00:19:48,640 --> 00:19:49,600 INFORMATION THAT'S ADDING ON TO 578 00:19:49,600 --> 00:19:50,240 THE INFORMATION THAT THE 579 00:19:50,240 --> 00:19:51,160 SEQUENCE ALIGNMENT IS TELLING 580 00:19:51,160 --> 00:19:51,600 US. 581 00:19:51,600 --> 00:19:53,320 AND THE REST OF THE TALK WILL 582 00:19:53,320 --> 00:19:55,600 REALLY BE TRYING TO UNDERSTAND 583 00:19:55,600 --> 00:19:56,800 WHAT'S THAT EXTRA INFORMATION 584 00:19:56,800 --> 00:19:59,200 THAT IT'S TELLING US AT LEAST 585 00:19:59,200 --> 00:20:01,200 OUR INITIAL GUESSES AS TO WHAT 586 00:20:01,200 --> 00:20:07,320 THAT EXTRA INFORMATION IS ABOUT. 587 00:20:07,320 --> 00:20:08,400 SO IF YOU LOOK AT WHERE THE MOST 588 00:20:08,400 --> 00:20:09,640 SENSITIVE RESIDUES ARE, THE ONES 589 00:20:09,640 --> 00:20:14,200 THAT YOU RILEY YOU REALLY CANNOT CHANGE T O 590 00:20:14,200 --> 00:20:15,760 ONE OR TWO MORE OTHER RESIDUES, 591 00:20:15,760 --> 00:20:17,040 YOU'LL FIND THESE RESIDUES 592 00:20:17,040 --> 00:20:19,920 COLORED BLEUL IN THIS BLUE IN THIS RESIDU E 593 00:20:19,920 --> 00:20:21,520 FORM A BELT AROUND THE DNA. 594 00:20:21,520 --> 00:20:23,440 THIS BELT IS VERY SATISFYING TO 595 00:20:23,440 --> 00:20:24,520 LOOK AT IN DETAIL. 596 00:20:24,520 --> 00:20:27,880 IT CONNECTS EACH ATP SHOWN IN 597 00:20:27,880 --> 00:20:29,760 RED TO DNA SHOWN IN THE CENTER 598 00:20:29,760 --> 00:20:34,560 IN BLACK, CONNECTS THE DNA AND 599 00:20:34,560 --> 00:20:36,160 THE ATP TO THE SLIDING CLAMP 600 00:20:36,160 --> 00:20:37,840 WHICH IS SHOWN UNDERNEATH, A 601 00:20:37,840 --> 00:20:39,200 WIRE DIAGRAM. 602 00:20:39,200 --> 00:20:42,600 THIS IS A CRYSTAL STRUCTURE DONE 603 00:20:42,600 --> 00:20:48,400 BY MY GROUP ABOUT 10 YEARS AGO. 604 00:20:48,400 --> 00:20:49,640 SO THE MOST CRITICAL RESIDUES 605 00:20:49,640 --> 00:20:52,400 ARE WHERE YOU EXPECT THEM TO BE. 606 00:20:52,400 --> 00:20:54,720 THIS BINDS ATP COOPERATIVELY TO 607 00:20:54,720 --> 00:20:56,240 THE DNA, TO THE SLIDING CLAMP. 608 00:20:56,240 --> 00:20:56,800 THEY INTEGRATE ALL THAT 609 00:20:56,800 --> 00:20:59,640 INFORMATION ALLOWING THE SYSTEM 610 00:20:59,640 --> 00:21:00,960 TO FIRE ATP HYDROLYSIS ONLY WHEN 611 00:21:00,960 --> 00:21:03,160 THIS IS CORRECTLY CONFIGURED. 612 00:21:03,160 --> 00:21:05,440 BUT THE REALLY STARTLING THING 613 00:21:05,440 --> 00:21:07,000 IN THIS DIAGRAM IS WHAT'S NOT 614 00:21:07,000 --> 00:21:07,800 THERE. 615 00:21:07,800 --> 00:21:09,400 EVERY SINGLE RESIDUE IN THIS 616 00:21:09,400 --> 00:21:12,960 STRUCTURE, IN THE SLIDING CLAMP, 617 00:21:12,960 --> 00:21:14,200 IN ALL SUBUNITS INCLUDING THE 618 00:21:14,200 --> 00:21:15,560 ONE COLORED GREEN WERE MUTATED 619 00:21:15,560 --> 00:21:17,200 IN SUBU'S EXPERIMENT. 620 00:21:17,200 --> 00:21:18,360 BUT THEY'RE NOT COLORED BLUE. 621 00:21:18,360 --> 00:21:19,640 AND WHAT THAT MEANS IS, ALL 622 00:21:19,640 --> 00:21:24,360 THOSE RESIDUES THAT ARE NOT 623 00:21:24,360 --> 00:21:25,400 COLORED, WHICH IS THE GREAT 624 00:21:25,400 --> 00:21:27,000 MAJORITY OF THE STRUCTURE, CAN, 625 00:21:27,000 --> 00:21:28,320 IN FACT, ACCEPT MANY, MANY 626 00:21:28,320 --> 00:21:30,680 SUBSTITUTIONS AND STILL KEEP 627 00:21:30,680 --> 00:21:30,920 WORKING. 628 00:21:30,920 --> 00:21:32,240 SO WHAT THIS DIAGRAM ACTUALLY 629 00:21:32,240 --> 00:21:33,560 REFLECTS IS SOMETHING WE ALREADY 630 00:21:33,560 --> 00:21:35,040 KNOW FROM THE MUTAGENESIS 631 00:21:35,040 --> 00:21:37,640 STUDIES OF SMALL PROTEINS THAT'S 632 00:21:37,640 --> 00:21:38,920 REFLECTING THE AMAZING TOLERANCE 633 00:21:38,920 --> 00:21:40,400 OF PROTEINS TO MUTATIONAL 634 00:21:40,400 --> 00:21:41,280 INSULT. 635 00:21:41,280 --> 00:21:44,120 BUT IT'S SAYING THAT EVEN FOR A 636 00:21:44,120 --> 00:21:45,920 LARGE SYSTEM LIKE THIS, DOING 637 00:21:45,920 --> 00:21:46,880 SOMETHING'S CRUCIAL AS 638 00:21:46,880 --> 00:21:50,200 REPLICATING THE DNA, IT CAN 639 00:21:50,200 --> 00:21:51,920 ACCEPT SINGLE POINT MUTATIONS. 640 00:21:51,920 --> 00:21:55,560 SOME OF THEM QUITE STARTLINGLY 641 00:21:55,560 --> 00:21:56,280 DIFFERENT FROM THE SEQUENCE 642 00:21:56,280 --> 00:21:57,160 WHILE KEEPING ON WORKING. 643 00:21:57,160 --> 00:22:03,240 NOW, WHEN WE LOOK AT THE 644 00:22:03,240 --> 00:22:04,560 RESIDUES REALLY SENSITIVE TO 645 00:22:04,560 --> 00:22:07,200 MUTATION, WE DID DISCOVER ONE 646 00:22:07,200 --> 00:22:11,440 THING, WHICH IS ACTUALLY 647 00:22:11,440 --> 00:22:12,400 IMPORTANT EVIDENCE IN THE 648 00:22:12,400 --> 00:22:13,280 STRUCTURE BUT I WANT TO POINT 649 00:22:13,280 --> 00:22:14,640 THIS OUT TO YOU JUST TO FINISH 650 00:22:14,640 --> 00:22:15,760 WRAPPING UP THE RESULTS OF THE 651 00:22:15,760 --> 00:22:16,640 FIRST ROUND OF EXPERIMENTS. 652 00:22:16,640 --> 00:22:19,440 AND THAT IS THAT WE KNOW THE 653 00:22:19,440 --> 00:22:21,360 SYSTEM IS HIGHLY COOPERATIVE 654 00:22:21,360 --> 00:22:25,720 WHEN IT COMES TO DNA BINDING AND 655 00:22:25,720 --> 00:22:26,280 ATP BINDING. 656 00:22:26,280 --> 00:22:28,800 SO YOU'D EXPECT SINCE THERE ARE 657 00:22:28,800 --> 00:22:31,000 FOUR ATP BINDING SITES AND THE 658 00:22:31,000 --> 00:22:32,600 ATP IS COLORED RED HERE, YOU'D 659 00:22:32,600 --> 00:22:35,760 EXPECT THE SYSTEM HAS ALLOSTERIC 660 00:22:35,760 --> 00:22:36,800 COMMUNICATION THAT GOES FROM ONE 661 00:22:36,800 --> 00:22:38,440 SITE TO THE OTHER, IT SURELY 662 00:22:38,440 --> 00:22:39,400 DOES, AND TO DNA. 663 00:22:39,400 --> 00:22:43,520 BUT WHAT YOU'LL SEAL IN SEE IN THIS VIEW 664 00:22:43,520 --> 00:22:45,000 IS THERE'S REALLY NO 665 00:22:45,000 --> 00:22:46,800 CONSERVATION OF SEQUENCE BETWEEN 666 00:22:46,800 --> 00:22:50,200 EACH ATP SIE. WE NOW SITE. 667 00:22:50,200 --> 00:22:51,600 WE NOW KNOW THAT'S BECAUSE 668 00:22:51,600 --> 00:22:53,200 SECONDARY ELEMENTS PLAY A 669 00:22:53,200 --> 00:22:54,240 CRITICAL ROLE IN THE 670 00:22:54,240 --> 00:22:55,360 COMMUNICATIONS, WE'VE DONE 671 00:22:55,360 --> 00:23:00,480 EXPERIMENTS TO ADDRESS THAT SO 672 00:23:00,480 --> 00:23:02,920 YOU DON'T NEED A SPECIFIC 673 00:23:02,920 --> 00:23:04,120 SEQUENCE, THERE'S ONLY ONE POINT 674 00:23:04,120 --> 00:23:07,280 OF STRICT CONSERVATION 675 00:23:07,280 --> 00:23:09,520 CONNECTING THE ATP BINDING SITES 676 00:23:09,520 --> 00:23:13,880 AND THAT'S GLUTAMINE 118. 677 00:23:13,880 --> 00:23:15,160 WE HADN'T NOTICED ITS IMPORTANCE 678 00:23:15,160 --> 00:23:15,480 BEFORE. 679 00:23:15,480 --> 00:23:18,480 IF YOU LOOK AT WHAT GLUTAMINE 680 00:23:18,480 --> 00:23:20,200 118 IS DOING, IT'S A GLUTAMINE 681 00:23:20,200 --> 00:23:21,680 SITE CHAIN THAT DOES WHAT 682 00:23:21,680 --> 00:23:23,520 GLUTAMINE IS SO GOOD AT DOING. 683 00:23:23,520 --> 00:23:25,000 GLUTAMINE CAN MIMIC -- IT GOT 684 00:23:25,000 --> 00:23:27,960 THE CAPACITY TO MIMIC BACKBONE 685 00:23:27,960 --> 00:23:29,480 HYDROGEN BONDING IN A BETA 686 00:23:29,480 --> 00:23:29,800 SHEET. 687 00:23:29,800 --> 00:23:32,200 SO WHAT IT'S ACTUALLY DOING IS 688 00:23:32,200 --> 00:23:34,600 IT'S STAPLING THIS PURPLE HELIX 689 00:23:34,600 --> 00:23:36,320 TO THIS YELLOW HELICAL STRUCTURE 690 00:23:36,320 --> 00:23:40,160 AND LOOP HERE. 691 00:23:40,160 --> 00:23:44,440 WHEN YOU SEE THIS, WE CALL THESE 692 00:23:44,440 --> 00:23:46,200 THREE HELICES, YELLOW, MAGENTA 693 00:23:46,200 --> 00:23:49,720 AND ORANGE, WE CALLED THEM THE 694 00:23:49,720 --> 00:23:51,200 CENTRAL COUPLER TOGETHER BECAUSE 695 00:23:51,200 --> 00:23:52,080 YOU'LL SEE WHAT'S HAPPENING IS 696 00:23:52,080 --> 00:23:53,400 THIS IS THE POINT WHERE THE 697 00:23:53,400 --> 00:23:55,160 CLAMP LOADER IS INTEGRATING ALL 698 00:23:55,160 --> 00:23:58,000 THE INFORMATION THAT IT NEEDS TO 699 00:23:58,000 --> 00:23:58,640 INTEGRATE. 700 00:23:58,640 --> 00:24:02,600 IN RED HERE IS THE DEAD BOX, OR 701 00:24:02,600 --> 00:24:05,200 DEXD OF THIS PARTICULAR VARIANT, 702 00:24:05,200 --> 00:24:08,040 AS PAR TART, GLUTAMATE X 703 00:24:08,040 --> 00:24:09,240 ASPARTATE AND IT'S LEADING INTO 704 00:24:09,240 --> 00:24:10,960 THE MAGENTA HELIX. 705 00:24:10,960 --> 00:24:13,120 THE GLUTAMINE IS COUPLING THAT 706 00:24:13,120 --> 00:24:14,400 MAGENTA HELIX TO THIS YELLOW 707 00:24:14,400 --> 00:24:17,240 STRUCTURE WHICH IS PRESENTING AN 708 00:24:17,240 --> 00:24:20,320 ARGININE CALLED THE ARGININE 709 00:24:20,320 --> 00:24:21,000 FINGER BORROWED FROM THE RAS 710 00:24:21,000 --> 00:24:21,400 LITERATURE. 711 00:24:21,400 --> 00:24:23,800 THE ARGININE FINGER IS POINTING 712 00:24:23,800 --> 00:24:25,280 TO A SUBUNIT NOT SHOWN HERE, THE 713 00:24:25,280 --> 00:24:26,440 ONE BEFORE THIS ONE IN THE 714 00:24:26,440 --> 00:24:27,640 ASSEMBLY, AND THE ARGININE IS 715 00:24:27,640 --> 00:24:28,800 DOING JUST WHAT THE ARGININE 716 00:24:28,800 --> 00:24:30,600 FINGER OF A RAS GAP DOES. 717 00:24:30,600 --> 00:24:33,040 IT'S POINTING RIGHT AT THE 718 00:24:33,040 --> 00:24:34,600 PHOSPHATE CENTER THAT'S GOING TO 719 00:24:34,600 --> 00:24:37,200 BE HYDROLYZED, AND IT HAS TO BE 720 00:24:37,200 --> 00:24:38,360 POSITIONED CORRECTLY FOR THE 721 00:24:38,360 --> 00:24:40,320 SUBUNIT BEFORE THIS ONE TO 722 00:24:40,320 --> 00:24:41,600 HYDROLYZE ATP. 723 00:24:41,600 --> 00:24:43,440 SO THIS STRUCTURE HERE IS 724 00:24:43,440 --> 00:24:45,160 COUPLING THE ARGININE FINGER TO 725 00:24:45,160 --> 00:24:47,280 THE DEAD BOX. 726 00:24:47,280 --> 00:24:49,600 AND THE MAGENTA HELIX IS ALSO 727 00:24:49,600 --> 00:24:50,360 TOUCHING DNA. 728 00:24:50,360 --> 00:24:52,000 AND THE ORANGE HELIX IS TOUCHING 729 00:24:52,000 --> 00:24:54,360 BOTH THE CLAMP AND THE DNA. 730 00:24:54,360 --> 00:24:55,680 SO IT'S INTEGRATING EVERYTHING 731 00:24:55,680 --> 00:24:58,000 AND THAT'S WHAT THE GLUTAMINE 732 00:24:58,000 --> 00:25:00,840 118 IS DOING. 733 00:25:00,840 --> 00:25:03,200 I COULD TELL YOU MORE ABOUT 734 00:25:03,200 --> 00:25:03,680 GLUTAMINE 118 BUT IN THE 735 00:25:03,680 --> 00:25:04,640 INTEREST OF PRESENTING NEW 736 00:25:04,640 --> 00:25:06,200 INFORMATION THAT ISN'T YET STUFF 737 00:25:06,200 --> 00:25:07,400 THAT WE'VE SOLIDIFIED AND 738 00:25:07,400 --> 00:25:08,760 PUBLISHED, I'M GOING TO MOVE ON 739 00:25:08,760 --> 00:25:10,120 BUT FIRST I'M JUST GOING TO SAY 740 00:25:10,120 --> 00:25:13,400 THAT THE POWER OF THIS PLATFORM 741 00:25:13,400 --> 00:25:15,800 IS SUCH THAT SUBU COULD ASK A 742 00:25:15,800 --> 00:25:17,040 VERY SIMPLE QUESTION, WHAT WOULD 743 00:25:17,040 --> 00:25:24,160 HAPPEN IF YOU MUTE TATED MUTATED GLUTAMIN E 744 00:25:24,160 --> 00:25:25,840 118 AND -- HOW QUICKLY COULD YOU 745 00:25:25,840 --> 00:25:27,000 GET FUNCTION BACK IN JUST ONE 746 00:25:27,000 --> 00:25:27,400 MUTATION. 747 00:25:27,400 --> 00:25:29,880 AND THE ANSWER IS, YOU COULD. 748 00:25:29,880 --> 00:25:31,960 SO WHAT THIS DIAGRAM SHOWS YOU 749 00:25:31,960 --> 00:25:34,600 IS THE RESULT OF A DEEP 750 00:25:34,600 --> 00:25:37,400 MUTAGENESIS SCREEN, IN WHICH 751 00:25:37,400 --> 00:25:38,720 GLUTAMINE 118 WAS FIXED TO 752 00:25:38,720 --> 00:25:39,080 ASPARAGINE. 753 00:25:39,080 --> 00:25:46,200 IT'S A SORT OF -- LEAST DAMAGES. 754 00:25:46,200 --> 00:25:47,200 THE ASPARAGINE IS THE LEAST 755 00:25:47,200 --> 00:25:47,600 DAMAGING. 756 00:25:47,600 --> 00:25:48,920 WHAT THIS DIAGRAM SHOWS YOU ARE 757 00:25:48,920 --> 00:25:52,080 THE RESULTS OF TWO EXPERIMENTS, 758 00:25:52,080 --> 00:25:52,960 TWO SATURATION MUTAGENESIS 759 00:25:52,960 --> 00:25:54,160 EXPERIMENTS CONDUCTED 760 00:25:54,160 --> 00:25:54,480 INDEPENDENTLY. 761 00:25:54,480 --> 00:25:57,680 SO YOU JUST HAVE FITNESS ON THE 762 00:25:57,680 --> 00:26:01,680 X AND Y AXIS ON A LOG 10 SCALE 763 00:26:01,680 --> 00:26:03,320 SO ZERO MEANS IT HAS THE FITNESS 764 00:26:03,320 --> 00:26:08,280 OF THE ORIGINAL Q118N MUTANT. 765 00:26:08,280 --> 00:26:09,720 NEGATIVE NUMBERS MEAN THE 766 00:26:09,720 --> 00:26:11,040 ADDITIONAL MUTATIONS JUST KILL 767 00:26:11,040 --> 00:26:13,400 THE CLAMP LOADER AND POSITIVE 768 00:26:13,400 --> 00:26:15,080 NUMBER MEAN YOU ACTUALLY GOT 769 00:26:15,080 --> 00:26:16,120 BACK ACTIVITY. 770 00:26:16,120 --> 00:26:18,200 SO 3 WOULD MEAN YOU GOT A 771 00:26:18,200 --> 00:26:18,880 THOUSAND FOLD TIMES MORE 772 00:26:18,880 --> 00:26:21,640 ACTIVITY THAN THE Q118N, AND SO 773 00:26:21,640 --> 00:26:24,040 IN IN FACT, WHAT'S SHOWN HERE IS 774 00:26:24,040 --> 00:26:27,800 A SINGLE MUTATION, Q118 AND 775 00:26:27,800 --> 00:26:29,640 GLYCINE 143N, THAT BRINGS BACK 776 00:26:29,640 --> 00:26:31,920 ACTIVITY IN TWO INDEPENDENT 777 00:26:31,920 --> 00:26:33,880 TRIALS. 778 00:26:33,880 --> 00:26:37,680 GLYCINE 143N IS A RESIDUE THAT'S 779 00:26:37,680 --> 00:26:38,320 VERY INTERESTING BECAUSE IT'S 780 00:26:38,320 --> 00:26:41,400 NOT ACTUALLY INTERESTING IN THE 781 00:26:41,400 --> 00:26:42,720 ORIGINAL SATURATION MUTAGENESIS 782 00:26:42,720 --> 00:26:43,360 EXPERIMENT. 783 00:26:43,360 --> 00:26:45,520 SO FIRST OF ALL, WHAT IT DOES, 784 00:26:45,520 --> 00:26:49,640 THIS GLYCINE RES DUAL RESIDUE 143 IS 785 00:26:49,640 --> 00:26:51,880 LOCATED CLOSE TO WHERE GLUTAMINE 786 00:26:51,880 --> 00:26:53,280 118 IS IN THE STRUCTURES, IT'S 787 00:26:53,280 --> 00:26:59,480 REPLACED BY ASPARE ASPARAGINE, WHAT THE 788 00:26:59,480 --> 00:27:02,400 SYSTEM DOES IS RESPOND BY 789 00:27:02,400 --> 00:27:04,360 PUTTING BACK AN ASPARAGINE WITH 790 00:27:04,360 --> 00:27:05,400 HYDROGEN BONDING CAPACITY RIGHT 791 00:27:05,400 --> 00:27:06,200 NEXT TO IT. 792 00:27:06,200 --> 00:27:08,920 WHAT'S INTERESTING IS TO LOOK AT 793 00:27:08,920 --> 00:27:09,960 WHERE THIS RESIDUE IS AND WHAT 794 00:27:09,960 --> 00:27:10,800 IT'S DOING. 795 00:27:10,800 --> 00:27:12,400 SO FIRST OF ALL, GLYCINE 143, 796 00:27:12,400 --> 00:27:14,800 THIS IS A RESIDUE THAT WILL COME 797 00:27:14,800 --> 00:27:15,800 BACK IN THINGS THAT I'M GOING TO 798 00:27:15,800 --> 00:27:18,800 BE DISCUSSING. 799 00:27:18,800 --> 00:27:20,440 GLYCINE 143 IS NOT AT A SITE OF 800 00:27:20,440 --> 00:27:21,880 CONSERVATION. 801 00:27:21,880 --> 00:27:23,240 OR AT A SITE OF MUTATIONAL 802 00:27:23,240 --> 00:27:25,200 SENSITIVITY. 803 00:27:25,200 --> 00:27:27,600 WHICH MAKES SENSE BECAUSE IF 804 00:27:27,600 --> 00:27:28,800 YOU'VE DAMAGED THE SYSTEM BY 805 00:27:28,800 --> 00:27:30,520 MAKING A MUTATION THAT CAUSES 806 00:27:30,520 --> 00:27:31,800 LOSS OF FUNCTION, THE RESPONSE 807 00:27:31,800 --> 00:27:35,280 WILL PROBABLY COME EASIER FROM A 808 00:27:35,280 --> 00:27:37,800 SYSTEM WHERE THE SYSTEM IS 809 00:27:37,800 --> 00:27:38,320 TOLERANT. 810 00:27:38,320 --> 00:27:39,560 SO THAT'S ONE THING. 811 00:27:39,560 --> 00:27:42,280 SO IT'S AT A MUTATIONALLY 812 00:27:42,280 --> 00:27:42,800 TOLERANT SITE. 813 00:27:42,800 --> 00:27:45,480 THE SECOND THING IS IT'S AN 814 00:27:45,480 --> 00:27:45,920 IDIOSYNCRATIC SITE. 815 00:27:45,920 --> 00:27:48,480 BY THAT, I MEAN THE T4 CLAMP 816 00:27:48,480 --> 00:27:49,680 LOADER HAS GLYCINE THERE, BUT 817 00:27:49,680 --> 00:27:52,040 THE POPULATION TENDS TO HAVE 818 00:27:52,040 --> 00:27:53,000 LYSINE OR ARGININE. 819 00:27:53,000 --> 00:27:54,280 AND THAT MAKES SENSE BECAUSE IF 820 00:27:54,280 --> 00:27:56,400 YOU SEE WHERE THAT 143 POSITION 821 00:27:56,400 --> 00:27:58,160 IS, IT'S RIGHT NEXT TO DNA. 822 00:27:58,160 --> 00:27:59,720 SO YOU CAN START TO SEE, I 823 00:27:59,720 --> 00:28:02,400 SHOWED YOU EARLIER ARGININE 824 00:28:02,400 --> 00:28:04,120 THAT'S CRITICAL IN THE T4 CLAMP 825 00:28:04,120 --> 00:28:05,760 LOADER WHICH IN THIS CLAMP 826 00:28:05,760 --> 00:28:07,840 LOADER AT ANOTHER POSITION, 827 00:28:07,840 --> 00:28:09,520 THERE ISN'T AN ARGININE OR 828 00:28:09,520 --> 00:28:11,000 LYSINE, IT'S FOR SOME REASON 829 00:28:11,000 --> 00:28:11,200 GLYCINE. 830 00:28:11,200 --> 00:28:14,360 SO YOU CAN SEE HOW THE SYSTEM IS 831 00:28:14,360 --> 00:28:15,360 IDIOSYNCRATIC IN RECOGNITION OF 832 00:28:15,360 --> 00:28:16,200 DNA AND THAT'S WHERE IT GETS 833 00:28:16,200 --> 00:28:21,720 BACK THE CAPACITY TO REPAIR A 834 00:28:21,720 --> 00:28:22,000 FUNCTION. 835 00:28:22,000 --> 00:28:22,960 SO WE WERE CURIOUS ABOUT THIS 836 00:28:22,960 --> 00:28:24,000 CAPACITY TO REPAIR FUNCTION AND 837 00:28:24,000 --> 00:28:25,880 I WANT TO NOW TURN OUR ATTENTION 838 00:28:25,880 --> 00:28:28,960 TO THE SUBUNIT AND THE T4 CLAMP 839 00:28:28,960 --> 00:28:32,560 LOADER WHICH WE CALL THE CLASP. 840 00:28:32,560 --> 00:28:34,200 IT'S JUST A SUBUNIT ALSO FOUND 841 00:28:34,200 --> 00:28:37,040 IN THE RFC COMPLEX IN 842 00:28:37,040 --> 00:28:37,480 EUKARYOTES. 843 00:28:37,480 --> 00:28:40,120 IF YOU LOOK AT THIS CRYSTAL 844 00:28:40,120 --> 00:28:41,560 STRUCTURE, I WOULD SAY AS A 845 00:28:41,560 --> 00:28:42,200 STRUCTURAL BIOLOGIST THAT IT'S 846 00:28:42,200 --> 00:28:45,320 THE MOST IMPORTANT SUBUNIT IN 847 00:28:45,320 --> 00:28:46,120 THE CLAMP LOADER. 848 00:28:46,120 --> 00:28:49,160 EVEN THOUGH IT'S NOT AN ATPASE 849 00:28:49,160 --> 00:28:49,720 SUBUNIT. 850 00:28:49,720 --> 00:28:51,280 SO LOOK AT WHAT IT DOING. 851 00:28:51,280 --> 00:28:52,120 IT'S ACTUALLY CONNECTING TO BOTH 852 00:28:52,120 --> 00:28:54,000 SIDES OF THE CLAMP. 853 00:28:54,000 --> 00:28:56,160 SO IT'S OPENED THE SLIDING CLAMP 854 00:28:56,160 --> 00:28:57,600 THE STRUCTURE, THE SLIDING CLAMP 855 00:28:57,600 --> 00:28:59,240 IN GREY IS OPEN AND IT'S 856 00:28:59,240 --> 00:29:00,880 TOUCHING BOTH SIDES OF THE OPEN 857 00:29:00,880 --> 00:29:01,800 SLIDING CLAMP. 858 00:29:01,800 --> 00:29:05,400 IT'S TOUCHING THE PRIMARY 859 00:29:05,400 --> 00:29:06,760 TEMPLATE JUNCTION AND IN FACT IT 860 00:29:06,760 --> 00:29:07,880 SPECIFICALLY RECOGNIZES THE 861 00:29:07,880 --> 00:29:09,000 PRIMARY TEMPLATE JUNCTION 862 00:29:09,000 --> 00:29:10,040 WITHOUT GOING INTO DETAIL, IT'S 863 00:29:10,040 --> 00:29:12,440 DOING SOMETHING THAT ALL 864 00:29:12,440 --> 00:29:14,400 HELICASES DO, USING A 865 00:29:14,400 --> 00:29:15,640 HYDROPHOBIC RESIDUE TO WEDGE IN 866 00:29:15,640 --> 00:29:21,320 BETWEEN THE STRANDS OF DNA, SO 867 00:29:21,320 --> 00:29:22,680 IT'S VERY, VERY IMPORTANT 868 00:29:22,680 --> 00:29:23,000 STRUCTURALLY. 869 00:29:23,000 --> 00:29:25,120 SO IT'S A DISAPPOINTMENT OR 870 00:29:25,120 --> 00:29:26,960 SHOCK TO LOOK AT THE RESULTS OF 871 00:29:26,960 --> 00:29:28,640 THE SATURATION MUTAGENESIS SHOWN 872 00:29:28,640 --> 00:29:30,920 HERE AS A HEAT MAP AND SEE THAT 873 00:29:30,920 --> 00:29:32,640 IN FACT IT'S NOT MUTATIONAL 874 00:29:32,640 --> 00:29:34,440 LISTENSTIVE. 875 00:29:34,440 --> 00:29:36,520 WHAT THIS HISTOGRAM DOES IS SHOW 876 00:29:36,520 --> 00:29:38,720 YOU THE DISTRIBUTION OF FITNESS 877 00:29:38,720 --> 00:29:40,000 EFFECTS FOR EVERY SINGLE POINT 878 00:29:40,000 --> 00:29:41,000 MUTATION ON THIS LOG 10 SCALE 879 00:29:41,000 --> 00:29:45,680 AND YOU'LL SEE BASICALLY MOST 880 00:29:45,680 --> 00:29:46,840 MUTATIONS DON'T DO ANYTHING EVEN 881 00:29:46,840 --> 00:29:49,280 THOUGH THE MOST DISTURBING ONES 882 00:29:49,280 --> 00:29:50,600 PROBABLY NOT VERY DISTURBING. 883 00:29:50,600 --> 00:29:53,200 SO THAT GOES COUNTER TO THE 884 00:29:53,200 --> 00:29:54,920 POINT THAT THIS GREEN STRUCTURE 885 00:29:54,920 --> 00:29:56,400 SO SO CENTRAL AND SO IMPORTANT 886 00:29:56,400 --> 00:29:58,640 AND IT SAYS, WHOA, IS IT THAT WE 887 00:29:58,640 --> 00:30:00,360 CAN'T READ WHAT THE STRUCTURE IS 888 00:30:00,360 --> 00:30:00,960 TELLING US? 889 00:30:00,960 --> 00:30:03,240 SO NOW WE WONDERED, WHAT WOULD 890 00:30:03,240 --> 00:30:04,800 HAPPEN IF WE TOOK THE CLAMP 891 00:30:04,800 --> 00:30:06,520 LOADER WHICH IS RUNNING UNDER 892 00:30:06,520 --> 00:30:09,600 IDEAL CONDITIONS IN E. COLI WITH 893 00:30:09,600 --> 00:30:11,240 LOTS OF LURIA BROTH AND 894 00:30:11,240 --> 00:30:12,000 EVERYTHING IS HAPPY. 895 00:30:12,000 --> 00:30:14,080 WHAT WOULD HAPPEN IF WE TIPPED 896 00:30:14,080 --> 00:30:15,200 IT OFF BALANCE? 897 00:30:15,200 --> 00:30:19,320 AND WHAT SUBU DID WAS TO MAKE A 898 00:30:19,320 --> 00:30:20,240 CHIMERIC CLAMP LOADER. 899 00:30:20,240 --> 00:30:22,680 SO THIS IS HERE IN PURPLE ARE 900 00:30:22,680 --> 00:30:27,000 THE SUBUNITS OF THE WILD TYPE T4 CLAMP LOA DER. 901 00:30:27,000 --> 00:30:29,680 ALL THESE PURPLE SUBUNITS ARE 902 00:30:29,680 --> 00:30:31,400 ENCODED BY ONE GENE, AND SO 903 00:30:31,400 --> 00:30:33,520 THERE ARE FOUR COPIES OF THAT 904 00:30:33,520 --> 00:30:42,360 GENE SO THERE ARE FOUR AAA+ 905 00:30:42,360 --> 00:30:42,800 ATPASES GENERATED. 906 00:30:42,800 --> 00:30:44,640 HERE ARE DIFFERENT CLAMP 907 00:30:44,640 --> 00:30:46,480 LOADERS, THERE'S SOME EUKARYOTIC 908 00:30:46,480 --> 00:30:49,720 ONES, SO BACTERIOPHAGE ONES SO 909 00:30:49,720 --> 00:30:51,080 HE CREATED CHIMERIC CLAMP 910 00:30:51,080 --> 00:30:54,520 LOADERS IN WHICH HE SWAMPED IN A 911 00:30:54,520 --> 00:30:56,600 AAA+ MODULE FROM ANOTHER CLAMP 912 00:30:56,600 --> 00:30:56,840 LOADER. 913 00:30:56,840 --> 00:30:59,640 THIS IS AN EXAMPLE OF A CHIMERIC 914 00:30:59,640 --> 00:31:02,800 CLAMP LOADER WITH A AAA+ THAT 915 00:31:02,800 --> 00:31:04,080 COMES FROM ANOTHER CLAMP LOADER. 916 00:31:04,080 --> 00:31:09,040 THE SO CALLED COLLAR DOMAINS ARE 917 00:31:09,040 --> 00:31:10,480 FROM T4 AND THE GREEN CLASP 918 00:31:10,480 --> 00:31:11,840 SUBUNIT IS FROM T4. 919 00:31:11,840 --> 00:31:14,200 AND THE SLIDING CLAMP IS FROM 920 00:31:14,200 --> 00:31:16,720 T4. 921 00:31:16,720 --> 00:31:19,960 NOW, WHAT HAPPENS IS THAT IF YOU 922 00:31:19,960 --> 00:31:21,160 SWAP IN EUKARYOTIC CLAMP LOADERS 923 00:31:21,160 --> 00:31:23,160 THAT ARE QUITE 924 00:31:23,160 --> 00:31:23,760 SEQUENCE-DIVERGENT, YOU GET 925 00:31:23,760 --> 00:31:24,400 COMPLEES LOSS OF FUNCTION. 926 00:31:24,400 --> 00:31:27,000 BY THAT, I MEAN SUBU CAN'T 927 00:31:27,000 --> 00:31:27,480 RECOVER ANY PHAGE. 928 00:31:27,480 --> 00:31:29,640 IF YOU CAN'T RECOVER ANY PHAGE 929 00:31:29,640 --> 00:31:31,000 TO SEQUENCE, YOU DON'T KNOW 930 00:31:31,000 --> 00:31:31,680 WHAT'S GOING ON. 931 00:31:31,680 --> 00:31:33,520 BUT FOR THIS PARTICULAR PHAGE 932 00:31:33,520 --> 00:31:35,640 CALLED RR2, WHEN HE SWAPS THAT 933 00:31:35,640 --> 00:31:38,920 IN AT 64% IDENTICAL IN SEQUENCE 934 00:31:38,920 --> 00:31:40,680 TO T4. 935 00:31:40,680 --> 00:31:42,080 VERY SIMILAR. 936 00:31:42,080 --> 00:31:44,720 WHEN HE SWAPS THAT IN, HE GETS A 937 00:31:44,720 --> 00:31:46,280 4,000 FOLD REDUCTION WHICH SEEPS 938 00:31:46,280 --> 00:31:47,640 LIKE A LOT BUT IT ACTUALLY 939 00:31:47,640 --> 00:31:49,520 ENOUGH TO PURIFY PHAGE AND 940 00:31:49,520 --> 00:31:51,840 ACTUALLY DOING A SECOND ROUND OF 941 00:31:51,840 --> 00:31:53,080 COMPETITION, HE'S ACTUALLY ABLE 942 00:31:53,080 --> 00:31:54,480 TO GROW UP THE WINNERS AND 943 00:31:54,480 --> 00:31:55,800 SEQUENCE THEM. 944 00:31:55,800 --> 00:31:58,640 WHEN HE SEQUENCES THEM, THE 945 00:31:58,640 --> 00:32:01,040 STHINGS 946 00:32:01,040 --> 00:32:02,320 THINGS THAT EMERGE -- HE'S NOW 947 00:32:02,320 --> 00:32:03,920 GOING TO MUTATE THE GREEN 948 00:32:03,920 --> 00:32:04,160 SUBUNIT. 949 00:32:04,160 --> 00:32:06,920 IN OTHER WORDS THE CHIMERAS 950 00:32:06,920 --> 00:32:10,200 REPLACE THE ATPASE -- THE SYSTEM 951 00:32:10,200 --> 00:32:13,560 IS NOW REALLY HOBBLED BUT HE 952 00:32:13,560 --> 00:32:15,080 MAKES MUTATIONS IN THE CLASP 953 00:32:15,080 --> 00:32:16,600 SUBUNIT AND ASKS HOW QUICKLY 954 00:32:16,600 --> 00:32:17,560 DOES THE SYSTEM RECOVER. 955 00:32:17,560 --> 00:32:19,320 THE GREAT THING IS THAT IT 956 00:32:19,320 --> 00:32:20,840 RECOVERS VERY QUICKLY. 957 00:32:20,840 --> 00:32:22,600 BUT BEFORE I SHOW YOU HOW MUCH 958 00:32:22,600 --> 00:32:24,680 IT RECOVERS, LET FOCUS ON THIS 959 00:32:24,680 --> 00:32:26,760 DIAGRAM HERE, WHICH IS A SMALL 960 00:32:26,760 --> 00:32:29,680 PART OF THE FITNESS MAP OF 961 00:32:29,680 --> 00:32:31,160 SINGLE SITE SATURATION 962 00:32:31,160 --> 00:32:32,520 MUTAGENESIS THAT IS ALL POSSIBLE 963 00:32:32,520 --> 00:32:34,600 KNEW 964 00:32:34,600 --> 00:32:35,080 MUTATIONS. 965 00:32:35,080 --> 00:32:37,880 OF IT'S A SMALL PART OF THE 966 00:32:37,880 --> 00:32:38,240 CLASP SUBUNIT. 967 00:32:38,240 --> 00:32:39,840 WHAT I WANT TO SHOW YOU NOW IS 968 00:32:39,840 --> 00:32:41,000 THE DEEP BLUE. 969 00:32:41,000 --> 00:32:42,760 THIS IS EXTREMELY GRATIFYING 970 00:32:42,760 --> 00:32:45,000 BECAUSE IF YOU GO AND ANALYZE 971 00:32:45,000 --> 00:32:46,680 THE DEEP BLUE, YOU'LL SEE THAT 972 00:32:46,680 --> 00:32:47,600 IT MAKES SENSE. 973 00:32:47,600 --> 00:32:49,240 SO OUR STRUCTURES WERE, IN FACT, 974 00:32:49,240 --> 00:32:52,480 TELLING US WHAT THE POINTS OF 975 00:32:52,480 --> 00:32:53,440 SENSITIVITY ARE. 976 00:32:53,440 --> 00:32:54,640 THESE TWO RESIDUES THAT ARE DEEP 977 00:32:54,640 --> 00:33:00,000 BLUE HERE ARE EXACTLY WHERE THE 978 00:33:00,000 --> 00:33:01,040 CLASP SUBUNIT TOUCHES THE 979 00:33:01,040 --> 00:33:01,760 SLIDING CLAMP. 980 00:33:01,760 --> 00:33:06,000 SO IN FACT, THE SENSITIVITY CAN 981 00:33:06,000 --> 00:33:07,400 NOW BE UNDERSTOOD IN TERMS OF 982 00:33:07,400 --> 00:33:08,560 THE STRUCTURAL DETAILS THAT WE 983 00:33:08,560 --> 00:33:09,720 KNOW FROM THE CRYSTAL STRUCTURE. 984 00:33:09,720 --> 00:33:11,280 BUT YOU CAN ALSO SEE THAT NOW 985 00:33:11,280 --> 00:33:15,000 THE SYSTEM HAS RED IN SEVERAL 986 00:33:15,000 --> 00:33:15,240 PLACES. 987 00:33:15,240 --> 00:33:15,920 AND IF YOU LOOK AT THE 988 00:33:15,920 --> 00:33:18,320 DISTRIBUTION OF FITNESS OVER THE 989 00:33:18,320 --> 00:33:21,040 WHOLE GREEN SUBUNIT, IT GOES 990 00:33:21,040 --> 00:33:23,000 FROM THIS VERY NARROW 991 00:33:23,000 --> 00:33:24,120 ESSENTIALLY TOLERANT 992 00:33:24,120 --> 00:33:24,760 DISTRIBUTION TO A DISTRIBUTION 993 00:33:24,760 --> 00:33:26,840 THAT SHOWS SUBSTANTIAL LOSS OF 994 00:33:26,840 --> 00:33:30,240 FUNCTION BUT ALSO GAIN OF 995 00:33:30,240 --> 00:33:31,120 FUNCTION IN MANY PLACES. 996 00:33:31,120 --> 00:33:33,000 YOU CAN NOW INTERPRET THE GAIN 997 00:33:33,000 --> 00:33:34,200 OF FUNCTION AND I'M JUST GOING 998 00:33:34,200 --> 00:33:37,080 TO SHOW YOU ONE EXAMPLE. 999 00:33:37,080 --> 00:33:40,640 SO THIS IS NOW LOOKING AT THE 1000 00:33:40,640 --> 00:33:43,800 CLAMP LOADER AND IN GREEN IS 1001 00:33:43,800 --> 00:33:45,440 THIS CLASP SUBUNIT. 1002 00:33:45,440 --> 00:33:46,240 HERE'S DNA. 1003 00:33:46,240 --> 00:33:52,200 AND HEERP HERE ARE TWO RESIDUES. 1004 00:33:52,200 --> 00:33:53,480 GLUTAMINE 26 IS TOUCHING THE 1005 00:33:53,480 --> 00:33:55,160 PHOSPHATE BACKBONE WITH DNA IN 1006 00:33:55,160 --> 00:34:01,720 OUR CRYSTAL STRUCTURE. 1007 00:34:01,720 --> 00:34:03,680 23 DOESN'T TOUCH THE BACKBONE. 1008 00:34:03,680 --> 00:34:06,400 IF YOU REPLACE THIS BY ARNLG 1009 00:34:06,400 --> 00:34:09,240 KNEE ARGININE 1010 00:34:09,240 --> 00:34:10,680 OR LYSINE, YOU GET A TENFOLD 1011 00:34:10,680 --> 00:34:12,520 INCREASE IN ACTIVITY. 1012 00:34:12,520 --> 00:34:13,960 IF YOU -- IN THE BACKGROUND OF 1013 00:34:13,960 --> 00:34:15,880 THE CHIMERIC CLAMP LOADER. 1014 00:34:15,880 --> 00:34:19,080 IF YOU COMBINE THEM, SUBU GETS 1015 00:34:19,080 --> 00:34:20,640 100 FOLD INCREASE IN ACTIVITY. 1016 00:34:20,640 --> 00:34:22,440 SO WHAT WE LEARNED FROM THIS IS 1017 00:34:22,440 --> 00:34:23,680 THAT THERE'S AN UNTAPPED 1018 00:34:23,680 --> 00:34:24,880 CAPACITY FOR OPTIMIZATION BUILT 1019 00:34:24,880 --> 00:34:26,400 INTO THE SYSTEM. 1020 00:34:26,400 --> 00:34:30,680 SO THAT IT'S REALLY ABLE TO 1021 00:34:30,680 --> 00:34:32,640 RESPOND VERY QUICKLY, BY QUICKLY 1022 00:34:32,640 --> 00:34:34,640 I MEAN A SINGLE POINT MUTATION 1023 00:34:34,640 --> 00:34:36,080 STARTS WAKING YOU UP. 1024 00:34:36,080 --> 00:34:37,520 THE REASON THIS HAPPENS IF WE 1025 00:34:37,520 --> 00:34:41,760 THINK ABOUT IT IS OBVIOUS VEES 1026 00:34:41,760 --> 00:34:44,080 THAT ANY SYSTEM DRIFT AWAY 1027 00:34:44,080 --> 00:34:48,120 THROUGH NEUTRAL MUTATIONAL 1028 00:34:48,120 --> 00:34:48,440 DRIFT. 1029 00:34:48,440 --> 00:34:53,120 FROM THE HIGHLY OPTIMIZED SYSTEM 1030 00:34:53,120 --> 00:34:55,760 WHEN IT'S OPERATING AND IT'S 1031 00:34:55,760 --> 00:34:57,760 REDUNDANT IN ITS FUNCTIONS AND 1032 00:34:57,760 --> 00:35:00,600 YOU DON'T SEE THE CAPACITY FOR 1033 00:35:00,600 --> 00:35:01,680 RESTORATION OF FUNCTION UNLESS 1034 00:35:01,680 --> 00:35:02,920 YOU STRESS THE SYSTEM. 1035 00:35:02,920 --> 00:35:04,680 SO THAT'S WHAT WE WANT TO LEARN 1036 00:35:04,680 --> 00:35:05,160 FROM THIS. 1037 00:35:05,160 --> 00:35:06,960 NOW I WANT TO END BY MOVING BACK 1038 00:35:06,960 --> 00:35:10,320 TO THE DEAD BOX AND SHOWING YOU 1039 00:35:10,320 --> 00:35:12,400 WHAT WE LEARNED THAT WAS 1040 00:35:12,400 --> 00:35:14,680 INTERESTING AND SURPRISING FOR 1041 00:35:14,680 --> 00:35:16,880 US FROM THE STUDIES OF THE DEAD 1042 00:35:16,880 --> 00:35:19,600 BOX. 1043 00:35:19,600 --> 00:35:20,800 SO I'VE EXPLAINED WHAT THE DEAD 1044 00:35:20,800 --> 00:35:23,880 BOX DOES AND I WANT TO TURN TO 1045 00:35:23,880 --> 00:35:25,720 THIS LAST ASPARTATE IN THE DEAD 1046 00:35:25,720 --> 00:35:28,640 BOX WHICH I SAID COORDINATES AN 1047 00:35:28,640 --> 00:35:30,400 INTERFACIAL RESIDUE, IT'S 1048 00:35:30,400 --> 00:35:33,200 ARGININE 122 COMING FROM A 1049 00:35:33,200 --> 00:35:34,920 NEIGHBORING DOMAIN. 1050 00:35:34,920 --> 00:35:38,480 THE DEAD BOX AS I SAID IS FOUND 1051 00:35:38,480 --> 00:35:40,200 IN RNA HELI CASES, THAT'S WHERE 1052 00:35:40,200 --> 00:35:41,560 THEY WERE FIRST DEFINED, AND 1053 00:35:41,560 --> 00:35:44,800 FROM THE YEARS OF WORK DONE IN 1054 00:35:44,800 --> 00:35:46,440 RNA HELI CASES, WE KNOW WHAT 1055 00:35:46,440 --> 00:35:48,760 THIS ASPARTATE IS DOING IS 1056 00:35:48,760 --> 00:35:52,080 COUPLING RNA RECOGNITION TO ATP 1057 00:35:52,080 --> 00:35:52,400 HYDROLYSIS. 1058 00:35:52,400 --> 00:35:53,720 SO WE INFER THAT'S WHAT IT MUST 1059 00:35:53,720 --> 00:35:56,160 BE DOING IN THE CLAMP LOADER AS 1060 00:35:56,160 --> 00:35:56,360 WELL. 1061 00:35:56,360 --> 00:35:58,840 AND HERE IS A DIAGRAM TAKEN FROM 1062 00:35:58,840 --> 00:36:01,760 A PICTURE IN -- OR TAKEN FROM A 1063 00:36:01,760 --> 00:36:04,280 PAPER PUBLISHED IN NATURE ABOUT 1064 00:36:04,280 --> 00:36:07,080 10 YEARS AGO, AND IT'S 1065 00:36:07,080 --> 00:36:11,760 EXPLAINING HOW A DEAD BOX HEL 1066 00:36:11,760 --> 00:36:14,200 CASE WORKS, BUT THE WAY TO 1067 00:36:14,200 --> 00:36:17,200 REALLY THINK ABOUT THEM ARE 1068 00:36:17,200 --> 00:36:20,800 THEY'RE TWO ATPASES ON THE SAME 1069 00:36:20,800 --> 00:36:22,600 CHANGE SO LIKE TWO CLAMP LOADER 1070 00:36:22,600 --> 00:36:22,880 SUBUNITS. 1071 00:36:22,880 --> 00:36:24,120 IN THIS DIAGRAM FROM THE PAPER, 1072 00:36:24,120 --> 00:36:27,320 YOU CAN SEE THAT ATP IS BOUND 1073 00:36:27,320 --> 00:36:29,040 AND RNA IS BOUND AND ONLY WHEN 1074 00:36:29,040 --> 00:36:32,000 BOTH ARE BOUND IS AN ACTIVE SITE 1075 00:36:32,000 --> 00:36:33,680 CONFIGURED APPROPRIATELY WHICH 1076 00:36:33,680 --> 00:36:38,480 WILL TRIGGER HYDROLYSIS. 1077 00:36:38,480 --> 00:36:41,800 AN ION PAIR FORMED BETWEEN THE 1078 00:36:41,800 --> 00:36:44,600 LAST D OF THE DEAD BOX MOTIF AND 1079 00:36:44,600 --> 00:36:46,360 AN ARGININE PRESENTED BY THE 1080 00:36:46,360 --> 00:36:48,680 SAME HELICAL ASSEMBLY I'VE BEEN 1081 00:36:48,680 --> 00:36:50,040 FOCUSING ON IN THE CLAMP LOADER. 1082 00:36:50,040 --> 00:36:52,360 THIS IS WHAT THE LAST D OF THE 1083 00:36:52,360 --> 00:36:53,680 DEAD BOX IS DOING. 1084 00:36:53,680 --> 00:36:55,600 SO WE ASKED, WHAT WOULD HAPPEN 1085 00:36:55,600 --> 00:36:59,600 IF WE MUTATED THE D AND THEN 1086 00:36:59,600 --> 00:37:01,360 ASKED HOW -- OH, SOMEBODY CLOSED 1087 00:37:01,360 --> 00:37:02,400 THE CLOCK THAT WAS THERE THAT I 1088 00:37:02,400 --> 00:37:05,600 WAS KEEPING MY EYE ON. 1089 00:37:05,600 --> 00:37:08,200 PLEASE PUT THAT BACK BECAUSE -- 1090 00:37:08,200 --> 00:37:09,360 HAVE YOU GIVEN ME 10 MORE 1091 00:37:09,360 --> 00:37:11,240 MINUTES OR WHAT DID YOU DO? 1092 00:37:11,240 --> 00:37:13,520 >> NO, I WAS TRYING TO ADJUST -- 1093 00:37:13,520 --> 00:37:14,440 YOU GO AHEAD. 1094 00:37:14,440 --> 00:37:16,080 >> I THINK THE TIME IS ACCURATE. 1095 00:37:16,080 --> 00:37:18,400 YOU DON'T NEED TO ADJUST IT. 1096 00:37:18,400 --> 00:37:19,360 IT 3:38 BY MY WATCH AND THAT 1097 00:37:19,360 --> 00:37:21,560 CLOCK BUT IT'S EASIER TO WATCH 1098 00:37:21,560 --> 00:37:22,880 THE CLOCK SO THAT I CAN END AND 1099 00:37:22,880 --> 00:37:24,400 LET EVERYBODY GO. 1100 00:37:24,400 --> 00:37:26,200 WITHOUT RESENTMENT AND ANGER. 1101 00:37:26,200 --> 00:37:27,880 SO DON'T PUT THAT AWAY. 1102 00:37:27,880 --> 00:37:33,000 SO YOU KNOW, SO WE WANTED TO ASK 1103 00:37:33,000 --> 00:37:34,600 WHAT WOULD HAPPEN IF YOU MUTATE 1104 00:37:34,600 --> 00:37:36,080 THE LAST ASPAR TATE AND SEE HOW 1105 00:37:36,080 --> 00:37:37,480 QUICKLY THINGS COME BACK. 1106 00:37:37,480 --> 00:37:39,400 SO WHAT SOMEBODY TOLD ME IS THAT 1107 00:37:39,400 --> 00:37:43,720 YOU CANNOT WAKE THE DEAD, BUT 1108 00:37:43,720 --> 00:37:49,200 YOU CAN WAKE THE WALKING DEAD. 1109 00:37:49,200 --> 00:37:51,400 SO WHAT KENDRA MARCUS WHO IS 1110 00:37:51,400 --> 00:37:52,520 ASKING THIS QUESTION DID WAS TO 1111 00:37:52,520 --> 00:37:56,040 TRY TO IDENTIFY A MUTATION IN 1112 00:37:56,040 --> 00:37:57,800 THE ASPARTATE THAT WOULD BE THE 1113 00:37:57,800 --> 00:37:58,680 WALKING DEAD. 1114 00:37:58,680 --> 00:38:02,000 AND I'D ALREADY POINTED THIS OUT 1115 00:38:02,000 --> 00:38:09,640 TO YOU IF YOU CHANGE AS PART 1116 00:38:09,640 --> 00:38:10,400 TATE 110 -- IF YOU THINK ABOUT 1117 00:38:10,400 --> 00:38:12,360 IT A LITTLE BIT, YOU CAN SEE 1118 00:38:12,360 --> 00:38:15,200 THAT THE REASON THIS IS THE CASE 1119 00:38:15,200 --> 00:38:18,800 IS THAT THE ASPARTATE CAN -- THE 1120 00:38:18,800 --> 00:38:21,080 CYSTINE CAN DEPROAT NATE, YOU'LL 1121 00:38:21,080 --> 00:38:23,800 GET S MINUS AND THAT'S A MIMIC 1122 00:38:23,800 --> 00:38:24,120 FOR ASPARTATE. 1123 00:38:24,120 --> 00:38:26,600 IN FACT, KENDRA DID THE CRYSTAL 1124 00:38:26,600 --> 00:38:28,640 STRUCTURE OF THE ASPARTATE TO 1125 00:38:28,640 --> 00:38:31,360 CYSTINE MUTANT CLAMP LOADER 1126 00:38:31,360 --> 00:38:33,520 COMPLEX BOUND TO DNA, FROM 1127 00:38:33,520 --> 00:38:35,000 LOOKING AT HOW THAT CYSTINE 1128 00:38:35,000 --> 00:38:36,280 COORDINATES THE ARGININE, WE'RE 1129 00:38:36,280 --> 00:38:37,800 PRETTY SURE THAT'S WHAT'S 1130 00:38:37,800 --> 00:38:38,040 HAPPENING. 1131 00:38:38,040 --> 00:38:39,720 SO THIS IS THE WALKING DEAD. 1132 00:38:39,720 --> 00:38:41,520 IT'S TAKEN A HIT IN ACTIVITY BUT 1133 00:38:41,520 --> 00:38:45,280 WE CAN ASK HOW QUICKLY DOES IT 1134 00:38:45,280 --> 00:38:45,840 RECOVER. 1135 00:38:45,840 --> 00:38:48,600 SO NOW THAT MUTATION IS FIXED 1136 00:38:48,600 --> 00:38:49,800 AND KENDRA MUTATES EVERYTHING 1137 00:38:49,800 --> 00:38:51,920 ELSE USING THIS ASSAY. 1138 00:38:51,920 --> 00:38:52,800 SO FIRST JUST TO SHOW YOU IT 1139 00:38:52,800 --> 00:38:55,000 REALLY IS THE WALKING DEAD, WHAT 1140 00:38:55,000 --> 00:38:59,480 THIS GRAPH IS SHOWING US IS TIME 1141 00:38:59,480 --> 00:39:01,800 ON THE X AXIS AND ATP HYDROLYSIS 1142 00:39:01,800 --> 00:39:03,480 ON THE Y AXIS. 1143 00:39:03,480 --> 00:39:07,600 IF YOU ADD THE CLAMP LOADER AND 1144 00:39:07,600 --> 00:39:11,880 DNA TOGETHER, YOU GET NO ATP 1145 00:39:11,880 --> 00:39:13,320 HYDROLYSIS. 1146 00:39:13,320 --> 00:39:16,680 YOU NEED TO LOAD THE CLAMP 1147 00:39:16,680 --> 00:39:19,000 LOADER, DNA AND CLAMP, YOU GET 1148 00:39:19,000 --> 00:39:20,960 ATP HYDROLYSIS. 1149 00:39:20,960 --> 00:39:22,640 ATP HYDROLYSIS IS ONLY ONE SMALL 1150 00:39:22,640 --> 00:39:24,680 WINDOW ON THE FULL FUNCTION OF 1151 00:39:24,680 --> 00:39:26,240 THE WINDOW BUT IT'S A USEFUL 1152 00:39:26,240 --> 00:39:26,400 ONE. 1153 00:39:26,400 --> 00:39:28,520 IF YOU MAKE THE AS PART TATE 110 1154 00:39:28,520 --> 00:39:29,760 TO C MUTATION YOU'LL SEE IT'S 1155 00:39:29,760 --> 00:39:30,480 THE WALKING DEAD. 1156 00:39:30,480 --> 00:39:32,000 IT'S NOT DEAD, IT'S NOT 1157 00:39:32,000 --> 00:39:34,200 COMPLETELY ACTIVE AND IT'S 1158 00:39:34,200 --> 00:39:35,560 INTERMEDIATE, SO NOW WE WILL FIX 1159 00:39:35,560 --> 00:39:37,280 THAT MUTATION AND LOOK AT HOW 1160 00:39:37,280 --> 00:39:39,240 THE SYSTEM RESPONDS. 1161 00:39:39,240 --> 00:39:42,000 AND THE RESULT OF THIS WAS 1162 00:39:42,000 --> 00:39:43,400 ACTUALLY SO DISAPPOINTING, IT 1163 00:39:43,400 --> 00:39:45,720 HELD ALL OF US WORKING AND 1164 00:39:45,720 --> 00:39:48,880 THINKING ABOUT THIS BACK I WOULD 1165 00:39:48,880 --> 00:39:50,600 SAY A YEAR, WHICH IS SHOWN IN 1166 00:39:50,600 --> 00:39:54,200 THIS DIAGRAM, WHICH IS THAT 1167 00:39:54,200 --> 00:39:55,600 THESE RED SPHERES OR SURFACES 1168 00:39:55,600 --> 00:39:57,800 SHOW YOU THE SITES WHERE THERE 1169 00:39:57,800 --> 00:39:59,880 ARE SINGLE POINT MUTATIONS THAT 1170 00:39:59,880 --> 00:40:03,480 WAKE UP THE WALKING 10, MEANING 1171 00:40:03,480 --> 00:40:04,680 D110C THE THE MAJOR CONCLUSION 1172 00:40:04,680 --> 00:40:07,480 YOU CAN DRAW FROM LOOKING AT 1173 00:40:07,480 --> 00:40:08,280 THIS DIAGRAM IS THAT THESE SITES 1174 00:40:08,280 --> 00:40:10,440 OF RESCUE MUTATIONS ALL OCCUR IN 1175 00:40:10,440 --> 00:40:12,400 REGIONS THAT ARE TOLERANT TO 1176 00:40:12,400 --> 00:40:13,440 MUTATION IN THE ORIGINAL CLAMP 1177 00:40:13,440 --> 00:40:14,040 LOADER. 1178 00:40:14,040 --> 00:40:15,400 SO THAT'S NICE, THAT'S 1179 00:40:15,400 --> 00:40:16,600 GRATIFYING BUT IT'S NOT VERY 1180 00:40:16,600 --> 00:40:17,120 EXCITING. 1181 00:40:17,120 --> 00:40:18,560 AND WHAT'S PARTICULARLY 1182 00:40:18,560 --> 00:40:23,040 FRUSTRATING ABOUT THIS -- THESE 1183 00:40:23,040 --> 00:40:24,560 RESULTS IS THAT WE'VE SPENT 1184 00:40:24,560 --> 00:40:26,960 MONTHS STARING AT THEM AND WE 1185 00:40:26,960 --> 00:40:28,000 JUST CAN'T UNDERSTAND WHAT THESE 1186 00:40:28,000 --> 00:40:29,400 MUTATIONS ARE DOING. 1187 00:40:29,400 --> 00:40:31,920 THEY JUST MAKE NO SENSE TO ME. 1188 00:40:31,920 --> 00:40:33,640 SO ONE OUT HERE IS TO SAY, WELL, 1189 00:40:33,640 --> 00:40:36,800 THERE'S NO DIRECT RELATIONSHIP 1190 00:40:36,800 --> 00:40:38,440 BETWEEN STRUCTURE AND THE FREE 1191 00:40:38,440 --> 00:40:39,440 ENERGY LANDSCAPE ON WHICH THE 1192 00:40:39,440 --> 00:40:40,600 PROTEIN OPERATES, SO THESE ARE 1193 00:40:40,600 --> 00:40:42,520 JUST CHANGING THE FREE ENERGY 1194 00:40:42,520 --> 00:40:43,720 LANDSCAPE BUT I FOUND THAT 1195 00:40:43,720 --> 00:40:46,200 UNSATISFACTORY BECAUSE I KNOW 1196 00:40:46,200 --> 00:40:47,600 THAT'S TRUE, BUT I'D STILL LIKE 1197 00:40:47,600 --> 00:40:48,720 TO UNDERSTAND HOW THESE 1198 00:40:48,720 --> 00:40:50,840 MUTATIONS ARE AFFECTING THE 1199 00:40:50,840 --> 00:40:52,200 FUNCTION AND THEY REALLY DO -- 1200 00:40:52,200 --> 00:40:53,880 THE ONES THAT KENDRA HAS 1201 00:40:53,880 --> 00:40:54,920 PURIFIED AND TESTED REALLY DO 1202 00:40:54,920 --> 00:40:57,760 AFFECT THE FUNCTION. 1203 00:40:57,760 --> 00:40:58,720 AS WE SHOW YOU HERE. 1204 00:40:58,720 --> 00:41:01,400 SO LET'S LOOK AT THIS ATPASE 1205 00:41:01,400 --> 00:41:02,080 ACTIVITY GRAPH FIRST. 1206 00:41:02,080 --> 00:41:06,040 SO AGAIN, THIS IS TIME VERSUS 1207 00:41:06,040 --> 00:41:08,440 ATP HYDROLYSIS AND THE WILD TYPE 1208 00:41:08,440 --> 00:41:10,920 IS IN BLUE -- IN BLACK AND WHEN 1209 00:41:10,920 --> 00:41:15,000 YOU ADD DNA AND THE CLAMP AND 1210 00:41:15,000 --> 00:41:16,880 ATP, IT HYDROLYZES ATP. 1211 00:41:16,880 --> 00:41:19,640 IN YELLOW IS D110C, WHICH I CALL 1212 00:41:19,640 --> 00:41:23,760 THE WALKING DEAD, WHICH HAS AN 1213 00:41:23,760 --> 00:41:25,000 ATTENUATED ATPASE ACTIVITY. 1214 00:41:25,000 --> 00:41:27,480 BUT IF YOU MAKE A SINGLE POINT 1215 00:41:27,480 --> 00:41:30,680 MUTATION, ONE OF THOSE RED 1216 00:41:30,680 --> 00:41:32,480 RESIDUES, YOU'VE WOKEN UP THE 1217 00:41:32,480 --> 00:41:37,320 WALKING DEAD. 1218 00:41:37,320 --> 00:41:39,200 THIS ATP ASSAY COMES BACK TO 1219 00:41:39,200 --> 00:41:44,600 WILD TYPE ATPASE ACTIVITY. 1220 00:41:44,600 --> 00:41:46,000 THIS GRAPH -- THE MUTATIONAL 1221 00:41:46,000 --> 00:41:47,040 FITNESS IS A LITTLE HARDER FOR 1222 00:41:47,040 --> 00:41:48,400 YOU TO TAKE IN BUT I'M JUST 1223 00:41:48,400 --> 00:41:49,720 GOING TO SHOW YOU A COUPLE 1224 00:41:49,720 --> 00:41:54,360 THINGS. 1225 00:41:54,360 --> 00:41:55,640 FIRST, IT'S SHOWING YOU THE 1226 00:41:55,640 --> 00:42:00,440 FITNESS OF VARIANCE, IN THE 1227 00:42:00,440 --> 00:42:03,400 BACKGROUND OF D110C, COMPLETELY 1228 00:42:03,400 --> 00:42:04,280 WILD TYPE CLAMP LOADER. 1229 00:42:04,280 --> 00:42:07,000 SO THIS IS PROLINE 50. 1230 00:42:07,000 --> 00:42:08,440 WHEN IT'S PROLINE, THAT MEANS 1231 00:42:08,440 --> 00:42:11,600 THE ONLY MUTATION IS D110C IS 1232 00:42:11,600 --> 00:42:12,880 LIGHT BLUE, THAT MEANS IT'S 1233 00:42:12,880 --> 00:42:14,080 TAKEN A LOSS OF FUNCTION. 1234 00:42:14,080 --> 00:42:15,520 THAT'S THE D110C FUNCTION, IN 1235 00:42:15,520 --> 00:42:17,200 OTHER WORDS. 1236 00:42:17,200 --> 00:42:20,640 WHY WOULD WHITE WOULD BE TOTAL WILD TYPE 1237 00:42:20,640 --> 00:42:23,000 PROTEIN WITHOUT THE D110C IS 1238 00:42:23,000 --> 00:42:24,400 ZERO OR WHITE IN THIS SCALE. 1239 00:42:24,400 --> 00:42:26,680 AND SO WHAT THAT MEANS IS THAT 1240 00:42:26,680 --> 00:42:28,320 IF YOU CHANGE PROLINE 50 TO 1241 00:42:28,320 --> 00:42:30,040 LYSINE, ACCORDING TO THIS 1242 00:42:30,040 --> 00:42:31,840 EXPERIMENT, YOU ACTUALLY -- NOT 1243 00:42:31,840 --> 00:42:33,000 ONLY DO YOU WAKE UP THE WALKING 1244 00:42:33,000 --> 00:42:34,600 DEAD, YOU ACTUALLY NOW -- THE 1245 00:42:34,600 --> 00:42:36,200 WALKING DEAD HAS WOKEN UP AND IS 1246 00:42:36,200 --> 00:42:37,320 RUNNING A MARATHON. 1247 00:42:37,320 --> 00:42:39,520 IT'S ACTUALLY A BETTER CLAMP 1248 00:42:39,520 --> 00:42:44,360 LOADER THAN THE FULLY WILD TYPE. 1249 00:42:44,360 --> 00:42:45,600 THIS IS THE ONE THING I'VE BEEN 1250 00:42:45,600 --> 00:42:46,680 ABLE TO RATIONALIZE A LITTLE 1251 00:42:46,680 --> 00:42:47,040 BIT. 1252 00:42:47,040 --> 00:42:49,360 THIS IS THE SITE OF PROLINE 50 1253 00:42:49,360 --> 00:42:51,480 IN THE SO CALLED FAMOUS P LOOP, 1254 00:42:51,480 --> 00:42:52,800 BECAUSE HERE'S THE PHOSPHATE, 1255 00:42:52,800 --> 00:42:55,400 THE PHOSPHATE BINDING LOOP. 1256 00:42:55,400 --> 00:42:56,280 HERE'S THE PROLINE. 1257 00:42:56,280 --> 00:42:58,440 THIS PROLINE WHICH IS ALSO A 1258 00:42:58,440 --> 00:43:00,200 SITE OF TOLERANT IDIOSYNCRATIC 1259 00:43:00,200 --> 00:43:01,960 SUBSTITUTION IS RIGHT NEXT TO A 1260 00:43:01,960 --> 00:43:02,440 GLUTAMATE. 1261 00:43:02,440 --> 00:43:04,040 SO YOU CAN SEE WHY MAKING IT A 1262 00:43:04,040 --> 00:43:05,920 LYSINE MIGHT GIVE YOU BACK 1263 00:43:05,920 --> 00:43:07,760 ACTIVITY BUT IT'S VERY 1264 00:43:07,760 --> 00:43:09,640 UNSATISFACTORY BECAUSE ARGININE 1265 00:43:09,640 --> 00:43:10,600 DOESN'T WORK. 1266 00:43:10,600 --> 00:43:12,600 BUT THE KEY TO UNDERSTANDING 1267 00:43:12,600 --> 00:43:14,240 WHAT ALL OF THESE MUTATIONS ARE 1268 00:43:14,240 --> 00:43:16,640 DOING IS TO LOOK AT THIS DIAGRAM 1269 00:43:16,640 --> 00:43:17,760 AND ACTUALLY APPRECIATE THAT 1270 00:43:17,760 --> 00:43:20,160 MANY, MANY, MANY SUBSTITUTIONS 1271 00:43:20,160 --> 00:43:22,600 ARE BETTER THAN THE ORIGINAL 1272 00:43:22,600 --> 00:43:27,720 WILD TYPE. 1273 00:43:27,720 --> 00:43:31,360 IN THE D110C BACKGROUND, PROLINE 1274 00:43:31,360 --> 00:43:34,480 TO LYSINE ISN'T THE ONLY 1275 00:43:34,480 --> 00:43:35,440 SUBSTITUTION THAT'S WAKING UP 1276 00:43:35,440 --> 00:43:36,000 THE WALKING DEAD. 1277 00:43:36,000 --> 00:43:37,840 YOU CAN ACQUIRE ACTIVITY TOWARDS 1278 00:43:37,840 --> 00:43:39,800 ORIGINAL WILD TYPE BY CHANGING 1279 00:43:39,800 --> 00:43:40,680 PROLINE TO MANY THINGS. 1280 00:43:40,680 --> 00:43:43,400 SO IT CAN'T JUST BE THIS ION 1281 00:43:43,400 --> 00:43:43,600 PAIR. 1282 00:43:43,600 --> 00:43:45,600 THIS WAS THE KEY THAT LET ME 1283 00:43:45,600 --> 00:43:47,640 GRIN 1284 00:43:47,640 --> 00:43:48,560 BEGIN TO UNDERSTAND WHAT THESE 1285 00:43:48,560 --> 00:43:49,600 RESCUE MISSIONS ARE DOING. 1286 00:43:49,600 --> 00:43:51,600 AND I UNDERSTOOD WHAT THEY'RE 1287 00:43:51,600 --> 00:43:53,000 DOING BECAUSE MOST OF MY LAB 1288 00:43:53,000 --> 00:43:56,360 WORKS ON SIGNALING PROTEINS LIKE 1289 00:43:56,360 --> 00:43:59,280 RAS AND EGF RECEPTOR. 1290 00:43:59,280 --> 00:44:00,840 WE STUDY MUTATIONS ALL THE TIME 1291 00:44:00,840 --> 00:44:05,200 BECAUSE THEY MANIFEST AS PATIENT 1292 00:44:05,200 --> 00:44:06,400 RESISTANCE MUTATIONS TO DRUGS. 1293 00:44:06,400 --> 00:44:07,240 BASICALLY THIS IS THE SIGNATURE 1294 00:44:07,240 --> 00:44:14,720 OF AN INHIBITORY INTERACTION 1295 00:44:14,720 --> 00:44:16,320 THAT'S BEING BROKEN BY THE 1296 00:44:16,320 --> 00:44:18,000 MUTATION, ALLOWING TO ESCAPE 1297 00:44:18,000 --> 00:44:19,480 FROM AUTO INHIBITION AND REGAIN 1298 00:44:19,480 --> 00:44:21,760 ACTIVITY THAT THE DAMAGE DONE BY 1299 00:44:21,760 --> 00:44:27,000 D110C HAD CAUSED TO INCUR. 1300 00:44:27,000 --> 00:44:29,960 THIS LETS US USE A PRINCIPLE WE 1301 00:44:29,960 --> 00:44:31,440 USE WHEN UNDERSTANDING KINASES 1302 00:44:31,440 --> 00:44:33,200 AND RAS AND THINGS LIKE THAT, 1303 00:44:33,200 --> 00:44:33,960 WHICH -- OH, I JUST WANTED TO 1304 00:44:33,960 --> 00:44:35,600 SHOW YOU, IF YOU LOOK AT THE 1305 00:44:35,600 --> 00:44:39,040 WHOLE HEAT MAP, JUST LOOK AT THE 1306 00:44:39,040 --> 00:44:41,600 LOWER GRAPH LIKE HERE. 1307 00:44:41,600 --> 00:44:43,600 YOU'LL SEE THAT MANY MUTATIONS 1308 00:44:43,600 --> 00:44:44,400 WAKE THE WALKING DEAD. 1309 00:44:44,400 --> 00:44:45,760 I DON'T WANT TO WALK THROUGH 1310 00:44:45,760 --> 00:44:51,520 THIS WHOLE COMPLICATED DIAGRAM. 1311 00:44:51,520 --> 00:44:58,440 THE PRINCIPLE WE USE TO 1312 00:44:58,440 --> 00:45:02,040 UNDERSTAND, I CALL THE 1313 00:45:02,040 --> 00:45:02,800 ANAKARANINA PRINCIPLE. 1314 00:45:02,800 --> 00:45:09,600 I READ A REVIEW BY LUIS JOHNSON, 1315 00:45:09,600 --> 00:45:10,680 INSPIRATION TO MANY OF US WHO 1316 00:45:10,680 --> 00:45:12,120 DIED SOME YEARS AGO BEFORE HER 1317 00:45:12,120 --> 00:45:15,080 TIME, AND IN HER OWE OBITUARY 1318 00:45:15,080 --> 00:45:17,000 PUBLISHED BY THE ROYAL SOCIETY, 1319 00:45:17,000 --> 00:45:20,400 THEY SAY -- THE WRITER SAYS SHE 1320 00:45:20,400 --> 00:45:21,600 WOULD BRING PROTEIN STRUCTURES 1321 00:45:21,600 --> 00:45:27,080 TO LIFE BY MAKING THEM POP LAT 1322 00:45:27,080 --> 00:45:30,080 CHARACTERS IN A NOVEL. 1323 00:45:30,080 --> 00:45:31,000 SHE'S QUOTING TO SAY HAPPY 1324 00:45:31,000 --> 00:45:32,680 FAMILIES ARE ALL ALIKE. 1325 00:45:32,680 --> 00:45:34,040 EVERY UNHAPPY FAMILY IS UNHAPPY 1326 00:45:34,040 --> 00:45:35,640 IN ITS OWN WAY. 1327 00:45:35,640 --> 00:45:37,000 AND THE PRINCIPLE HERE IS THAT 1328 00:45:37,000 --> 00:45:39,480 WHEN YOU HAVE AN ENZYME SYSTEM, 1329 00:45:39,480 --> 00:45:42,000 THE CATALYTIC CENTER HAS TO BE 1330 00:45:42,000 --> 00:45:44,120 CONSERVED IN THE HAPPY STATE, 1331 00:45:44,120 --> 00:45:44,960 WHICH IS THE ON STATE, BECAUSE 1332 00:45:44,960 --> 00:45:48,920 IT HAS TO OBEY THE DICTATES OF 1333 00:45:48,920 --> 00:45:49,600 CHEMISTRY. 1334 00:45:49,600 --> 00:45:50,320 BUT IF EVOLUTION WANTS TO 1335 00:45:50,320 --> 00:45:52,520 REGULATE THAT ENZYME, ALL THAT 1336 00:45:52,520 --> 00:45:55,120 EVOLUTION HAS TO DO TO CREATE AN 1337 00:45:55,120 --> 00:45:56,440 AUTO INHIBITED STATE THAT'S OFF 1338 00:45:56,440 --> 00:45:59,800 IS TO DO SOMETHING THAT MAKES 1339 00:45:59,800 --> 00:46:00,280 THE ENZYME UNHAPPY. 1340 00:46:00,280 --> 00:46:01,600 THE WAY EVOLUTION WILL DO THAT 1341 00:46:01,600 --> 00:46:03,480 FOR A DIVERGENT SET OF PROTEINS 1342 00:46:03,480 --> 00:46:05,840 WILL BE UNIQUE FOR EVERY 1343 00:46:05,840 --> 00:46:06,960 DIVERGENT PROTEIN OR EVERY 1344 00:46:06,960 --> 00:46:07,440 SUBCLASS. 1345 00:46:07,440 --> 00:46:09,360 AND WE SEE THAT IN KINASES, 1346 00:46:09,360 --> 00:46:12,320 THAT'S A LECTURE FOR ANOTHER 1347 00:46:12,320 --> 00:46:12,600 DAY. 1348 00:46:12,600 --> 00:46:16,680 SO WE STARTED THINKING ABOUT THE 1349 00:46:16,680 --> 00:46:19,400 ANNA KARENIN A PROCESS, WE'RE 1350 00:46:19,400 --> 00:46:20,600 MISSING A STRUCTURE. 1351 00:46:20,600 --> 00:46:21,600 THE STRUCTURE WE'RE MISSING IS 1352 00:46:21,600 --> 00:46:23,400 AN OFF-STATE OF THE CLAMP 1353 00:46:23,400 --> 00:46:25,240 LOADER. 1354 00:46:25,240 --> 00:46:28,600 AND TODAY, WE HAVE THE POWER OF 1355 00:46:28,600 --> 00:46:30,480 CRYO EM THAT WE CAN DEPLOY TO 1356 00:46:30,480 --> 00:46:32,960 ANSWER A QUESTION LIKE THIS. 1357 00:46:32,960 --> 00:46:34,880 WITH CRYSTALLOGRAPHY, WE COULD 1358 00:46:34,880 --> 00:46:36,240 BARELY OBTAIN THE CRYSTAL 1359 00:46:36,240 --> 00:46:37,520 STRUCTURES I'VE SHOWN YOU 1360 00:46:37,520 --> 00:46:38,800 BECAUSE -- I'VE ONLY SHOWN YOU 1361 00:46:38,800 --> 00:46:40,040 GLIMPSES OF BECAUSE THE CLAMP 1362 00:46:40,040 --> 00:46:41,880 LOADER IS SO DIFFICULT TO 1363 00:46:41,880 --> 00:46:43,200 CRYSTALLIZE. 1364 00:46:43,200 --> 00:46:44,560 WE DEFINITELY HAD NO VIEW OF THE 1365 00:46:44,560 --> 00:46:46,040 STRUCTURE FOR THE T4 SYSTEM AND 1366 00:46:46,040 --> 00:46:48,000 WE COULDN'T USED WHATEVER PEOPLE 1367 00:46:48,000 --> 00:46:49,840 HAD GUESSED ABOUT FOR OTHER 1368 00:46:49,840 --> 00:46:50,960 SYSTEMS TO UNDERSTAND THESE 1369 00:46:50,960 --> 00:46:52,640 MUTATIONS. 1370 00:46:52,640 --> 00:47:01,800 SO FORTUNATELY, THEY STARTED 1371 00:47:01,800 --> 00:47:03,080 APPLYING CRYO-EM TO DETERMINE 1372 00:47:03,080 --> 00:47:04,320 STRUCTURES OF UNCROSS LINKED 1373 00:47:04,320 --> 00:47:05,440 CLAMP LOADERS. 1374 00:47:05,440 --> 00:47:06,640 REALLY AS CLOSE TO THE NATIVE 1375 00:47:06,640 --> 00:47:08,960 STATE AS WE CAN. 1376 00:47:08,960 --> 00:47:11,440 THE FIRST RESULT SHOWN HERE IS 1377 00:47:11,440 --> 00:47:12,840 THE STRUCTURE OF THE HAPPY STATE 1378 00:47:12,840 --> 00:47:14,680 OF THE CLAMP LOADERS. 1379 00:47:14,680 --> 00:47:16,640 BOUND TO DNA, BOUND TO ATP 1380 00:47:16,640 --> 00:47:17,440 ANALOG, BOUND TO THE CLAMP. 1381 00:47:17,440 --> 00:47:19,440 AND IT LOOKS JUST LIKE THE 1382 00:47:19,440 --> 00:47:20,800 CRYSTAL STRUCTURE WE'VE 1383 00:47:20,800 --> 00:47:23,200 PUBLISHED MORE THAN 10 YEARS 1384 00:47:23,200 --> 00:47:23,360 AGO. 1385 00:47:23,360 --> 00:47:24,480 SO THAT'S A RELIEF FOR US, WE 1386 00:47:24,480 --> 00:47:28,200 DON'T HAVE TO REWRITE ANY OF OUR 1387 00:47:28,200 --> 00:47:28,640 CONCLUSIONS. 1388 00:47:28,640 --> 00:47:30,440 BUT HE WAS ALSO ABLE, VERY 1389 00:47:30,440 --> 00:47:34,360 QUICKLY, TO DETERMINE STRUCTURES 1390 00:47:34,360 --> 00:47:35,400 WHERE ONE OR THE OTHER THING IS 1391 00:47:35,400 --> 00:47:36,720 MISSING SO THE SYSTEM IS 1392 00:47:36,720 --> 00:47:37,040 UNHAPPY. 1393 00:47:37,040 --> 00:47:38,640 WHENEVER HE DOES THAT, HE GETS 1394 00:47:38,640 --> 00:47:39,880 THE SAME STRUCTURE BACK. 1395 00:47:39,880 --> 00:47:43,120 WHEN HE OMITS DNA, WHEN HE OMITS 1396 00:47:43,120 --> 00:47:46,400 THE CLAMP, WHEN HE USES ATP, 1397 00:47:46,400 --> 00:47:48,280 WHEN HE PUTS THE D110C MUTATION 1398 00:47:48,280 --> 00:47:49,600 IN, HE GETS THE STRUCTURE ON THE 1399 00:47:49,600 --> 00:47:49,920 RIGHT. 1400 00:47:49,920 --> 00:47:51,480 AND THIS IS AN UNHAPPY 1401 00:47:51,480 --> 00:47:52,720 STRUCTURE, IN FACT, IT'S A 1402 00:47:52,720 --> 00:47:57,880 STRUCTURE THAT'S VERY STABLE, A 1403 00:47:57,880 --> 00:47:59,560 DOESN'T NEED A CLAMP AND IT'S IN 1404 00:47:59,560 --> 00:48:00,600 A CONFIGURATION WHERE IT CAN'T 1405 00:48:00,600 --> 00:48:01,040 BIND DNA. 1406 00:48:01,040 --> 00:48:06,520 TO SHOW YOU THAT, HERE'S AN 1407 00:48:06,520 --> 00:48:07,840 ANIMATION INTERPOLATION BETWEEN 1408 00:48:07,840 --> 00:48:09,360 THE OFF OR UNHAPPY STATE AND THE 1409 00:48:09,360 --> 00:48:09,680 ACTIVE STATE. 1410 00:48:09,680 --> 00:48:10,720 IT'S ACTUALLY FASCINATING TO 1411 00:48:10,720 --> 00:48:13,840 LOOK AT THIS, YOU'LL SEE HELICES 1412 00:48:13,840 --> 00:48:15,440 UNWINDING AND THE REAL POINT I 1413 00:48:15,440 --> 00:48:16,680 WANTED TO MAKE FROM THIS IS THAT 1414 00:48:16,680 --> 00:48:19,680 THERE MUST BE AN ENERGY COST TO 1415 00:48:19,680 --> 00:48:20,800 GOING FROM THE INACTIVE STATE TO 1416 00:48:20,800 --> 00:48:23,080 THE ACTIVE STATE, THAT ENERGY 1417 00:48:23,080 --> 00:48:25,720 COST IS PAID BY DNA BUT ALSO BY 1418 00:48:25,720 --> 00:48:27,760 THE ARGININE ASPARTATE 1419 00:48:27,760 --> 00:48:29,000 INTERACTION SO PRESUMING WHAT'S 1420 00:48:29,000 --> 00:48:31,080 HAPPENING IS THESE MUTATIONS ARE 1421 00:48:31,080 --> 00:48:36,440 LOWERING THAT ENERGY COST. 1422 00:48:36,440 --> 00:48:37,600 WHAT'S REALLY EXCITING, IF WE GO 1423 00:48:37,600 --> 00:48:39,680 IN AND LOOK AT THE STRUCTURAL 1424 00:48:39,680 --> 00:48:42,040 CHANGES HAPPENING INSIDE ONE OF 1425 00:48:42,040 --> 00:48:43,880 THE ATPASES, OF COURSE THESE 1426 00:48:43,880 --> 00:48:44,560 GLOBAL CHANGES ARE ALSO 1427 00:48:44,560 --> 00:48:46,800 IMPORTANT, BUT IF WE LOOK AT ONE 1428 00:48:46,800 --> 00:48:49,760 OF THE STRUCTURAL CHANGES 1429 00:48:49,760 --> 00:48:52,040 HAPPENING INSIDE THE ATPASE, WE 1430 00:48:52,040 --> 00:48:53,200 CAN SEE THINGS WE CAN APPRECIATE 1431 00:48:53,200 --> 00:48:54,280 FROM THE STUDIES OF RAS WHICH 1432 00:48:54,280 --> 00:48:56,120 GUIDE SO MUCH THINKING IN MY 1433 00:48:56,120 --> 00:48:57,040 LAB. 1434 00:48:57,040 --> 00:48:59,080 SO TO SET THAT UP, HERE'S ONE 1435 00:48:59,080 --> 00:49:01,000 SUBUNIT OF THE CLAMP LOADER IN 1436 00:49:01,000 --> 00:49:03,920 THE ASSEMBLY, IN THE HAPPY 1437 00:49:03,920 --> 00:49:06,200 STATE. 1438 00:49:06,200 --> 00:49:09,160 HERE'S AN ATP ANALOG BACK HERE, 1439 00:49:09,160 --> 00:49:10,600 AND HERE IS THE TERMINAL 1440 00:49:10,600 --> 00:49:11,760 PHOSPHATE COLORED YELLOW AND 1441 00:49:11,760 --> 00:49:13,960 BLUE, HAPPENS TO BE -- FLUORIDE. 1442 00:49:13,960 --> 00:49:16,160 HERE'S A RESIDUE CALLED 1443 00:49:16,160 --> 00:49:17,040 ASPARAGINE 139 THAT'S ATTACKING 1444 00:49:17,040 --> 00:49:20,800 THE PHOSPHATE. 1445 00:49:20,800 --> 00:49:22,760 ASPARAGINE 139 IS EQUIVALENT TO 1446 00:49:22,760 --> 00:49:24,480 GLUTAMINE 61, THE SITE OF THE 1447 00:49:24,480 --> 00:49:26,000 THREE FAMOUS CANCER MUTATIONS IN 1448 00:49:26,000 --> 00:49:28,160 RAS. 1449 00:49:28,160 --> 00:49:29,240 WHAT ASPARAGINE 139 IS DOING IS 1450 00:49:29,240 --> 00:49:33,840 THE SAME, IT'S ON SWITCH 2, AND 1451 00:49:33,840 --> 00:49:35,400 IT'S DEPROAT NATEING A WATER 1452 00:49:35,400 --> 00:49:36,960 MOLECULE OR ACTIVATING A WATER 1453 00:49:36,960 --> 00:49:37,960 MOLECULE FOR ATTACKING THE 1454 00:49:37,960 --> 00:49:40,560 PHOSPHATE. 1455 00:49:40,560 --> 00:49:41,720 THIS IS A CONFIGURATION THAT'S 1456 00:49:41,720 --> 00:49:42,600 PERFECTLY POISED TO DO THAT. 1457 00:49:42,600 --> 00:49:47,320 WE'VE TRAPPED IT USING ATB 1458 00:49:47,320 --> 00:49:48,040 BERYLLIUM FLUORIDE. 1459 00:49:48,040 --> 00:49:52,080 YOU'LL SEE TWO ARGININE 1460 00:49:52,080 --> 00:49:53,400 RESIDUES, IT IS INTERACTING WITH 1461 00:49:53,400 --> 00:49:54,800 THE ATP IN THE SUBUNIT BEFORE, 1462 00:49:54,800 --> 00:49:59,400 HERE IT IS, AND THIS IS ARGININE 1463 00:49:59,400 --> 00:50:00,600 122, WHICH IS INTERACTING WITH 1464 00:50:00,600 --> 00:50:02,160 THE ASPARTATE OF THE DEAD BOX IN 1465 00:50:02,160 --> 00:50:03,920 THE PREVIOUS SUBUNIT. 1466 00:50:03,920 --> 00:50:04,800 SO IT'S ALL SET UP. 1467 00:50:04,800 --> 00:50:06,400 AND IF YOU GO THROUGH THE 1468 00:50:06,400 --> 00:50:08,280 SUBUNIT, THERE'S ANOTHER 1469 00:50:08,280 --> 00:50:10,160 ARGININE IN THE AAA PLUSES 1470 00:50:10,160 --> 00:50:12,360 CALLED SENSOR 2 AND IT'S 1471 00:50:12,360 --> 00:50:13,440 INTERACTING WITH THE PHOSPHATE 1472 00:50:13,440 --> 00:50:14,600 BUT NOW SETTING UP THE 1473 00:50:14,600 --> 00:50:15,560 INTERACTION WITH THE NEXT ATP. 1474 00:50:15,560 --> 00:50:16,760 SO THIS IS THE STRUCTURE WE 1475 00:50:16,760 --> 00:50:17,200 UNDERSTAND. 1476 00:50:17,200 --> 00:50:20,880 NOW IF YOU NO YOU LOOK AT AN 1477 00:50:20,880 --> 00:50:22,680 INTERPLAITION BETWEEN THE OFF 1478 00:50:22,680 --> 00:50:25,720 STATE DRIVED FROM THE 1479 00:50:25,720 --> 00:50:27,800 3.2 ANGSTROM CRY YOE EM 1480 00:50:27,800 --> 00:50:28,880 STRUCTURE, YOU START TO SEE TO 1481 00:50:28,880 --> 00:50:32,040 THOSE OF US WHO STUDY THESE 1482 00:50:32,040 --> 00:50:33,120 REALLY MARVELOUS CHANGES. 1483 00:50:33,120 --> 00:50:36,240 SO FOR EXAMPLE HERE, THAT'S THE 1484 00:50:36,240 --> 00:50:37,680 HAPPY STATE. 1485 00:50:37,680 --> 00:50:40,560 YOU SEE ASPARGINE 139 PULLED 1486 00:50:40,560 --> 00:50:41,560 AWAY BECAUSE IN THE OFF STATE, 1487 00:50:41,560 --> 00:50:43,600 THERE ARE NO FLANKING 1488 00:50:43,600 --> 00:50:44,200 INTERACTIONS. 1489 00:50:44,200 --> 00:50:46,000 THEY'RE ALL BROKEN IN THE OFF 1490 00:50:46,000 --> 00:50:46,360 STATE. 1491 00:50:46,360 --> 00:50:48,920 SO THIS IS REALLY OFF BECAUSE 1492 00:50:48,920 --> 00:50:50,800 THE CATALYTIC RESIDUE HAS BEEN 1493 00:50:50,800 --> 00:50:51,160 PULLED OUT. 1494 00:50:51,160 --> 00:50:52,200 AND THE OTHER THINGS YOU COULD 1495 00:50:52,200 --> 00:50:54,480 LOOK AT, HERE'S GLUTAMINE 118 1496 00:50:54,480 --> 00:50:56,040 RESIDUE, I SPENT A LOT OF TIME 1497 00:50:56,040 --> 00:50:57,720 DISCUSSING EARLIER, BUT SUDDENLY 1498 00:50:57,720 --> 00:51:00,840 WHAT YOU SEE IS THAT THESE 1499 00:51:00,840 --> 00:51:02,080 RESCUE MUTATIONS, THE MOST 1500 00:51:02,080 --> 00:51:03,440 IMPORTANT ONES SHOWN AT RED 1501 00:51:03,440 --> 00:51:04,640 SPHERE, ARE IN REGIONS WHERE THE 1502 00:51:04,640 --> 00:51:06,600 STRUCTURE IS CHANGING. 1503 00:51:06,600 --> 00:51:08,200 SO I DON'T KNOW WHAT THEY'RE 1504 00:51:08,200 --> 00:51:13,680 DOING THROUGH THE INNER JE TICS, THE ENERG ETICS, 1505 00:51:13,680 --> 00:51:14,600 BUT I SEE THEY'RE LOCATED IN 1506 00:51:14,600 --> 00:51:15,400 SITES WHERE THE STRUCTURE 1507 00:51:15,400 --> 00:51:17,360 CHANGES BETWEEN OFF AND ON. 1508 00:51:17,360 --> 00:51:19,360 SO ALL THEY HAVE TO DO IS ALTER 1509 00:51:19,360 --> 00:51:20,680 THAT ENERGETIC BALANCE OR THE 1510 00:51:20,680 --> 00:51:22,440 LANDSCAPE OR THE BARRIER, AND WE 1511 00:51:22,440 --> 00:51:24,440 CAN UNDERSTAND WHY THEY CAUSE 1512 00:51:24,440 --> 00:51:28,080 THE WALKING DEAD TO WAKE UP. 1513 00:51:28,080 --> 00:51:29,880 SO WITH THAT, I'LL END WHAT 1514 00:51:29,880 --> 00:51:31,280 WE'VE LEARNED FROM OUR STUDIES 1515 00:51:31,280 --> 00:51:36,160 IS THAT THERE IS, OF COURSE, RES 1516 00:51:36,160 --> 00:51:38,400 RESIDENT IN EACH OF THESE 1517 00:51:38,400 --> 00:51:39,640 PROTEINS ENORMOUS CAPACITY FOR 1518 00:51:39,640 --> 00:51:43,320 SEQUENCE CHANGE. 1519 00:51:43,320 --> 00:51:45,240 WHILE THE PHAGE IS REPLICATING 1520 00:51:45,240 --> 00:51:45,760 DNA. 1521 00:51:45,760 --> 00:51:49,720 THAT'S REALLY THE MOST IMPORTANT 1522 00:51:49,720 --> 00:51:50,880 FUNCTION. 1523 00:51:50,880 --> 00:51:51,960 HYDROGEN BONDED STRUCTURAL 1524 00:51:51,960 --> 00:51:54,200 ELEMENTS MAINTAIN COUPLING 1525 00:51:54,200 --> 00:52:00,520 ACROSS INTERFACES AND AND I INLT I 1526 00:52:00,520 --> 00:52:04,640 INTRODUCED THE ANNA CARE NINA 1527 00:52:04,640 --> 00:52:05,440 PRINCIPLE, IT'S DOING SO 1528 00:52:05,440 --> 00:52:06,000 DIFFERENTLY BECAUSE THE OFF 1529 00:52:06,000 --> 00:52:11,320 STATES CAN BE DIFFERENT. 1530 00:52:11,320 --> 00:52:12,600 I THINK THE STRUCTURES CHANGE 1531 00:52:12,600 --> 00:52:14,600 AND AS THEY CHANGE, THEY'RE 1532 00:52:14,600 --> 00:52:16,600 MOVING ALONG DIFFERENT 1533 00:52:16,600 --> 00:52:17,600 EVOLUTIONARY TRAJECTORIES. 1534 00:52:17,600 --> 00:52:20,360 IT'S PART OF WHAT HAS ENABLED 1535 00:52:20,360 --> 00:52:22,000 MANY THINGS -- THE DIVERSITY OF 1536 00:52:22,000 --> 00:52:23,040 THESE STRUCTURES, WHICH I'VE 1537 00:52:23,040 --> 00:52:25,640 SHOWN YOU THREE HERE, CLAMP 1538 00:52:25,640 --> 00:52:31,720 LOADER, CLPX UNFOLDASE USING THE 1539 00:52:31,720 --> 00:52:32,800 SAME ELEMENTS TO CONTROL THEIR 1540 00:52:32,800 --> 00:52:33,120 ACTION. 1541 00:52:33,120 --> 00:52:34,760 WITH THAT I'LL END, AND ONCE 1542 00:52:34,760 --> 00:52:36,640 AGAIN THANK SUBU SUBRAMANIAN, 1543 00:52:36,640 --> 00:52:40,120 KENDRA MARCUS, KENT GORDAY, 1544 00:52:40,120 --> 00:52:44,600 YOUNG JAN JUAN AND CHRISTINE GEE 1545 00:52:44,600 --> 00:52:45,200 FOR BEING INSTRUMENTAL IN 1546 00:52:45,200 --> 00:52:46,680 LETTING THIS WORK TAKE PLACE AND 1547 00:52:46,680 --> 00:52:47,040 BE DONE. 1548 00:52:47,040 --> 00:52:49,680 SO THANK YOU. 1549 00:52:49,680 --> 00:52:54,360 [APPLAUSE] 1550 00:52:54,360 --> 00:52:55,800 >> THANK YOU FOR THE GREAT TALK. 1551 00:52:55,800 --> 00:52:58,200 I WAS CURIOUS, IN YOUR RESCUE 1552 00:52:58,200 --> 00:53:01,640 EXPERIMENT WITH THE D110C, DO 1553 00:53:01,640 --> 00:53:04,640 YOU EVER HAVE DOUBLE MUTATIONS 1554 00:53:04,640 --> 00:53:05,880 FOR EXAMPLE THAT RESCUE MORE 1555 00:53:05,880 --> 00:53:07,040 EFFICIENTLY THAN THE SINGLE 1556 00:53:07,040 --> 00:53:07,280 MUTATIONS? 1557 00:53:07,280 --> 00:53:09,600 >> SO THE QUESTION IS, WITH THE 1558 00:53:09,600 --> 00:53:13,720 D110C, HAVE WE STUDIED DOUBLE 1559 00:53:13,720 --> 00:53:14,000 MUTATIONS. 1560 00:53:14,000 --> 00:53:16,200 AND SO THE SIMPLE ANSWER IS NO, 1561 00:53:16,200 --> 00:53:18,400 BECAUSE THE WAY THE EXPERIMENT 1562 00:53:18,400 --> 00:53:22,480 WAS DONE WAS TO ONLY INTRODUCE 1563 00:53:22,480 --> 00:53:26,720 ONE MUTATION AT A TIME. 1564 00:53:26,720 --> 00:53:27,760 SO -- I ALLUDED TO THE FACT IN 1565 00:53:27,760 --> 00:53:29,920 THE RESCUE OF THE SO-CALLED 1566 00:53:29,920 --> 00:53:32,000 CLASP SUBUNITS, SUBU DID FIND HE 1567 00:53:32,000 --> 00:53:36,960 COULD COMBINE TWO OF THE 1568 00:53:36,960 --> 00:53:37,600 MUTATIONS GIVEN ADDITIONAL. 1569 00:53:37,600 --> 00:53:39,560 SO AS YOU CAN IMAGINE, THE 1570 00:53:39,560 --> 00:53:40,640 DIVERSITY OF POSSIBLE SEQUENCES 1571 00:53:40,640 --> 00:53:42,440 EXPLODES WHEN YOU START 1572 00:53:42,440 --> 00:53:44,160 COMBINING MUTATIONS, SO ONE OF 1573 00:53:44,160 --> 00:53:45,280 THE THAINGS WE'RE PROBABLY GOING 1574 00:53:45,280 --> 00:53:46,800 TO DO IS NOT SIT AND COMBINE 1575 00:53:46,800 --> 00:53:48,840 THESE MUTATIONS BUT USE VARIOUS 1576 00:53:48,840 --> 00:53:51,720 METHODS FOR GENERATING 1577 00:53:51,720 --> 00:53:55,440 ALTERNATIVE SEQUENCES THAT ARE 1578 00:53:55,440 --> 00:53:57,560 VASTLY -- NOT COMBINING JUST TWO 1579 00:53:57,560 --> 00:53:58,960 MUTATIONS, BUT MIXING UP LOTS OF 1580 00:53:58,960 --> 00:54:00,800 THINGS AND THEN -- BECAUSE THIS 1581 00:54:00,800 --> 00:54:01,760 IS A HIGH-THROUGHPUT EXPERIMENT, 1582 00:54:01,760 --> 00:54:03,560 WE CAN TEST THOUSANDS OF 1583 00:54:03,560 --> 00:54:08,240 VARIANTS IN AN EXPERIMENT, AND 1584 00:54:08,240 --> 00:54:10,120 FROM THAT GLEAN SOMETHING ABOUT 1585 00:54:10,120 --> 00:54:11,080 COUPLING BETWEEN RESIDUES. 1586 00:54:11,080 --> 00:54:11,640 >> THANK YOU. 1587 00:54:11,640 --> 00:54:15,800 >> JOHN, THANKS FOR A PHENOMENAL 1588 00:54:15,800 --> 00:54:16,160 TALK. 1589 00:54:16,160 --> 00:54:17,680 SO I WAS WONDERING ABOUT SORT OF 1590 00:54:17,680 --> 00:54:18,120 TWO THINGS. 1591 00:54:18,120 --> 00:54:20,840 ONE IS, I'M THINKING ABOUT THE 1592 00:54:20,840 --> 00:54:22,720 WORK THAT HAS BEEN DONE IN TERMS 1593 00:54:22,720 --> 00:54:23,880 OF THE STATISTICAL COUPLING 1594 00:54:23,880 --> 00:54:24,280 ANALYSIS -- 1595 00:54:24,280 --> 00:54:25,880 >> SORRY, I JUST WANT TO 1596 00:54:25,880 --> 00:54:28,280 INTERRUPT BECAUSE YOU MENTIONED 1597 00:54:28,280 --> 00:54:30,640 HIM AND I SHOULD REALLY HAVE PUT 1598 00:54:30,640 --> 00:54:33,040 HIM ON THE SLIDE BECAUSE HE'S 1599 00:54:33,040 --> 00:54:34,960 RESPONSIBLE FOR TRANSFERRING -- 1600 00:54:34,960 --> 00:54:36,200 HE'S THE PERSON WHO TAUGHT MY 1601 00:54:36,200 --> 00:54:38,080 LAB HOW TO DO THESE EXPERIMENTS 1602 00:54:38,080 --> 00:54:39,760 AND SUBU SUBRAMANIAN IS THE 1603 00:54:39,760 --> 00:54:41,240 UPPER LEFT-HAND CORNER HERE WAS 1604 00:54:41,240 --> 00:54:44,080 A GRADUATE STUDENT WITH RAMARAN. 1605 00:54:44,080 --> 00:54:47,400 SO WE REALLY OWE RAMA FOR HIS 1606 00:54:47,400 --> 00:54:48,080 INTELLECTUAL LEADERSHIP IN 1607 00:54:48,080 --> 00:54:49,040 GETTING US TO THIS POINT. 1608 00:54:49,040 --> 00:54:49,800 >> RIGHT. 1609 00:54:49,800 --> 00:54:51,760 SO I IMAGINE THAT THE -- IN THE 1610 00:54:51,760 --> 00:54:56,200 FIRST PART, THE CONSERVE CLUSTER 1611 00:54:56,200 --> 00:54:57,320 YOU SHOW THAT WOULD PROBABLY 1612 00:54:57,320 --> 00:54:59,160 COME OUT OF A STATISTICAL 1613 00:54:59,160 --> 00:54:59,560 COUPLING ANALYSIS. 1614 00:54:59,560 --> 00:55:00,280 >> YES. 1615 00:55:00,280 --> 00:55:02,000 >> I'M CURIOUS, THE LAST PATCH 1616 00:55:02,000 --> 00:55:03,720 OF THE SCATTERED RESIDUE THAT 1617 00:55:03,720 --> 00:55:05,760 YOU SHOWED, WOULD THEY ALSO SHOW 1618 00:55:05,760 --> 00:55:06,640 UP IN AN -- 1619 00:55:06,640 --> 00:55:07,320 >> THEY WOULD NOT. 1620 00:55:07,320 --> 00:55:08,560 AND YOUR QUESTION IS A GREAT ONE 1621 00:55:08,560 --> 00:55:10,200 BECAUSE IT LETS ME FURTHER 1622 00:55:10,200 --> 00:55:11,720 EXPAND WHAT I THINK IS 1623 00:55:11,720 --> 00:55:12,320 CONCEPTUALLY THE IMPORTANCE OF 1624 00:55:12,320 --> 00:55:14,600 WHAT I CALL THE ANNA KARENINA 1625 00:55:14,600 --> 00:55:16,760 PRINCIPLE. 1626 00:55:16,760 --> 00:55:18,320 SO ALL HAPPY FAMILIES HAVE TO BE 1627 00:55:18,320 --> 00:55:19,760 HAPPY IN THE SAME WAY. 1628 00:55:19,760 --> 00:55:22,320 AND SO WHEN YOU DO A SEQUENCE 1629 00:55:22,320 --> 00:55:24,640 CONSERVATION OF A LARGE FAMILY, 1630 00:55:24,640 --> 00:55:26,280 THE SEQUENCE CONSERVATION IS 1631 00:55:26,280 --> 00:55:28,880 GOING TO REFLECT THE HAPPINESS 1632 00:55:28,880 --> 00:55:29,320 STATE. 1633 00:55:29,320 --> 00:55:31,320 SO YOU SEE THE BLUE BUILT WHICH 1634 00:55:31,320 --> 00:55:32,960 IS REALLY COUPLING CATALYSIS. 1635 00:55:32,960 --> 00:55:35,360 NOW YOU USE THE ANNA KARENINA 1636 00:55:35,360 --> 00:55:37,120 PRINCIPLE AND YOU LEARN THAT 1637 00:55:37,120 --> 00:55:38,400 EACH UNHAPPY FAMILY IS UNHAPPY 1638 00:55:38,400 --> 00:55:41,760 IN ITS OWN WAY. 1639 00:55:41,760 --> 00:55:54,160 I REFER TO THAT AS I HAD YOE AS I HAD YOE SIN AS IDIOSYNCRASY. 1640 00:55:54,160 --> 00:55:56,200 IF YOU HAVE A LARGE DATABASE, 1641 00:55:56,200 --> 00:55:57,040 SOME FAMILIES WILL BE HAPPY IN 1642 00:55:57,040 --> 00:55:58,040 THE SAME WAY. 1643 00:55:58,040 --> 00:56:00,440 FROM OUR WORK ON SIGNALING, OUR 1644 00:56:00,440 --> 00:56:01,800 WORK ON SIGNALING IS REALLY WHAT 1645 00:56:01,800 --> 00:56:03,040 LED US TO JUMP TO THIS 1646 00:56:03,040 --> 00:56:04,240 CONCLUSION, IS TO CONSIDER 1647 00:56:04,240 --> 00:56:05,720 THE -- AND I'M JUST GOING TO SAY 1648 00:56:05,720 --> 00:56:06,720 THIS BECAUSE SIGNALING IS REALLY 1649 00:56:06,720 --> 00:56:09,440 WHAT MOST OF THE LAB DOES, THE 1650 00:56:09,440 --> 00:56:13,000 KINASES THAT HAVE SH2, SH3 AND 1651 00:56:13,000 --> 00:56:15,800 KINASE ARE ANCIENT FAMILY OF 1652 00:56:15,800 --> 00:56:17,920 SIGNALING KINASES. 1653 00:56:17,920 --> 00:56:23,040 BUT THE SUBMEMBERS OF THIS, THE 1654 00:56:23,040 --> 00:56:25,400 BTK KINASE HAVE REALLY DIFFERENT 1655 00:56:25,400 --> 00:56:27,240 WAYS IN WHICH THEY USE THE 1656 00:56:27,240 --> 00:56:29,040 SH2 AND SH3 DOMAINS TO SWITCH 1657 00:56:29,040 --> 00:56:30,000 OFF THE KINASE. 1658 00:56:30,000 --> 00:56:33,400 SO THEY'RE UNHAPPY IN DIFFERENT 1659 00:56:33,400 --> 00:56:33,600 WAYS. 1660 00:56:33,600 --> 00:56:39,760 OF COURSE IF YOU ENTER THE SARC 1661 00:56:39,760 --> 00:56:41,880 KINASE LINEAGE YOU'LL FIND WHAT 1662 00:56:41,880 --> 00:56:42,880 MAKES THEM -- 1663 00:56:42,880 --> 00:56:44,080 >> I HAVE A SECOND QUESTION. 1664 00:56:44,080 --> 00:56:45,920 THIS IS ABOUT THE GLYCINE THAT 1665 00:56:45,920 --> 00:56:49,560 BASICALLY GETS MUTATED TO SORT 1666 00:56:49,560 --> 00:56:51,760 OF THEN RESCUE THE FIRST 1667 00:56:51,760 --> 00:56:53,560 MUTATION, IN THE FIRST PART OF 1668 00:56:53,560 --> 00:56:54,240 YOUR TALK. 1669 00:56:54,240 --> 00:56:56,800 SO MY QUESTION IS THAT IF YOU 1670 00:56:56,800 --> 00:56:59,640 CONSTRAIN THAT -- IF YOU NOW DO 1671 00:56:59,640 --> 00:57:01,000 AN EXPERIMENT WHERE YOU DON'T -- 1672 00:57:01,000 --> 00:57:04,840 THAT YOU DON'T LET THE GLYCINE 1673 00:57:04,840 --> 00:57:06,240 MAKE A CHANGE INTO THE -- I 1674 00:57:06,240 --> 00:57:08,040 THINK IT WAS CHANGING INTO A 1675 00:57:08,040 --> 00:57:09,480 GLUTAMINE, HAVE YOU DONE 1676 00:57:09,480 --> 00:57:10,800 EXPERIMENTS LIKE THAT AND NOW -- 1677 00:57:10,800 --> 00:57:14,320 SO ESSENTIALLY IT'S -- TO WHAT 1678 00:57:14,320 --> 00:57:16,360 EXTENT CAN IT ACTUALLY RECOVER, 1679 00:57:16,360 --> 00:57:19,400 IF YOU START CONSTRAINING TWO 1680 00:57:19,400 --> 00:57:19,880 RESIDUES? 1681 00:57:19,880 --> 00:57:21,200 I DON'T KNOW IF I MAKE MY 1682 00:57:21,200 --> 00:57:22,280 QUESTION CLEAR. 1683 00:57:22,280 --> 00:57:23,760 >> WE HAVEN'T DONE THE 1684 00:57:23,760 --> 00:57:24,880 EXPERIMENT QUITE THE WAY YOU'RE 1685 00:57:24,880 --> 00:57:27,800 SAYING, BUT WE KNOW FIRST FROM 1686 00:57:27,800 --> 00:57:29,320 JUST THE NATURAL SEQUENCE 1687 00:57:29,320 --> 00:57:30,720 VARIATION IS THAT THERE ARE 1688 00:57:30,720 --> 00:57:32,680 PROTEINS THAT DON'T USE THAT 1689 00:57:32,680 --> 00:57:36,240 GLUTAMINE AND DON'T -- SO NATURE 1690 00:57:36,240 --> 00:57:37,560 IS VERY DIVERSE IN COMING UP 1691 00:57:37,560 --> 00:57:38,120 WITH SOLUTIONS. 1692 00:57:38,120 --> 00:57:39,480 AND THE SECOND THING I'D SAY IS 1693 00:57:39,480 --> 00:57:41,000 THAT WE CAN, OF COURSE, THINK AS 1694 00:57:41,000 --> 00:57:43,520 I SAID EARLIER OF COMBINING THIS 1695 00:57:43,520 --> 00:57:45,120 WITH THAT WITH THIS BUT WE 1696 00:57:45,120 --> 00:57:46,440 HAVEN'T QUITE GOTTEN GOING WITH 1697 00:57:46,440 --> 00:57:46,640 THOSE. 1698 00:57:46,640 --> 00:57:48,040 >> THANK YOU. 1699 00:57:48,040 --> 00:57:49,600 Q.JUST 1700 00:57:49,600 --> 00:57:51,040 >> JUST BECAUSE THE COMBINATIONS 1701 00:57:51,040 --> 00:57:51,400 ARE SO LARGE. 1702 00:57:51,400 --> 00:57:52,080 >> OKAY. 1703 00:57:52,080 --> 00:57:56,000 SO FROM ONLINE, TOM SCHNEIDER 1704 00:57:56,000 --> 00:57:57,840 ASKS, HOW MUCH ATP IS USED PER 1705 00:57:57,840 --> 00:58:00,640 STEP OR REMOVAL, QUESTION MARK, 1706 00:58:00,640 --> 00:58:01,680 OF THE CLAMP LOADER? 1707 00:58:01,680 --> 00:58:02,880 >> YES. 1708 00:58:02,880 --> 00:58:05,880 I'M ASKED HOW MUCH ATP IS USED 1709 00:58:05,880 --> 00:58:09,200 IN THE CYCLE OF WORK, NOT OUR 1710 00:58:09,200 --> 00:58:10,680 WORK BUT MIKE O'DONNELL HAS 1711 00:58:10,680 --> 00:58:12,440 SUGGESTED IN PAPERS -- THAT'S A 1712 00:58:12,440 --> 00:58:13,200 DIFFICULT QUESTION TO ANSWER 1713 00:58:13,200 --> 00:58:15,000 BECAUSE WHAT YOU END UP DOING IS 1714 00:58:15,000 --> 00:58:16,320 MAKING MUTATIONS AND SO YOU 1715 00:58:16,320 --> 00:58:18,080 CAN -- THE ANSWER IS THAT YOU 1716 00:58:18,080 --> 00:58:19,440 CAN GET A FUNCTIONING CLAMP 1717 00:58:19,440 --> 00:58:23,040 LOADER IF ONLY ONE ATP IS 1718 00:58:23,040 --> 00:58:23,360 HYDROLYZED. 1719 00:58:23,360 --> 00:58:27,640 SORT OF HOBBLING ALONG. 1720 00:58:27,640 --> 00:58:28,640 >> ANY MORE QUESTIONS? 1721 00:58:28,640 --> 00:58:30,840 ONE MORE COMING DOWN? 1722 00:58:30,840 --> 00:58:31,960 >> THERE'S SOMEBODY COMING DOWN 1723 00:58:31,960 --> 00:58:34,720 HERE OR THERE. 1724 00:58:34,720 --> 00:58:38,560 NO, PLEASE. 1725 00:58:38,560 --> 00:58:42,720 >> ABOUT THE GLYCINE MUTATION, 1726 00:58:42,720 --> 00:58:45,680 YOU KNOW IT'S NOT ONLY THE -- 1727 00:58:45,680 --> 00:58:47,880 BUT IT'S ALSO THE VIBRATIONS AND 1728 00:58:47,880 --> 00:58:50,640 THE MOTION OF THIS, THE GLYCINE 1729 00:58:50,640 --> 00:58:52,040 DIFFERENTLY IS BASICALLY 1730 00:58:52,040 --> 00:58:55,120 ALLOWING TO BE MORE FLEXIBLE 1731 00:58:55,120 --> 00:58:55,760 THAN THAT. 1732 00:58:55,760 --> 00:58:57,640 >> YES, OF COURSE THE GLYCINE 1733 00:58:57,640 --> 00:58:58,840 INTRODUCES FLEXIBILITY WHICH IS 1734 00:58:58,840 --> 00:59:02,640 REMOVED BY THE MUTATION THAT 1735 00:59:02,640 --> 00:59:07,360 RESCUES AND THE DYNAMICS 1736 00:59:07,360 --> 00:59:07,920 CANNOT -- 1737 00:59:07,920 --> 00:59:14,680 >> ALSO THE -- IS GLYCOSYLATED. 1738 00:59:14,680 --> 00:59:17,480 >> THE T4 DNA IS GLU COST LATED 1739 00:59:17,480 --> 00:59:18,720 AND THAT'S ONE OF THE THINGS 1740 00:59:18,720 --> 00:59:19,800 THAT THE PHAGE MACHINERY DOES 1741 00:59:19,800 --> 00:59:21,840 AFTER IT INFECTS THE PHAGE AND 1742 00:59:21,840 --> 00:59:23,400 ACTUALLY THAT'S THE REASON WE 1743 00:59:23,400 --> 00:59:25,480 HAD TO PUT IN A CRISPR SYSTEM, 1744 00:59:25,480 --> 00:59:26,440 BECAUSE THAT PREVENTS US -- YOU 1745 00:59:26,440 --> 00:59:28,440 SEE THE PHAGE INFECTS THE 1746 00:59:28,440 --> 00:59:30,680 E. COLI, THEN I DIDN'T GET ASKED 1747 00:59:30,680 --> 00:59:35,200 THE QUESTION WHY DIDN'T WE JUST 1748 00:59:35,200 --> 00:59:36,120 SEQUENCE THE E. COLI THAT'S LEFT 1749 00:59:36,120 --> 00:59:36,960 OR SOMETHING LIKE THAT. 1750 00:59:36,960 --> 00:59:39,040 BUT THE THING IS THE PHAGE COMES 1751 00:59:39,040 --> 00:59:41,080 IN AND SO DAMAGES THE DNA, THE 1752 00:59:41,080 --> 00:59:42,760 HOST DNA. 1753 00:59:42,760 --> 00:59:44,360 AND THAT'S THE REASON WE PUT IN 1754 00:59:44,360 --> 00:59:46,280 THE CRISPR SYSTEM TO GET THE 1755 00:59:46,280 --> 00:59:48,120 PHAGE OUT. 1756 00:59:48,120 --> 00:59:50,640 SO FULLY APPRECIATE THAT. 1757 00:59:50,640 --> 00:59:50,840 YEAH. 1758 00:59:50,840 --> 00:59:51,720 >> A QUESTION ABOUT THE FIRST 1759 00:59:51,720 --> 00:59:53,720 PART OF THE TALK. 1760 00:59:53,720 --> 00:59:56,840 WHEN YOU DID THE FIRST 1761 00:59:56,840 --> 00:59:57,840 MUTATIONAL ANALYSIS, SOME OF THE 1762 00:59:57,840 --> 01:00:01,240 MUTANTS ACTUALLY ENHANCE THE 1763 01:00:01,240 --> 01:00:03,200 PROPERTIES, THE REPLICATION OF 1764 01:00:03,200 --> 01:00:06,000 THE T4, SO THAT MEANS THAT 1765 01:00:06,000 --> 01:00:07,480 NATURE, IF EVOLUTION, YOU KNOW, 1766 01:00:07,480 --> 01:00:09,680 WENT ON LONGER, COULD ACTUALLY 1767 01:00:09,680 --> 01:00:12,680 FIND MORE EFFICIENT T4s THAT 1768 01:00:12,680 --> 01:00:12,920 COULD -- 1769 01:00:12,920 --> 01:00:15,720 >> YES, NATURE COULD FIND MORE 1770 01:00:15,720 --> 01:00:16,760 EFFICIENT T4 WHICH IS SURELY THE 1771 01:00:16,760 --> 01:00:17,680 CASE BECAUSE WE'RE SO QUICKLY 1772 01:00:17,680 --> 01:00:19,240 ABLE TO CHANGE THE FUNCTION 1773 01:00:19,240 --> 01:00:21,440 AFTER A MUTATION. 1774 01:00:21,440 --> 01:00:24,240 SO THAT LEADS INTO DIFFICULT 1775 01:00:24,240 --> 01:00:27,320 QUESTIONS OF WHAT IS ACTUALLY 1776 01:00:27,320 --> 01:00:30,200 THE CONDITION AL SPECTRUM UNDER 1777 01:00:30,200 --> 01:00:31,360 WHICH THE PHAGE IS ACTUALLY 1778 01:00:31,360 --> 01:00:33,520 BEING SELECTED. 1779 01:00:33,520 --> 01:00:37,840 >> AND ONE OF THEM WAS THE 1780 01:00:37,840 --> 01:00:43,200 PHENYL RESIDUE INTO DEXD MOTIF 1781 01:00:43,200 --> 01:00:43,400 TO -- 1782 01:00:43,400 --> 01:00:44,840 >> LIKE WHY WOULD THAT WORK 1783 01:00:44,840 --> 01:00:46,120 BETTER THAN WILD TYPE. 1784 01:00:46,120 --> 01:00:49,400 SO WE DON'T KNOW. 1785 01:00:49,400 --> 01:00:50,840 WE SHOULD MAKE THAT. 1786 01:00:50,840 --> 01:00:53,040 BUT MAYBE I'M TRYING TO 1787 01:00:53,040 --> 01:00:54,240 COMMUNICATE THE SENSE THAT THESE 1788 01:00:54,240 --> 01:00:55,640 DATA ARE RICH AND CAN BE 1789 01:00:55,640 --> 01:00:57,200 EXPLOITED IN MANY WAYS. 1790 01:00:57,200 --> 01:00:57,520 >> ABSOLUTELY. 1791 01:00:57,520 --> 01:00:57,840 THANK YOU. 1792 01:00:57,840 --> 01:00:59,120 >> THANK YOU. 1793 01:00:59,120 --> 01:01:00,360 >> ALL RIGHT. 1794 01:01:00,360 --> 01:01:03,040 I THINK WITH THAT, LET'S THANK 1795 01:01:03,040 --> 01:01:05,200 JOHN FOR A REALLY, REALLY 1796 01:01:05,200 --> 01:01:06,120 INTRIGUING AND ENGAGING TALK. 1797 01:01:06,120 --> 01:01:06,680 [APPLAUSE] 1798 01:01:06,680 --> 01:03:01,080 >> THANK YOU ALL.