1 00:00:07,605 --> 00:00:10,341 >> GOOD MORNING, EVERYONE, AND 2 00:00:10,341 --> 00:00:12,443 WELCOME TO ANOTHER GREAT EDITION 3 00:00:12,443 --> 00:00:14,312 OF THE BIOWULF SEMINAR SERIES. 4 00:00:14,312 --> 00:00:16,581 IN FACT, THIS YEAR WE ARE 5 00:00:16,581 --> 00:00:20,518 CELEBRATING THE 25TH ANNIVERSARY 6 00:00:20,518 --> 00:00:23,120 OF BIOWULF. 7 00:00:23,120 --> 00:00:25,756 [APPLAUSE] 8 00:00:25,756 --> 00:00:28,059 AND WE HAVE A GREAT LINEUP OF 9 00:00:28,059 --> 00:00:30,394 SCIENTIFIC TALKS FOR THIS YEAR'S 10 00:00:30,394 --> 00:00:30,728 SEMINAR. 11 00:00:30,728 --> 00:00:33,431 WE ALSO HAVE SPECIAL VISITORS 12 00:00:33,431 --> 00:00:36,300 ATTENDING OUR TALK TODAY. 13 00:00:36,300 --> 00:00:38,803 AND TODAY SPECIFICALLY, WE ARE 14 00:00:38,803 --> 00:00:42,206 HONORED TO HAVE DR. JULIANA 15 00:00:42,206 --> 00:00:46,377 MARTINEZ FIESCO AS OUR SPEAKER, 16 00:00:46,377 --> 00:00:48,679 SHE RECEIVED HER Ph.D. FROM 17 00:00:48,679 --> 00:00:50,114 FLORIDA STATE UNIVERSITY WHERE 18 00:00:50,114 --> 00:00:53,050 SHE STUDIED GLOBAL KINASE 19 00:00:53,050 --> 00:00:54,585 MECHANISM OF ACTION AND 20 00:00:54,585 --> 00:00:57,221 ACTIVATION AND ALSO TRAINED AS A 21 00:00:57,221 --> 00:00:58,823 BIOCHEMIST. 22 00:00:58,823 --> 00:01:03,461 IN 2017 SHE JOINED 23 00:01:03,461 --> 00:01:05,763 DR. PING JEN'S LAB IN THE KINASE 24 00:01:05,763 --> 00:01:06,964 SECTION -- CENTRAL KINASE 25 00:01:06,964 --> 00:01:09,367 SECTION IN NATIONAL CANCER 26 00:01:09,367 --> 00:01:10,268 INSTITUTE? 27 00:01:10,268 --> 00:01:10,534 FREDERICK. 28 00:01:10,534 --> 00:01:11,769 THIS WAS FOR HER POSTDOCTORAL 29 00:01:11,769 --> 00:01:14,205 TRAINING, WHERE SHE ALSO STUDIED 30 00:01:14,205 --> 00:01:17,008 A SET OF KINASES AND TRAINED AS 31 00:01:17,008 --> 00:01:19,644 A STRUCTURAL BIOLOGIST. 32 00:01:19,644 --> 00:01:21,312 SHE RECENTLY TRANSITIONED TO A 33 00:01:21,312 --> 00:01:24,782 STAFF SCIENTIST POSITION WITH 34 00:01:24,782 --> 00:01:27,318 PI -- PINGJEN'S LAB, HER 35 00:01:27,318 --> 00:01:28,653 SPECIFIC INTEREST IS TO 36 00:01:28,653 --> 00:01:29,620 UNDERSTAND THE STRUCTURE 37 00:01:29,620 --> 00:01:31,689 FUNCTION RELATIONSHIP IN ENZYMES 38 00:01:31,689 --> 00:01:34,225 TO UNDERSTAND THEIR MECHANISM OF 39 00:01:34,225 --> 00:01:35,993 ACTION, TO GAIN SIGHT INTO 40 00:01:35,993 --> 00:01:39,864 DIFFERENT DISEASES. 41 00:01:39,864 --> 00:01:43,134 SHE IS AN AVE. INDIVIDUAL -- 42 00:01:43,134 --> 00:01:45,002 USER AND HER TALK TODAY IS 43 00:01:45,002 --> 00:01:48,572 TITLED STRUCTURAL INSIGHTS INTO 44 00:01:48,572 --> 00:01:52,310 THE MONOMER ARE DIMER TRANSITION 45 00:01:52,310 --> 00:01:57,448 MEDIATING BY RAS BINDING. 46 00:01:57,448 --> 00:02:00,084 [APPLAUSE] 47 00:02:00,084 --> 00:02:00,251 HAD. 48 00:02:00,251 --> 00:02:02,720 >> GOOD MORNING, EVERYONE, THANK 49 00:02:02,720 --> 00:02:04,355 YOU FOR THE OPPORTUNITY SHARE 50 00:02:04,355 --> 00:02:05,356 OUR WORK TODAY. 51 00:02:05,356 --> 00:02:07,858 AS HE MENTIONED, TODAY I'M GOING 52 00:02:07,858 --> 00:02:09,193 TO SHARE SOME STRUCTURAL WORK WE 53 00:02:09,193 --> 00:02:11,896 HAVE DONE IN THE LAB IN 54 00:02:11,896 --> 00:02:15,199 SPECIFICALLY IN BRAF PROTEINS 55 00:02:15,199 --> 00:02:18,569 AND WE SPECIFICALLY CHOSE THIS 56 00:02:18,569 --> 00:02:21,305 PROJECT BECAUSE WE REALLY RELY 57 00:02:21,305 --> 00:02:23,407 ON BIOWULF RESOURCES TO BE ABLE 58 00:02:23,407 --> 00:02:25,476 TO DO OUR ANALYSIS ESPECIALLY 59 00:02:25,476 --> 00:02:28,846 THE STRUCTURAL WORK PART. 60 00:02:28,846 --> 00:02:30,948 SO I JUST WANT TO MAKE SURE THIS 61 00:02:30,948 --> 00:02:31,916 IS WORKING OKAY. 62 00:02:31,916 --> 00:02:35,419 AS YOU MAY KNOW, THE WILL RAS 63 00:02:35,419 --> 00:02:37,054 PATHWAY PLAYS A VERY IMPORTANT 64 00:02:37,054 --> 00:02:38,255 ROLE IN THE TRANSMISSION OF 65 00:02:38,255 --> 00:02:39,690 GROWTH SIGNALS BUT ACCOUNTS ALSO 66 00:02:39,690 --> 00:02:42,193 A CONTRIBUTOR CONTRIBUTOR OF 67 00:02:42,193 --> 00:02:43,427 HUMAN DISEASE STATES, HAVING 68 00:02:43,427 --> 00:02:45,062 SPECIFICALLY MUTATIONS IN RAS 69 00:02:45,062 --> 00:02:47,698 PROTEINS AND BRAF -- AND RAS 70 00:02:47,698 --> 00:02:48,899 PROTEINS BEING THE MAJOR DRIVERS 71 00:02:48,899 --> 00:02:50,668 OF MANY DIFFERENT TYPES OF 72 00:02:50,668 --> 00:02:50,901 CANCERS. 73 00:02:50,901 --> 00:02:54,505 BECAUSE OF THIS, THESE PROTEINS 74 00:02:54,505 --> 00:02:57,341 ARE THE TARGET SPORE THERAPEUTIC 75 00:02:57,341 --> 00:02:58,442 INTERVENTION, BUT IN ORDER TO DO 76 00:02:58,442 --> 00:03:01,078 THAT, IT'S VERY IMPORTANT TO 77 00:03:01,078 --> 00:03:02,279 UNDERSTAND THE MECHANISTIC 78 00:03:02,279 --> 00:03:04,782 DETAILS OF HOW THE SIGNALS ARE 79 00:03:04,782 --> 00:03:06,550 TRANSMITTED THROUGH THE PATHWAY. 80 00:03:06,550 --> 00:03:09,420 SO OUR LAB HAS BEEN FOCUSED ON 81 00:03:09,420 --> 00:03:11,122 UNDERSTANDING THE STRUCTURE OF 82 00:03:11,122 --> 00:03:12,923 ONE OF THESE PROTEINS IN ORDER 83 00:03:12,923 --> 00:03:15,526 TO UNDERSTAND HOW THEY AK -- HOW 84 00:03:15,526 --> 00:03:17,228 THE ACTIVATION OF THESE PROTEINS 85 00:03:17,228 --> 00:03:19,029 HAPPEN AND HOW THE SIGNALS ARE 86 00:03:19,029 --> 00:03:22,967 TRANSMITTED THROUGH THE PATHWAY. 87 00:03:22,967 --> 00:03:25,069 SO WE ARE MAINLY FOCUSED ON THE 88 00:03:25,069 --> 00:03:28,005 STRUCTURAL BIOLOGY PART OF THE 89 00:03:28,005 --> 00:03:29,774 PATHWAY, SO WE WANT TO DETERMINE 90 00:03:29,774 --> 00:03:31,876 THE DIFFERENT STRUCTURES OF BRAF 91 00:03:31,876 --> 00:03:34,612 PROTEINS IN DIFFERENT COMPLEXES. 92 00:03:34,612 --> 00:03:37,448 SO BRAF PROTEINS, THERE ARE 93 00:03:37,448 --> 00:03:41,652 THREE MEMBERS IN THE BRAF 94 00:03:41,652 --> 00:03:41,886 FAMILY. 95 00:03:41,886 --> 00:03:45,122 WE HAVE ARAF PROTEIN, BRAF AND 96 00:03:45,122 --> 00:03:48,893 CRAF PROTEINS, BUT BRAF AND 97 00:03:48,893 --> 00:03:51,729 CRA -- AND CRAF PROTEINS THE 98 00:03:51,729 --> 00:03:53,164 MAIN DRIVERS MUCH HUMAN DISEASES 99 00:03:53,164 --> 00:03:53,931 SPECIFICALLY CANCER. 100 00:03:53,931 --> 00:03:55,132 ALL THE MEMBERS OF THE FAMILY 101 00:03:55,132 --> 00:03:56,200 HAVE A SIMILAR DOMAIN 102 00:03:56,200 --> 00:03:56,901 ORGANIZATION, SO YOU CAN THINK 103 00:03:56,901 --> 00:04:01,906 OF IT AS IF IT HAS AN END 104 00:04:01,906 --> 00:04:03,774 TERMINAL REGULATORY DOMAIN AND 105 00:04:03,774 --> 00:04:05,743 FROM THIS HAS A DOMAIN THAT IS 106 00:04:05,743 --> 00:04:07,711 CALLED THE RAS BINDING DOMAIN 107 00:04:07,711 --> 00:04:14,952 THAT WE'RE GOING TO CALL IN THIS 108 00:04:14,952 --> 00:04:17,021 PRESENTATION RBD AND CYSTEINE 109 00:04:17,021 --> 00:04:18,556 RICH DOMAIN, CRD. 110 00:04:18,556 --> 00:04:20,191 THIS IS THE REGULATORY PART OF 111 00:04:20,191 --> 00:04:21,025 THE PROTEIN. 112 00:04:21,025 --> 00:04:23,928 AT THE C TERMINALS WE HAVE THE 113 00:04:23,928 --> 00:04:25,129 KINASE DOMAIN AND THIS IS THE 114 00:04:25,129 --> 00:04:27,198 PART OF THE PROTEIN THAT 115 00:04:27,198 --> 00:04:29,733 ACTUALLY CARRIES OUT THE 116 00:04:29,733 --> 00:04:36,073 CATALYTIC FUNCTION SO THE -- SO 117 00:04:36,073 --> 00:04:37,808 THE NEXT PROTEIN IN THE CHAIN 118 00:04:37,808 --> 00:04:40,110 WHICH IS MEK. 119 00:04:40,110 --> 00:04:42,613 THESE PROTEINS ARE FOUND IN AN 120 00:04:42,613 --> 00:04:43,914 INHIBITORY, OUTER INHIBITORY 121 00:04:43,914 --> 00:04:45,716 STATE AND IT'S BASICALLY 122 00:04:45,716 --> 00:04:47,251 INTERACTIONS BETWEEN THE N 123 00:04:47,251 --> 00:04:49,887 TERMINAL REGULATORY DOMAINS AND 124 00:04:49,887 --> 00:04:54,158 THE C TERMINAL DOMAIN THAT 125 00:04:54,158 --> 00:04:55,559 MAINTAIN THE STATE BUT YOU CAN 126 00:04:55,559 --> 00:05:01,398 ALSO SEE THAT INTERACTION WITH 127 00:05:01,398 --> 00:05:02,700 14-3-2 PROTEINS THAT FORM A 128 00:05:02,700 --> 00:05:05,102 DIMER AND HELP STABILIZE THE 129 00:05:05,102 --> 00:05:06,403 OUTER STATE. 130 00:05:06,403 --> 00:05:08,739 ONCE THE PROTEIN RECEIVES 131 00:05:08,739 --> 00:05:11,475 SIGNALS TO ACTIVATE THE PATHWAY, 132 00:05:11,475 --> 00:05:16,947 WHAT HAS HAPPENED IS THAT THE 133 00:05:16,947 --> 00:05:19,984 PROTEIN, THE BRAF PROTEIN IN 134 00:05:19,984 --> 00:05:23,420 THIS CASE INTERACTS WITH PROTEIN 135 00:05:23,420 --> 00:05:24,488 THROUGH THE BINDING DOMAIN OF 136 00:05:24,488 --> 00:05:26,156 THE RBD DOMAIN. 137 00:05:26,156 --> 00:05:27,391 WHEN THIS INTERACTION HAPPENS, 138 00:05:27,391 --> 00:05:29,293 WE ARE GOING TO HAVE SOME 139 00:05:29,293 --> 00:05:30,127 CONFORMATIONAL CHANGES IN THE 140 00:05:30,127 --> 00:05:32,596 PROTEIN THAT ARE GOING TO 141 00:05:32,596 --> 00:05:34,798 PRODUCE DIMER ZAIFTION THE 142 00:05:34,798 --> 00:05:36,433 PROTEIN -- DIMERIZATION OF THE 143 00:05:36,433 --> 00:05:38,636 PROTEIN AND IN TURN PRODUCE 144 00:05:38,636 --> 00:05:39,603 ACTIVATION OF THE PROTEIN. 145 00:05:39,603 --> 00:05:41,472 SO IT HAS BEEN KNOWN FOR 146 00:05:41,472 --> 00:05:46,844 DIFFERENT STUDIES, MAINLY ARE 147 00:05:46,844 --> 00:05:48,812 CRYSTAL STRUCTURES OF JUST 148 00:05:48,812 --> 00:05:51,949 CONTAINING THE KINASE DOMAIN OF 149 00:05:51,949 --> 00:05:56,820 THE R PROTEINS, NEED TO DIMER 150 00:05:56,820 --> 00:05:58,589 EYES IN ORDER TO BECOME ACTIVE. 151 00:05:58,589 --> 00:05:59,557 THERE ARE SOME STRUCTURAL 152 00:05:59,557 --> 00:06:01,525 ARRANGEMENTS THAT NEED TO HAPPEN 153 00:06:01,525 --> 00:06:03,894 WHEN RAS BINDING TO RAF IN ORDER 154 00:06:03,894 --> 00:06:05,362 FOR THE DIMERIZATION TO TAKE 155 00:06:05,362 --> 00:06:07,097 PLACE AND THEN THE PATHWAY CAN 156 00:06:07,097 --> 00:06:12,469 BE ACTIVATED. 157 00:06:12,469 --> 00:06:13,637 SO WHAT WE REALLIMENT TO 158 00:06:13,637 --> 00:06:15,406 UNDERSTAND IS HOW THE BINDING OF 159 00:06:15,406 --> 00:06:18,142 RAS TO RAF IS GOING TO PRODUCE 160 00:06:18,142 --> 00:06:19,510 THE TRANSITION THAT IS REQUIRED 161 00:06:19,510 --> 00:06:24,281 TO GO FROM MONOMER TO DIMER. 162 00:06:24,281 --> 00:06:29,219 SO WE WANTED TO INVESTIGATE THIS 163 00:06:29,219 --> 00:06:30,120 RAILROAD STRUCTURALLY. 164 00:06:30,120 --> 00:06:31,188 BEFORE I CONTINUE EXPLAINING 165 00:06:31,188 --> 00:06:32,489 WHAT WE DID, EU79 TO MENTION 166 00:06:32,489 --> 00:06:35,859 THAT THIS WORK HAS BEEN DONE IN 167 00:06:35,859 --> 00:06:38,929 COLLABORATION WITH DR. MAURICE 168 00:06:38,929 --> 00:06:41,231 SONT AT THE LABORATORY OF 169 00:06:41,231 --> 00:06:42,900 DEVELOPMENTAL SIGNALLING AND 170 00:06:42,900 --> 00:06:44,368 FOLLOWED DEBBIE DUR RANT, SO 171 00:06:44,368 --> 00:06:45,803 THEY ARE VERY EXPERTS LIKE IN 172 00:06:45,803 --> 00:06:49,039 ALL THE RAS AND RAF BIOLOGY AND 173 00:06:49,039 --> 00:06:51,108 OUR LAB HAS MORE EXPERTISE IN 174 00:06:51,108 --> 00:06:54,745 THE -- AND OUR LAB HAS MORE 175 00:06:54,745 --> 00:06:55,479 EXPERTISE IN THE STRUCTURAL 176 00:06:55,479 --> 00:06:57,615 BIOLOGY PART, SO IT WAS A VERY 177 00:06:57,615 --> 00:06:58,248 COMPLEMENTARY WORK BETWEEN THE 178 00:06:58,248 --> 00:06:59,049 TWO LABS. 179 00:06:59,049 --> 00:07:04,922 WE MAINLY USE A ELECTRIC CALLED 180 00:07:04,922 --> 00:07:05,923 CRYOMICROSCOPY TO SIGN THE 181 00:07:05,923 --> 00:07:07,791 STRUCTURES OF BRAF PROTEIN IN 182 00:07:07,791 --> 00:07:08,892 THESE DIFFERENT COMPLEXES AND WE 183 00:07:08,892 --> 00:07:10,394 WERE ABLE TO DETERMINE THREE 184 00:07:10,394 --> 00:07:12,796 DIFFERENT STRUCTURES. 185 00:07:12,796 --> 00:07:16,767 TWO STRUCTURES THAT CORRESPONDS 186 00:07:16,767 --> 00:07:23,440 TO RAF IN ARE THE STATE, ONE HAS 187 00:07:23,440 --> 00:07:26,477 MEK, ONE DOESN'T HAVE MEK AND 188 00:07:26,477 --> 00:07:27,911 DIMER STRUCTURE OF THE PROTEIN. 189 00:07:27,911 --> 00:07:29,446 BRING MOVE FORWARD TO SHOW YOU 190 00:07:29,446 --> 00:07:31,215 THE STRUCTURE, I JUST WANTED TO 191 00:07:31,215 --> 00:07:34,151 GIVE A BRIEF DESCRIPTION OF WHAT 192 00:07:34,151 --> 00:07:36,253 THE CRYO-EM PROCESS LOOKS LIKE. 193 00:07:36,253 --> 00:07:38,522 SO NORMALLY YOU YOU'RE PHI YOUR 194 00:07:38,522 --> 00:07:39,523 PROTEIN, YOU WANT TO HAVE YOUR 195 00:07:39,523 --> 00:07:42,026 PROTEIN TO BE HIGHLY PURE AND BE 196 00:07:42,026 --> 00:07:43,761 VERY HOMOGENEOUS, THEN YOU APPLY 197 00:07:43,761 --> 00:07:46,430 THIS PROTEIN IN WHAT IS CALLED A 198 00:07:46,430 --> 00:07:46,964 CRYO-EM GRID. 199 00:07:46,964 --> 00:07:50,701 SO THE CRYO-EM GRID IS JUST 200 00:07:50,701 --> 00:07:53,504 WHERE YOUR PROTEIN IS GOING TO 201 00:07:53,504 --> 00:07:56,940 BE DEPOSITED SO BUT NORMALLY 202 00:07:56,940 --> 00:07:58,809 CARBON, YOU'LL SEE THIS CARBON 203 00:07:58,809 --> 00:08:00,144 LAYER HAS SOME TINY HOLES. 204 00:08:00,144 --> 00:08:01,578 SO THE IDEA IS THAT WHEN YOU 205 00:08:01,578 --> 00:08:04,715 APPLY THE SAMPLE TO THIS GRID, 206 00:08:04,715 --> 00:08:06,850 YOU CAN BLOT THE EXCESS SOLUTION 207 00:08:06,850 --> 00:08:09,453 AND THEN YOU FREEZE IT VERY 208 00:08:09,453 --> 00:08:09,653 FAST. 209 00:08:09,653 --> 00:08:10,954 WHAT YOU END UP HAVING IS THAT 210 00:08:10,954 --> 00:08:13,290 THE IN HOLES, IN THE CRYO-EM 211 00:08:13,290 --> 00:08:15,826 GRID, YOU FORM A VERY FINE LAYER 212 00:08:15,826 --> 00:08:18,128 OF ICE, AND IN THIS LAYER OF 213 00:08:18,128 --> 00:08:19,296 ICE, YOU HAVE DIFFERENT 214 00:08:19,296 --> 00:08:21,298 PARTICLES OF YOUR PROTEIN, SO 215 00:08:21,298 --> 00:08:22,533 YOU MAYBE CAN APPRECIATE IT 216 00:08:22,533 --> 00:08:24,368 BETTER IN THIS DIAGRAM HERE. 217 00:08:24,368 --> 00:08:26,770 SO YOU'RE GOING TO HAVE YOUR 218 00:08:26,770 --> 00:08:28,639 PROTEIN LIKE IN THIS VERY TINY 219 00:08:28,639 --> 00:08:30,307 LAYER OF ICE, AND THE PROTEIN IS 220 00:08:30,307 --> 00:08:33,343 GOING TO BE LIKE SITTING IN 221 00:08:33,343 --> 00:08:34,211 DIFFERENT ORIENTATIONS. 222 00:08:34,211 --> 00:08:36,413 SO WHAT YOU DO IS THAT YOU IMAGE 223 00:08:36,413 --> 00:08:39,249 THESE PARTICLES IN THIS ICE WITH 224 00:08:39,249 --> 00:08:42,019 AN ELECTRON BEAM AND THEN YOU 225 00:08:42,019 --> 00:08:43,620 DETECT, OBVIOUSLY WITH ELECTRON 226 00:08:43,620 --> 00:08:44,621 DETECTOR, AND WHAT YOU 227 00:08:44,621 --> 00:08:45,723 UNDERSTAND UP COLLECT SG A 228 00:08:45,723 --> 00:08:50,194 SERIES OF IMAGES THAT CONTAIN 2D 229 00:08:50,194 --> 00:08:51,628 PROJECTIONS OF YOUR PARTICLE, IN 230 00:08:51,628 --> 00:08:53,697 THIS CASE YOUR PROTEIN. 231 00:08:53,697 --> 00:08:57,000 AND WHAT YOU DO IS THAT YOU 232 00:08:57,000 --> 00:08:59,403 EX-TRABLGHT THESE 2D CLASSES OR 233 00:08:59,403 --> 00:09:01,238 2D I AM EXPHADGES TRY TO USE 234 00:09:01,238 --> 00:09:03,307 THIS 2D IMAGE TO BACK PROJECT 235 00:09:03,307 --> 00:09:04,975 AND RECONSTRUCT THE 236 00:09:04,975 --> 00:09:07,244 THREE-DIMENSIONAL STRUCTURE OF 237 00:09:07,244 --> 00:09:09,246 THE PROTEIN. 238 00:09:09,246 --> 00:09:11,081 TH T THIS IS WHERE YOU NEED A 239 00:09:11,081 --> 00:09:12,349 LOT OF COMPUTATIONAL RESOURCES 240 00:09:12,349 --> 00:09:13,851 TO DO THAT AND THIS IS WHERE 241 00:09:13,851 --> 00:09:14,818 BIOWULF WAS VERY IMPORTANT FOR 242 00:09:14,818 --> 00:09:17,755 US IN THIS PROJECT. 243 00:09:17,755 --> 00:09:18,655 SO PROBABLY MOST OF THE PEOPLE 244 00:09:18,655 --> 00:09:20,924 HERE KNOW THAT BIOWULF HAS LIKE 245 00:09:20,924 --> 00:09:23,227 A LOT OF RESOURCES THAT ARE 246 00:09:23,227 --> 00:09:26,430 DEDICATED TO THE CRYO-EM DATA 247 00:09:26,430 --> 00:09:26,697 ANALYSIS. 248 00:09:26,697 --> 00:09:29,533 I THINK I COUNT PROBABLY 20 249 00:09:29,533 --> 00:09:31,268 DIFFERENT SCIENTIFIC PROGRAMS 250 00:09:31,268 --> 00:09:32,436 THAT YOU HAVE IN BIOWULF, AND 251 00:09:32,436 --> 00:09:40,844 FOR THIS PROJECT, WE MAINLY USE 252 00:09:40,844 --> 00:09:44,248 RE -- RELION. 253 00:09:44,248 --> 00:09:45,783 WE DID THREE DIFFERENT PURE 254 00:09:45,783 --> 00:09:45,983 FIXES. 255 00:09:45,983 --> 00:09:48,519 THERE ARE TWO PURE FIXES THAT WE 256 00:09:48,519 --> 00:09:51,355 USE -- PURIFICATIONS THAT WE 257 00:09:51,355 --> 00:09:51,522 USE. 258 00:09:51,522 --> 00:09:54,324 ALL THE PURIFICATIONS WERE DONE 259 00:09:54,324 --> 00:09:55,759 FROM MAMMALIAN CELLS THAT WERE 260 00:09:55,759 --> 00:09:59,029 ALREADY EXPRESSING A FORM OF THE 261 00:09:59,029 --> 00:10:05,936 BRAF PROTEIN THAT HAD HALO T 262 00:10:05,936 --> 00:10:06,170 TAG. 263 00:10:06,170 --> 00:10:07,671 THE PROTEINS YOU'LL SEE IN THE 264 00:10:07,671 --> 00:10:09,206 COMPLEX WERE ENDOGENOUS PROTEINS 265 00:10:09,206 --> 00:10:10,741 IN THESE MAMMALIAN CELLS. 266 00:10:10,741 --> 00:10:12,476 SO WE HAVE TWO PURIFICATIONS 267 00:10:12,476 --> 00:10:15,479 THAT WERE DONE IN SERUM DEPLETED 268 00:10:15,479 --> 00:10:17,648 CELLS AND WE ISOLATED FROM THESE 269 00:10:17,648 --> 00:10:19,149 TWO SAMPLES PROTEIN COME PLEKS 270 00:10:19,149 --> 00:10:21,652 THAT CONTAIN JUST ONE MONOMER OF 271 00:10:21,652 --> 00:10:24,188 BRAF AND THEN WHEN WE TREATED 272 00:10:24,188 --> 00:10:25,923 THE SAMPLE -- OR THE CELLS, 273 00:10:25,923 --> 00:10:28,458 SORRY, WITH BRAF INHIBITOR THAT 274 00:10:28,458 --> 00:10:30,394 PATROLMAN THE DIMER FORMATION, 275 00:10:30,394 --> 00:10:37,734 WE WERE ABLE TO ISOLATE A -- -- 276 00:10:37,734 --> 00:10:40,504 SO YOU CAN SEE HERE, ON THE 277 00:10:40,504 --> 00:10:43,440 RIGHT IS THE SDS GELS THAT 278 00:10:43,440 --> 00:10:45,209 ALLOWED US TO IDENTIFIES WHAT 279 00:10:45,209 --> 00:10:46,210 PROTEINS WERE PRESENT AND ALSO 280 00:10:46,210 --> 00:10:48,078 THE PURITY OF THE COMPLEX THAT 281 00:10:48,078 --> 00:10:48,812 WERE PURIFIED. 282 00:10:48,812 --> 00:10:51,348 SO YOU CAN SEE HERE, IN THE 283 00:10:51,348 --> 00:10:54,418 FIRST TWO GELS THAT I'M SHOWING 284 00:10:54,418 --> 00:10:59,957 HERE, WHERE WE ISOLATED THE 285 00:10:59,957 --> 00:11:01,391 MONOMERIC OF WE HAVE THE 286 00:11:01,391 --> 00:11:05,629 PRESENCE OF BRAF AND 14-3-3 AND 287 00:11:05,629 --> 00:11:06,730 ALL THE PRESENCE OF MEK. 288 00:11:06,730 --> 00:11:08,398 IN THE OTHER ONE WHEN WE TREATED 289 00:11:08,398 --> 00:11:10,167 THE CELLS WITH THE KINASE 290 00:11:10,167 --> 00:11:12,236 INHIBITOR SPECIFICALLY BRAF 291 00:11:12,236 --> 00:11:14,638 INHIBITOR, WE MAINLY ONLY HAVE 292 00:11:14,638 --> 00:11:17,708 BRAF AND 14-3-3. 293 00:11:17,708 --> 00:11:19,877 SO WE'LL TAKE A LOOK FIRST AT 294 00:11:19,877 --> 00:11:22,846 THE SAMPLE WHERE WE PURIFY THE 295 00:11:22,846 --> 00:11:24,615 DIMERIC FORM OF BRAF, WE CAN 296 00:11:24,615 --> 00:11:27,117 SEE, I'M SHOWING YOU HERE 297 00:11:27,117 --> 00:11:28,418 REPRESENTATIVE TO THE CLASSES OR 298 00:11:28,418 --> 00:11:32,689 TO THE IMAGES THAT WE USED TO 299 00:11:32,689 --> 00:11:34,892 RECONSTRUCT, TO FIND A 300 00:11:34,892 --> 00:11:36,059 RESOLUTION OF THE STRUCTURE OF 301 00:11:36,059 --> 00:11:37,294 THE BRAF. 302 00:11:37,294 --> 00:11:39,496 SO YOU CAN SEE HERE THAT WE HAVE 303 00:11:39,496 --> 00:11:40,898 THIS BIG BLOCK HERE WHERE WE CAN 304 00:11:40,898 --> 00:11:42,966 FIT THE KINASE DOMAIN, AND HERE 305 00:11:42,966 --> 00:11:46,603 AT THE BOTTOM, WHERE WE CAN FIT 306 00:11:46,603 --> 00:11:47,404 14-3-3 PROTEINS. 307 00:11:47,404 --> 00:11:50,107 SO THIS IS WHAT IS SHOWN YOU 308 00:11:50,107 --> 00:11:52,910 HERE IN THESE REPRESENTATIONS. 309 00:11:52,910 --> 00:11:54,478 SO THE FIRST THING I WANT TO 310 00:11:54,478 --> 00:11:57,214 MENTION IS THAT IN THE DIMER 311 00:11:57,214 --> 00:12:00,384 FORM, THE N TERMINAL REGION, THE 312 00:12:00,384 --> 00:12:05,322 REGULATORY REGION THAT CONTAIN 313 00:12:05,322 --> 00:12:10,027 THE R DOMAIN AND CDD DOMAIN WERE 314 00:12:10,027 --> 00:12:12,429 NOT VISIBLE, SO THE PRESENCE OF 315 00:12:12,429 --> 00:12:14,798 THE MEMBRANE WAS REQUIRED TO 316 00:12:14,798 --> 00:12:15,933 STABILIZE THAT IN THE DIMER 317 00:12:15,933 --> 00:12:17,701 OTHERWISE IT SEEMS TO BE VERY 318 00:12:17,701 --> 00:12:18,902 FLEXIBLE AND WE WERE ABLE TO 319 00:12:18,902 --> 00:12:20,337 RECONSTRUCTED IT. 320 00:12:20,337 --> 00:12:20,604 LY. 321 00:12:20,604 --> 00:12:24,808 WE CAN SEE HERE THAT WE FORM IN 322 00:12:24,808 --> 00:12:26,677 THIS COMPLEX THE CHARACTERISTIC 323 00:12:26,677 --> 00:12:31,181 BACK TO BACK DIMER, I SHOWED 324 00:12:31,181 --> 00:12:37,955 BEFORE WAS DETERMINED BY X-RAY, 325 00:12:37,955 --> 00:12:39,923 AND WE CAN SHOW THE PRESENCE IN 326 00:12:39,923 --> 00:12:45,162 THE DIMER AND WE CAN ALSO SEE 327 00:12:45,162 --> 00:12:48,465 THAT THIS -- STRUCTURE IS 328 00:12:48,465 --> 00:12:51,201 PRESENT THIS AN ACTIVE 329 00:12:51,201 --> 00:12:51,535 CONFIRMATION. 330 00:12:51,535 --> 00:12:52,402 WE CAN MAINLY SEE THAT BECAUSE 331 00:12:52,402 --> 00:12:57,541 OF THE POSITION OF THESE 332 00:12:57,541 --> 00:13:00,277 HELICESR THESE HEL I CES CAN 333 00:13:00,277 --> 00:13:01,311 ADOPT VERY SPECIFIC FORMATION 334 00:13:01,311 --> 00:13:02,713 WHEN THE PROTEIN IS ACTIVE AND 335 00:13:02,713 --> 00:13:04,114 WHEN THE PROTEIN IS INACTIVE. 336 00:13:04,114 --> 00:13:05,649 IN THIS DIMER STRUCTURE, WE 337 00:13:05,649 --> 00:13:09,686 DEFINITELY SEE IT IN 338 00:13:09,686 --> 00:13:11,021 CONFIRMATION -- IN THE 339 00:13:11,021 --> 00:13:11,688 CONFORMATION INDICATING THAT THE 340 00:13:11,688 --> 00:13:13,290 PROTEIN IS ACTIVE. 341 00:13:13,290 --> 00:13:19,529 WE CAN SEE EACH -- TREN GAUGE 342 00:13:19,529 --> 00:13:23,834 EACH COPY OF THE 14-3-3-3 WHICH 343 00:13:23,834 --> 00:13:28,705 IS A DIMER, EACH INTERACTS WITH 344 00:13:28,705 --> 00:13:30,907 14-3-3 WITH THE PHOSPHORYLATION 345 00:13:30,907 --> 00:13:35,612 SIDE WHICH IS -- WHICH IS PS729. 346 00:13:35,612 --> 00:13:36,813 WE WANT TO SEE HOW WE CAN GET TO 347 00:13:36,813 --> 00:13:38,882 THIS DIMER CONFORMATION GOING 348 00:13:38,882 --> 00:13:41,518 FROM THE MONOMER CONFORMATION. 349 00:13:41,518 --> 00:13:43,920 ONE THING I FORGOT TO MENTION IS 350 00:13:43,920 --> 00:13:45,789 THAT IT INVOLVES ACTIVE SITES OF 351 00:13:45,789 --> 00:13:48,191 THE KINASE DOMAIN, WE SEE THE 352 00:13:48,191 --> 00:13:49,559 PRESENCE OF THE KINASE INHIBITOR 353 00:13:49,559 --> 00:13:52,796 THAT WE USE TO FORM THE COMPLEX, 354 00:13:52,796 --> 00:13:55,332 AND THIS IS AN ATP ANALOG 355 00:13:55,332 --> 00:13:57,501 BINDING TO THE ACTIVE SIDE OF 356 00:13:57,501 --> 00:13:59,269 THE PROTEIN, SO THIS INDICATES 357 00:13:59,269 --> 00:14:04,241 THAT PROTOMERS OF THE BRAF CAN 358 00:14:04,241 --> 00:14:05,842 PERFORM CAT LIS AT THE SAME TIME 359 00:14:05,842 --> 00:14:07,778 SO BOTH ACTIVE CONFORMATIONS OF 360 00:14:07,778 --> 00:14:08,678 THE PROTEIN. 361 00:14:08,678 --> 00:14:12,082 SO IF WE LOOK AT THE MONOMER 362 00:14:12,082 --> 00:14:12,949 STRUCTURES, HERE AGAIN I'M 363 00:14:12,949 --> 00:14:15,452 SHOWING YOU THE 2D CLASSES THAT 364 00:14:15,452 --> 00:14:18,655 WERE USED TO RECONSTRUCT THE 365 00:14:18,655 --> 00:14:20,457 THREE-DIMENSIONAL STRUCTURE OF 366 00:14:20,457 --> 00:14:23,026 THE PROTEIN R TWO COMPLEXES, ONE 367 00:14:23,026 --> 00:14:24,961 HAS THE PRESENCE OF MEK WHICH 368 00:14:24,961 --> 00:14:30,834 WAS RESOLVED AT 3.27 -- AT 3.7 369 00:14:30,834 --> 00:14:32,335 RESOLUTION AND OTHER ONE ABSENCE 370 00:14:32,335 --> 00:14:34,571 OF MEK, WHICH IS 4.1. 371 00:14:34,571 --> 00:14:36,807 YOU CAN SEE ABSENCE OF MEK IS A 372 00:14:36,807 --> 00:14:38,208 LITTLE LOWER SO IT MAY INDICATE 373 00:14:38,208 --> 00:14:40,077 THAT THE PRESENCE OF MEK HELPS 374 00:14:40,077 --> 00:14:41,611 TO STABILIZE THE CONFORMATION OF 375 00:14:41,611 --> 00:14:43,914 THE COMPLEX. 376 00:14:43,914 --> 00:14:48,752 SO IF WE SEE THE ATOMIC 377 00:14:48,752 --> 00:14:51,154 STRUCTURE, WE CAN SEE THAT WE 378 00:14:51,154 --> 00:14:53,356 HAVE AT THE CENTER OF THE -- 379 00:14:53,356 --> 00:14:55,926 FIRST OF ALL, BOTH STRUCTURES 380 00:14:55,926 --> 00:14:58,695 ARE ALMOST THE SAME IN THE 381 00:14:58,695 --> 00:15:01,331 PRESENCE OR ABSENCE OF MEK. 382 00:15:01,331 --> 00:15:04,267 THE KINASE DOMAIN AND RBD AND 383 00:15:04,267 --> 00:15:06,903 CRD ARE IN THE SAME CONFORMATION 384 00:15:06,903 --> 00:15:08,438 ORIENTATIONS, IT SEEMS THAT THE 385 00:15:08,438 --> 00:15:10,240 MEK HELPS TO STABILIZE THE 386 00:15:10,240 --> 00:15:10,607 COMPLEX. 387 00:15:10,607 --> 00:15:12,609 OTHERWISE, THEY ARE LIKE 388 00:15:12,609 --> 00:15:12,876 IDENTICAL. 389 00:15:12,876 --> 00:15:16,079 WE CAN SEE THAT THE CRD DOMAIN 390 00:15:16,079 --> 00:15:17,948 IS AT THE CENTER OF THE 391 00:15:17,948 --> 00:15:20,484 INHIBITORY STATE, SHOWN HERE IN 392 00:15:20,484 --> 00:15:21,585 VIOLET, AND WHAT YOU CAN SEE 393 00:15:21,585 --> 00:15:23,653 HERE IS THAT IT MAKES 394 00:15:23,653 --> 00:15:26,289 INTERACTIONS WITH THE 14-3-3 395 00:15:26,289 --> 00:15:27,924 DIMER AND WITH THE KINASE 396 00:15:27,924 --> 00:15:28,258 DOMAIN. 397 00:15:28,258 --> 00:15:30,660 SO IT SEEMS THAT IT REALLY HELPS 398 00:15:30,660 --> 00:15:32,496 TO MAINTAIN, AT THE CENTER OF 399 00:15:32,496 --> 00:15:38,969 THE OUTER STATE OF THE PROTEIN. 400 00:15:38,969 --> 00:15:40,837 WHEN MEK IS PRESENT WE CAN SEE 401 00:15:40,837 --> 00:15:42,706 THAT IT INTERACTS WON'T KINASE 402 00:15:42,706 --> 00:15:48,411 DOMAIN OF THE BRAF, SO IT'S LIKE 403 00:15:48,411 --> 00:15:49,713 KINASE-KINASE INTERACTION BUT 404 00:15:49,713 --> 00:15:51,681 MEK DOESN'T INTERACT WITH THE 405 00:15:51,681 --> 00:15:54,084 REGULATORY PART OF THE BRAF 406 00:15:54,084 --> 00:15:54,384 PROTEIN. 407 00:15:54,384 --> 00:15:56,286 IF WE LOOK AT THE KINASE DOMAIN 408 00:15:56,286 --> 00:15:57,587 OF THE PROTEIN, WHICH IS SHOWN 409 00:15:57,587 --> 00:16:00,423 HERE IN BLUE F WE JUST FOCUS TO 410 00:16:00,423 --> 00:16:02,159 THAT PART, THE FIRST THING THAT 411 00:16:02,159 --> 00:16:04,394 WE CAN OBSERVE IS THAT IN OUR 412 00:16:04,394 --> 00:16:06,396 STRUCTURES, THE ATP BINDING 413 00:16:06,396 --> 00:16:08,465 POCKET IS EMPTY, SO THERE IS NO 414 00:16:08,465 --> 00:16:11,434 ATP BOUND IN OUR STRUCTURES, AND 415 00:16:11,434 --> 00:16:13,737 WHERE WE CAN SEE THAT THIS 416 00:16:13,737 --> 00:16:16,439 KINASE DOMAIN ADOPTS THE 417 00:16:16,439 --> 00:16:17,240 INACTIVE CONFORMATION. 418 00:16:17,240 --> 00:16:18,642 SO AGAIN, WE CAN SEE THAT 419 00:16:18,642 --> 00:16:21,811 BECAUSE THE POSITION OF THESE 420 00:16:21,811 --> 00:16:24,748 ALPHA C HELIX SHOWING IN RED IS 421 00:16:24,748 --> 00:16:26,416 IN WHAT IS CALLED THE OUT 422 00:16:26,416 --> 00:16:28,084 POSITION, SO IT IS ACTIVE 423 00:16:28,084 --> 00:16:29,019 CONFORMATION AND MAINLY IT IS 424 00:16:29,019 --> 00:16:31,988 BECAUSE WHEN THESE ALPHA HELIX 425 00:16:31,988 --> 00:16:34,724 IS IN THE OUT CONFORMATION WHAT 426 00:16:34,724 --> 00:16:37,827 IS CALLED REGULATORY SPINE IS 427 00:16:37,827 --> 00:16:39,796 BROKEN BECAUSE THIS IS 428 00:16:39,796 --> 00:16:40,330 MISALIGNED. 429 00:16:40,330 --> 00:16:42,098 THE OTHER THING AWE OBSERVED IS 430 00:16:42,098 --> 00:16:43,767 THAT EVEN THOUGH OUR STRUCTURE 431 00:16:43,767 --> 00:16:47,470 WAS DONE IN THE ABSENCE -- OR 432 00:16:47,470 --> 00:16:50,307 WAS IN THE ABSENCE OF MEK, 433 00:16:50,307 --> 00:16:51,841 ALMOST THE SAME CONFORMATION 434 00:16:51,841 --> 00:16:54,578 THAT WHEN ATP ANALOGS ARE BOUND 435 00:16:54,578 --> 00:16:55,779 TO THE PROTEIN. 436 00:16:55,779 --> 00:16:57,647 AND THIS WAS IMPORTANT BECAUSE 437 00:16:57,647 --> 00:17:00,517 IN THIS STRUCTURE, WE CAN SEE 438 00:17:00,517 --> 00:17:05,055 THAT WHAT WE CALL THE N LOBE AND 439 00:17:05,055 --> 00:17:07,557 C LOBE END UP LIKE A MORE 440 00:17:07,557 --> 00:17:10,827 COMPACT CONFORMATION AND IN THIS 441 00:17:10,827 --> 00:17:11,595 CONFORMATION, IT'S IMPORTANT TO 442 00:17:11,595 --> 00:17:16,099 FORM A DIMER ARE OCCLUDED, SO 443 00:17:16,099 --> 00:17:18,168 THERE CAN BE NOT FORMATION OF A 444 00:17:18,168 --> 00:17:19,069 DIMER FORM OF THE PROTEIN. 445 00:17:19,069 --> 00:17:21,771 SO THIS INDICATES THAT OUR 446 00:17:21,771 --> 00:17:23,673 STRUCTURES TRULY REPRESENT AN 447 00:17:23,673 --> 00:17:24,674 INHIBITORY STATE OF THE PROTEIN, 448 00:17:24,674 --> 00:17:28,278 AND IT IS AN OUTER STATE BECAUSE 449 00:17:28,278 --> 00:17:29,879 THEY ARE ALL N TERMINAL 450 00:17:29,879 --> 00:17:31,147 INTERACTIONS THAT ARE HELPING TO 451 00:17:31,147 --> 00:17:34,651 MAINTAIN THIS CONFORMATION. 452 00:17:34,651 --> 00:17:36,653 SO VERY IMPORTANT CHARACTERISTIC 453 00:17:36,653 --> 00:17:39,022 OF OUR STRUCTURE IS THAT WE WERE 454 00:17:39,022 --> 00:17:42,492 ABLE TO SOLVE THE POSITION OF 455 00:17:42,492 --> 00:17:44,928 THE LAST BINDING DOMAIN. 456 00:17:44,928 --> 00:17:46,263 SO LAST BINDING DOMAIN HERE 457 00:17:46,263 --> 00:17:47,664 SHOWN IN ORANGE, AND AS YOU CAN 458 00:17:47,664 --> 00:17:50,934 SEE HERE, THE RAS BINDING DOMAIN 459 00:17:50,934 --> 00:17:52,569 SITS ADJACENT TO THE KINASE 460 00:17:52,569 --> 00:17:59,075 DOMAIN, AND ON TOP OF ONE OF THE 461 00:17:59,075 --> 00:18:00,243 14-3-3PROTOMERS, AND THEY FORM 462 00:18:00,243 --> 00:18:02,145 KIND OF AN EXTENSIVE INTERACTION 463 00:18:02,145 --> 00:18:10,053 WITH AN AREA OF AROUND 464 00:18:10,053 --> 00:18:11,554 400 AMSTRONS AND COMPLEMENT TEAR 465 00:18:11,554 --> 00:18:12,889 RI BETWEEN THE BINDING DOMAIN 466 00:18:12,889 --> 00:18:14,524 AND THE 14-3-3. 467 00:18:14,524 --> 00:18:16,359 IF WE LOOK CLOSER TO THIS 468 00:18:16,359 --> 00:18:19,329 INTERFACE, WE CAN SEE THAT THE 469 00:18:19,329 --> 00:18:21,064 ALPHA-1 IN THE RAS BINDING 470 00:18:21,064 --> 00:18:22,632 DOMAIN THAT IS ESTABLISHING SOME 471 00:18:22,632 --> 00:18:25,001 CONTACTS WITH THE ALPHA 8, ALPHA 472 00:18:25,001 --> 00:18:28,104 9 AND LOOP 8 IN 14-3-3. 473 00:18:28,104 --> 00:18:29,739 SPECIFICALLY, WHEN WE LOOK AT 474 00:18:29,739 --> 00:18:32,676 THE RESIDUES THAT ARE LOCATED, 475 00:18:32,676 --> 00:18:35,011 THE 14-3-3 SITE OF THE 476 00:18:35,011 --> 00:18:36,413 INTERFACE, WE CAN SEE THAT ALL 477 00:18:36,413 --> 00:18:39,249 THE RESIDUES THAT ARE AT THIS 478 00:18:39,249 --> 00:18:41,651 INTERFACE ARE CONSERVED BETWEEN 479 00:18:41,651 --> 00:18:44,054 THE DIFFERENT ISOFORMS OF THE 480 00:18:44,054 --> 00:18:44,621 14-3-3 FAMILY. 481 00:18:44,621 --> 00:18:47,557 AS YOU MAY KNOW, 14-3-3 HAS 482 00:18:47,557 --> 00:18:49,526 SEVEN DIFFERENT ISOFORMS BUT ALL 483 00:18:49,526 --> 00:18:52,262 THE RESIDUES IN THIS INTERFACE 484 00:18:52,262 --> 00:18:53,930 ARE CONSERVED BETWEEN THESE 485 00:18:53,930 --> 00:18:56,199 DIFFERENT 7 ISOFORMS, SO THIS 486 00:18:56,199 --> 00:18:57,834 INDICATES THAT REGARDLESS OF THE 487 00:18:57,834 --> 00:19:02,772 COMPOSITION OF THE 14-3-3 CAN, 488 00:19:02,772 --> 00:19:04,107 THIS INTERFACE CAN BE 489 00:19:04,107 --> 00:19:04,607 ESTABLISHED. 490 00:19:04,607 --> 00:19:06,409 THAT'S IMPORTANT BECAUSE THE 491 00:19:06,409 --> 00:19:07,777 14-3-3 TENDS ON DIMER EYES, SO 492 00:19:07,777 --> 00:19:09,979 YOU CAN HAVE THE -- DIMERIZE, SO 493 00:19:09,979 --> 00:19:13,016 YOU CAN HAVE THE COMPLEX 494 00:19:13,016 --> 00:19:14,651 DIFFERENT 14-3-3. 495 00:19:14,651 --> 00:19:16,920 HOWEVER, WHEN WE LOOK AT THE RAS 496 00:19:16,920 --> 00:19:18,621 BINDING DOMAIN RESIDUES WE SEE 497 00:19:18,621 --> 00:19:20,390 THEY ARE NOT FULLY CONSERVED IN 498 00:19:20,390 --> 00:19:22,826 THE FAMILY OF THE RAF PROTEINS, 499 00:19:22,826 --> 00:19:25,695 SPECIFICALLY THERE ARE THESE TWO 500 00:19:25,695 --> 00:19:29,199 MECHANISMS, 187 AND 186 IN THE 501 00:19:29,199 --> 00:19:30,667 BRAF THAT THEY ARE DIFFERENT 502 00:19:30,667 --> 00:19:40,977 FROM CRAF OR ARAF. 503 00:19:43,513 --> 00:19:48,685 SO THESE TWO RESIDUESES, 186 AND 504 00:19:48,685 --> 00:19:50,553 187 WE CAN SEE THEY CAN HAVE 505 00:19:50,553 --> 00:19:51,855 MULTIPLE POTENTIAL INTERACTIONS 506 00:19:51,855 --> 00:19:53,123 WITH 14-3-3. 507 00:19:53,123 --> 00:19:55,358 SO EVEN THOUGH WE KNOW THAT THE 508 00:19:55,358 --> 00:19:57,427 CRT HERE IN VIOLET IS KIND OF 509 00:19:57,427 --> 00:20:00,497 LIKE THE KEY COMPONENT OF THE 510 00:20:00,497 --> 00:20:02,365 OUTER HELIX BECAUSE IT MAKES 511 00:20:02,365 --> 00:20:03,800 INTERACTIONS WITH THE KINASE 512 00:20:03,800 --> 00:20:05,969 DOMAIN AND THE 14-3-3, WE WANTED 513 00:20:05,969 --> 00:20:09,239 TO INVESTIGATE IF THE RAS 514 00:20:09,239 --> 00:20:13,443 BINDING DOMAIN OF RBD ORANGE 515 00:20:13,443 --> 00:20:15,512 ALSO DROIBTD THE MAINTAINING OF 516 00:20:15,512 --> 00:20:17,280 THIS OUTER HELIX STATE BY 517 00:20:17,280 --> 00:20:19,115 INTERACT WITH 14-3-3. 518 00:20:19,115 --> 00:20:20,283 SPECIFICALLY WE WANTED TO SEE IF 519 00:20:20,283 --> 00:20:25,588 THESE TWO RESIDUES 186 AND 187 520 00:20:25,588 --> 00:20:27,357 WERE IMPORTANT FOR MAINTAINING 521 00:20:27,357 --> 00:20:28,358 THIS OUTER STATE. 522 00:20:28,358 --> 00:20:32,729 SO IN ORDER TO DO THAT, WE TOOK 523 00:20:32,729 --> 00:20:34,364 MUTATIONAL APPROACH IN WHICH WE 524 00:20:34,364 --> 00:20:38,735 MUTATE THESE TWO RESIDUES TO 525 00:20:38,735 --> 00:20:41,604 FIRST THE RESIDUES AND TO OTHER 526 00:20:41,604 --> 00:20:44,541 MEMBERS. 527 00:20:44,541 --> 00:20:45,008 FAMILY. 528 00:20:45,008 --> 00:20:45,975 -- MEMBERS OF THE FAMILY, 529 00:20:45,975 --> 00:20:47,844 WHETHER IT'S PRESENT THIS ARAF 530 00:20:47,844 --> 00:20:51,581 OR CRAF AND ALSO TO A SMALLER 531 00:20:51,581 --> 00:20:54,184 AMINO ACID, LARGER ACIDS OR 532 00:20:54,184 --> 00:20:55,618 TENDED TO CHARGE RESIDUES, AND 533 00:20:55,618 --> 00:20:59,122 WE WANTED TO SEE IF BY DOING 534 00:20:59,122 --> 00:21:01,191 THESE MUTATIONS BY MODULATING 535 00:21:01,191 --> 00:21:02,725 THIS INTERFACE WE COULD SEE A 536 00:21:02,725 --> 00:21:04,594 DIFFERENCE IN OUTER INHIBITION. 537 00:21:04,594 --> 00:21:07,330 SO WITHOUT GOING INTO TOO MUCH 538 00:21:07,330 --> 00:21:08,965 DETAILS ON THE ASSAY, WHAT WE DO 539 00:21:08,965 --> 00:21:11,935 IS THAT WE USE A ACID THAT 540 00:21:11,935 --> 00:21:13,236 ALLOWS US TO SEE WHAT WAS THE 541 00:21:13,236 --> 00:21:15,305 DEGREE OF OUTER INHIBITION IN 542 00:21:15,305 --> 00:21:16,206 THE PROTEIN. 543 00:21:16,206 --> 00:21:22,779 WE ALSO USED A FOCUS FORM, HOW 544 00:21:22,779 --> 00:21:24,247 ACTIVE THE CELLS ARE BASED ON 545 00:21:24,247 --> 00:21:25,315 THE GROWTH OF THE CELLS. 546 00:21:25,315 --> 00:21:27,150 BASICALLY WHAT WE CAN SEE HERE 547 00:21:27,150 --> 00:21:29,786 IS THAT MOTD LAITD RESIDUES AT 548 00:21:29,786 --> 00:21:31,321 THIS INTERFACE TRANSLATED IN 549 00:21:31,321 --> 00:21:32,822 MODULATION IN OUTER INHIBITION 550 00:21:32,822 --> 00:21:34,691 AND ACTIVITY OF THE PROTEIN, AND 551 00:21:34,691 --> 00:21:37,227 THAT ALSO CORRELATED WITH THE 552 00:21:37,227 --> 00:21:42,332 LEVELS OF APPROXIMATE PHOSFORMIC 553 00:21:42,332 --> 00:21:43,766 IN THE CELLS, REMEMBER THAT MEK 554 00:21:43,766 --> 00:21:45,735 IS THE SUBSTRATE FOR RAF. 555 00:21:45,735 --> 00:21:47,270 SO ALL OF THIS INDICATED THAT 556 00:21:47,270 --> 00:21:49,706 EVEN THE CRT'S, THE KEY 557 00:21:49,706 --> 00:21:51,674 COMPONENT TO MAINTAIN THE OUTER 558 00:21:51,674 --> 00:21:53,743 INHIBITORY STATE WE CAN SAY THAT 559 00:21:53,743 --> 00:21:55,945 THE RAS BINDING DOMAIN ALSO 560 00:21:55,945 --> 00:21:57,780 DROIBTD THE MAINTENANCE OF THE 561 00:21:57,780 --> 00:21:59,682 OUTER INHIBITORY STATE OF THE 562 00:21:59,682 --> 00:22:00,149 PROTEIN. 563 00:22:00,149 --> 00:22:07,724 BUT OBVIOUSLY THE -- BUT OBVIOUN 564 00:22:07,724 --> 00:22:09,425 OF THE BINDING DOMAIN IS TO BIND 565 00:22:09,425 --> 00:22:11,060 TO RAS SO WE WANTED TO SEE WHAT 566 00:22:11,060 --> 00:22:14,030 HAPPENS IF WE ADD RAS TO IT 567 00:22:14,030 --> 00:22:15,698 COMPLEX AND IF THAT CAN GIVE US 568 00:22:15,698 --> 00:22:16,900 SOME INDICATION OF HOW THIS WILL 569 00:22:16,900 --> 00:22:18,868 HAVE TO TRANSITION FROM THE 570 00:22:18,868 --> 00:22:20,303 MONOMER TO THE DIMER STATE. 571 00:22:20,303 --> 00:22:23,239 SO AS YOU CAN SEE HERE, WE HAVE 572 00:22:23,239 --> 00:22:25,208 THE STRUCTURE THAT WE SAW FOR 573 00:22:25,208 --> 00:22:27,176 THE OUTER INHIBITORY STATE AND 574 00:22:27,176 --> 00:22:30,647 WE KIND OF MODEL THE BINDING OF 575 00:22:30,647 --> 00:22:32,782 THE RAS PROTEIN INTO THIS 576 00:22:32,782 --> 00:22:34,183 MONOMER STRUCTURE. 577 00:22:34,183 --> 00:22:38,488 SO WE DID THIS MODEL, BASICALLY 578 00:22:38,488 --> 00:22:40,123 USING FOUR RESIDUE THAT IS ARE 579 00:22:40,123 --> 00:22:41,858 PRESENT IN THE RAS BINDING 580 00:22:41,858 --> 00:22:44,294 DOMAIN THAT THEY FORM IONIC 581 00:22:44,294 --> 00:22:45,595 INTERACTIONS WITH RAS. 582 00:22:45,595 --> 00:22:48,231 SO THESE ARE VERY WELL KNOWN 583 00:22:48,231 --> 00:22:49,732 ESTABLISHED AND STUDIED 584 00:22:49,732 --> 00:22:50,833 INTERACTIONS BETWEEN THE TWO 585 00:22:50,833 --> 00:22:51,167 PROTEINS. 586 00:22:51,167 --> 00:22:53,703 SO WE USE THIS MODULATOR BINDING 587 00:22:53,703 --> 00:22:57,407 OF RAS TO RAF, AND WE ALSO 588 00:22:57,407 --> 00:23:01,144 WANTED TO SEE IF OUR ISOLATED 589 00:23:01,144 --> 00:23:02,879 OUTER COMPLEX WERE ABLE TO BIND 590 00:23:02,879 --> 00:23:04,213 TO RAS. 591 00:23:04,213 --> 00:23:07,050 SO WE DID ASSAYS AND WE CAN 592 00:23:07,050 --> 00:23:09,352 DETERMINE THAT OUR OUTER 593 00:23:09,352 --> 00:23:10,486 INHIBITORY COMPLEXES INTERACTS 594 00:23:10,486 --> 00:23:12,989 WITH HIGH AFFINITY WITH PURIFIED 595 00:23:12,989 --> 00:23:13,957 RAS PROTEIN. 596 00:23:13,957 --> 00:23:18,261 WE ALSO DID SOME EXPERIMENTS 597 00:23:18,261 --> 00:23:20,363 WHERE OUR ALPHA HELIX COMPLEX 598 00:23:20,363 --> 00:23:27,904 WERE ABLE TO PULL DOWN R AS -- 599 00:23:27,904 --> 00:23:29,439 K-RAS AND WE CAN ABOLISH 600 00:23:29,439 --> 00:23:31,708 INTERACTION BETWEEN RAF AND RAS. 601 00:23:31,708 --> 00:23:34,143 SO I'M GOING BACK TO THE 602 00:23:34,143 --> 00:23:35,678 MODELING OF THE BINDING, WHEN WE 603 00:23:35,678 --> 00:23:38,615 MODELED THE K-RAS BINDING TO THE 604 00:23:38,615 --> 00:23:40,149 RAS BINDING DOMAIN, WHAT WE CAN 605 00:23:40,149 --> 00:23:41,884 SEE HERE IS THAT WHEN THE 606 00:23:41,884 --> 00:23:44,854 INTERACTION BETWEEN KRAS AND RPD 607 00:23:44,854 --> 00:23:46,255 IS ESTABLISHED, THERE IS GOING 608 00:23:46,255 --> 00:23:47,924 TO BE -- RBD IS ESTABLISHED, 609 00:23:47,924 --> 00:23:49,459 THERE WILL BE A CLASHING BETWEEN 610 00:23:49,459 --> 00:23:52,128 THE KRAS AND 14-3-3 THAT IS 611 00:23:52,128 --> 00:23:53,930 BINDING TO THE RAS BINDING 612 00:23:53,930 --> 00:23:56,132 DOMAIN AND THERE WILL ALSO BE AN 613 00:23:56,132 --> 00:23:57,000 ELECTROSTATIC REPULSION BETWEEN 614 00:23:57,000 --> 00:24:00,103 THE KRAS AND THE 14-3-3 BECAUSE 615 00:24:00,103 --> 00:24:02,138 THEY ARE BOTH NEGATIVELY 616 00:24:02,138 --> 00:24:02,472 CHARGED. 617 00:24:02,472 --> 00:24:04,874 SO BASICALLY, WHAT THIS TELLS US 618 00:24:04,874 --> 00:24:07,510 IS THAT WHEN RAS BINDS TO THE 619 00:24:07,510 --> 00:24:09,812 OUTER HELIX, THERE WILL BE RAF, 620 00:24:09,812 --> 00:24:11,347 THERE NEEDS TO BE ORGANIZATION 621 00:24:11,347 --> 00:24:13,383 OF THE 14-3-3 BINDING IN ORDER 622 00:24:13,383 --> 00:24:18,788 FOR RAS COMPLETELY BIND TO BRAF. 623 00:24:18,788 --> 00:24:20,757 THERE WAS ANOTHER INTERESTING 624 00:24:20,757 --> 00:24:23,059 OBSERVATION, WHEN WE DID THIS 625 00:24:23,059 --> 00:24:25,261 MODELING OF THIS BINDING, IS 626 00:24:25,261 --> 00:24:28,097 THAT WHEN RAS BIND TO THE RBD, 627 00:24:28,097 --> 00:24:29,766 THESE TWO MECHANISMS THAT WE 628 00:24:29,766 --> 00:24:31,000 OBSERVED BEFORE WERE IMPORTANT 629 00:24:31,000 --> 00:24:32,902 FOR THE BINDING BETWEEN THE RAS 630 00:24:32,902 --> 00:24:35,138 BINDING DOMAIN AND THE 14-3-3, 631 00:24:35,138 --> 00:24:38,307 THEY COME IN CLOSE PROXIMITY TO 632 00:24:38,307 --> 00:24:40,043 RAS SO WE WANTED TO INVESTIGATE 633 00:24:40,043 --> 00:24:45,081 IF THESE TWO RESIDUES WERE ALSO 634 00:24:45,081 --> 00:24:46,949 INVOLVED TO THE RAS BINDING ONCE 635 00:24:46,949 --> 00:24:49,585 THE 14-3-3 IS MOVED OUT OF THE 636 00:24:49,585 --> 00:24:49,752 WAY. 637 00:24:49,752 --> 00:24:54,524 IN ORDER TO DO THAT, WE PERFORM 638 00:24:54,524 --> 00:24:56,025 ANOTHER TYPE OF ASSAY THAT 639 00:24:56,025 --> 00:24:59,529 ALLOWS US TO STUDY INTERACTION 640 00:24:59,529 --> 00:25:01,831 BETWEEN RAS PROTEIN AND BRAF 641 00:25:01,831 --> 00:25:03,466 PROTEIN AND WITH A SERIES OF 642 00:25:03,466 --> 00:25:05,234 MUTATIONS OF THESE TWO RESIDUE 643 00:25:05,234 --> 00:25:07,336 AND IS WE SAW IF THERE WAS AN 644 00:25:07,336 --> 00:25:08,971 EFFECT BETWEEN THE BINDING OF 645 00:25:08,971 --> 00:25:11,474 RAS AND RAF AND IN FACT WE SEE 646 00:25:11,474 --> 00:25:13,109 THAT AGAIN WHEN WE MODULATE 647 00:25:13,109 --> 00:25:15,078 THESE TWO MECHANISMS, WE SEE A 648 00:25:15,078 --> 00:25:16,312 DIFFERENT DEGREE OF BINDING 649 00:25:16,312 --> 00:25:18,681 BETWEEN RAS AND RAF INDICATING 650 00:25:18,681 --> 00:25:21,117 THAT THESE TWO RESIDUES IN FACT 651 00:25:21,117 --> 00:25:21,784 PARTICIPATE IN THE BINDING 652 00:25:21,784 --> 00:25:27,824 BETWEEN THE TWO PROTEINS. 653 00:25:27,824 --> 00:25:29,992 ALSO WITH ASSAYS PULL DOWN 654 00:25:29,992 --> 00:25:31,094 EXPERIMENTS WHERE WE OBSERVE THE 655 00:25:31,094 --> 00:25:32,829 SAME THING, THAT THESE TWO 656 00:25:32,829 --> 00:25:33,896 RESIDUES ARE IMPORTANT FOR THE 657 00:25:33,896 --> 00:25:35,131 BINDING OF THE TWO PROTEINS. 658 00:25:35,131 --> 00:25:36,466 SO BASICALLY, WHAT THIS IS 659 00:25:36,466 --> 00:25:39,168 TELLING US IS THAT THESE TWO 660 00:25:39,168 --> 00:25:42,672 MECHANISMS IN THE ALPHA-1 HELIX 661 00:25:42,672 --> 00:25:44,741 IN THE RAS BINDING DOMAIN HAVE 662 00:25:44,741 --> 00:25:47,410 KIND OF A DUAL FUNCTION. 663 00:25:47,410 --> 00:25:49,378 IN THE OUTER INHIBITORY STATE, 664 00:25:49,378 --> 00:25:51,881 THEY BIND WITH 14-3-3 AND HELP 665 00:25:51,881 --> 00:25:54,050 TO MAINTAIN THE OUTER INHIBITORY 666 00:25:54,050 --> 00:25:57,253 STATE, BUT WHEN RAS COME AND 667 00:25:57,253 --> 00:26:03,659 BINDS TO THE RAS PROTEIN AND THE 668 00:26:03,659 --> 00:26:05,895 14-3-3 MOVES MOVES OUT OF THE 669 00:26:05,895 --> 00:26:07,830 WAY, THERE'S ALSO INTERACTION 670 00:26:07,830 --> 00:26:09,866 WITH THE RAS PROTEIN, MAKING 671 00:26:09,866 --> 00:26:11,267 INTERACTION BETWEEN THE TWO 672 00:26:11,267 --> 00:26:11,534 PROTEINS. 673 00:26:11,534 --> 00:26:13,536 SO IF WE PULL ALL OF THIS 674 00:26:13,536 --> 00:26:15,505 TOGETHER, WE KIND OF PROPOSE A 675 00:26:15,505 --> 00:26:17,373 MODEL ON HOW THE ACTIVATION OF 676 00:26:17,373 --> 00:26:20,109 THE BRAF PROTEINS CAN HAPPEN 677 00:26:20,109 --> 00:26:23,179 AFTER THE RAS PROTEIN 678 00:26:23,179 --> 00:26:23,479 SOUTHBOUND. 679 00:26:23,479 --> 00:26:26,649 SO WE START WITH THE CELLS WHEN 680 00:26:26,649 --> 00:26:30,219 THE TWO PROTEINS ARE IN INACT 681 00:26:30,219 --> 00:26:34,857 INACTIVE, KRAS BOUND TO GDP, WE 682 00:26:34,857 --> 00:26:38,060 HAVE BRAF FLEEN OUTER INHIBITORY 683 00:26:38,060 --> 00:26:39,162 STATE MAINLY BETWEEN 684 00:26:39,162 --> 00:26:40,797 INTERACTIONS BETWEEN N TERMINAL 685 00:26:40,797 --> 00:26:42,431 REGULATORY AND C TERMINAL 686 00:26:42,431 --> 00:26:44,100 CATALYTIC DOMAIN AND WITH 687 00:26:44,100 --> 00:26:46,169 INTERACTIONS WITH 14-3-3. 688 00:26:46,169 --> 00:26:49,005 ONCE RAS IS ACTIVATED THE 689 00:26:49,005 --> 00:26:51,874 BINDING OF GDP IT IS GOING 690 00:26:51,874 --> 00:26:54,143 RECRUIT AND BRING BRAF TO THE 691 00:26:54,143 --> 00:26:55,511 PLASMA MEMBRANE, SPECIFICALLY 692 00:26:55,511 --> 00:26:57,213 FOR THE INITIAL IONIC 693 00:26:57,213 --> 00:27:00,516 INTERACTIONS BETWEEN KRAS AND 694 00:27:00,516 --> 00:27:00,783 RBD. 695 00:27:00,783 --> 00:27:01,984 WHEN THIS INTERACTION HAPPENS, 696 00:27:01,984 --> 00:27:04,887 THERE IS GOING TO BE A CLASH AND 697 00:27:04,887 --> 00:27:06,856 ELECTROSTATIC REPULSION BETWEEN 698 00:27:06,856 --> 00:27:09,392 THE RAS AND THE 14-3-3. 699 00:27:09,392 --> 00:27:12,795 SO THIS 14-3-3 HAS TO BE 700 00:27:12,795 --> 00:27:13,196 REORGANIZED. 701 00:27:13,196 --> 00:27:15,498 WHEN THAT HAPPENS, IT ALLOWS FOR 702 00:27:15,498 --> 00:27:19,001 THE KINASE DOMAIN TO REORGANIZE 703 00:27:19,001 --> 00:27:22,205 AND FORM THE DIMER, THE RESIDUE 704 00:27:22,205 --> 00:27:24,273 THAT FORM THE DIMER INTERFACE 705 00:27:24,273 --> 00:27:26,342 BECOME EXPOSED AND THEY CAN'T 706 00:27:26,342 --> 00:27:27,109 OBVIOUSLY REORGANIZE AND FORM 707 00:27:27,109 --> 00:27:27,877 THE DIMER. 708 00:27:27,877 --> 00:27:33,816 AND THEN THE PROTEIN BECOMES 709 00:27:33,816 --> 00:27:35,651 ACTIVE. 710 00:27:35,651 --> 00:27:37,954 SO THAT'S THE MODEL WE PROPOSED. 711 00:27:37,954 --> 00:27:39,188 NOW MOVING FORWARD WHERE WE'RE 712 00:27:39,188 --> 00:27:42,792 TRYING TO UNDERSTAND REGULATORY 713 00:27:42,792 --> 00:27:47,630 OUTER INHIB -- INFIN HI BASE IS 714 00:27:47,630 --> 00:27:49,599 SHARED BY OTHER MEMBERS OF THE 715 00:27:49,599 --> 00:27:51,701 FAMILY, ESPECIALLY IF IT HAS 716 00:27:51,701 --> 00:27:54,303 SAME OUTER FORMATION OF BRAF AND 717 00:27:54,303 --> 00:27:55,938 PARTICULARLY IF THE DIMER 718 00:27:55,938 --> 00:27:56,973 FORMATION HAPPENS IN THE SAME 719 00:27:56,973 --> 00:27:57,206 WAY. 720 00:27:57,206 --> 00:27:58,441 THAT'S WHAT WE ARE TRYING 721 00:27:58,441 --> 00:28:00,877 INVESTIGATE NOW. 722 00:28:00,877 --> 00:28:02,545 SO WE BASICALLY COME TO THE END 723 00:28:02,545 --> 00:28:03,412 OF MY PRESENTATION. 724 00:28:03,412 --> 00:28:04,814 I WANT TO THANK ALL THE MEMBERS 725 00:28:04,814 --> 00:28:08,284 OF MY GROUP, PRINCIPALLY MY PI 726 00:28:08,284 --> 00:28:13,389 WHICH IS DR. PING ZING, 727 00:28:13,389 --> 00:28:16,559 DR. MORRISON AND DAVID IN THE 728 00:28:16,559 --> 00:28:19,829 LABORATORY OF CELL AND 729 00:28:19,829 --> 00:28:23,132 DEVELOPMENTAL BIOLOGY AND ALL 730 00:28:23,132 --> 00:28:29,138 THE IF RESOURCES, AND ALSO OUR 731 00:28:29,138 --> 00:28:31,474 CRYO-EM CENTER AND BIOWULF FOR 732 00:28:31,474 --> 00:28:32,842 ALL THE COMPUTATIONAL RESOURCES 733 00:28:32,842 --> 00:28:36,445 THAT ALLOW US TO STUDY THE 734 00:28:36,445 --> 00:28:38,281 STRUCTURE OF THE PROTEINS, AND 735 00:28:38,281 --> 00:28:40,216 THE CENTERS FOR BIOSTRUCTURAL 736 00:28:40,216 --> 00:28:42,485 PHYSICS RESOURCES FOR ALL THE 737 00:28:42,485 --> 00:28:43,286 RESOURCES FOR THE 738 00:28:43,286 --> 00:28:44,253 CHARACTERIZATION OF THE COMPLEX, 739 00:28:44,253 --> 00:28:45,354 AND ALL OF YOU FOR YOUR 740 00:28:45,354 --> 00:28:48,424 ATTENTION. 741 00:28:48,424 --> 00:28:58,601 [APPLAUSE] 742 00:29:10,446 --> 00:29:20,823 >> (AWAY FROM MICROPHONE). 743 00:29:30,466 --> 00:29:32,435 >> SO ALL THE GROWTH FACTOR 744 00:29:32,435 --> 00:29:34,737 SIGNALS THAT INITIATE THE 745 00:29:34,737 --> 00:29:35,938 CASCADE OF THE SIGNALLING 746 00:29:35,938 --> 00:29:40,843 PATHWAYS FROM THE TRANSITION OF 747 00:29:40,843 --> 00:29:44,914 KRAS FROM GTB TO GDP BINDING 748 00:29:44,914 --> 00:29:47,416 STATE SO ONCE THE PROTEIN IS IN 749 00:29:47,416 --> 00:29:52,154 THE GDP STATE IT CAN TBIEND THE 750 00:29:52,154 --> 00:29:54,023 RAF PROTEINS, OTHERWISE THEY 751 00:29:54,023 --> 00:29:55,758 WON'T BIND. 752 00:29:55,758 --> 00:29:57,426 OKAY? 753 00:29:57,426 --> 00:30:07,870 >> (AWAY FROM MICROPHONE). 754 00:30:11,440 --> 00:30:13,376 >> YEAH, ALL THE DOMAINS WE CAN 755 00:30:13,376 --> 00:30:15,945 HAVE A STARTING POINT BASICALLY 756 00:30:15,945 --> 00:30:17,580 FROM IT CHRYSALIS STRUCTURES, SO 757 00:30:17,580 --> 00:30:21,751 LIKE THE RAS DOMAIN, KINASE 758 00:30:21,751 --> 00:30:23,919 DOMAIN, 14-3-3 PROTEINS THEY 759 00:30:23,919 --> 00:30:26,555 WERE ALL DOMAINS THAT HAVE 760 00:30:26,555 --> 00:30:28,524 ALREADY BEEN DEPOSITED IN THE 761 00:30:28,524 --> 00:30:30,393 PPD SO WE DRINTD IS TO DO FROM 762 00:30:30,393 --> 00:30:31,394 SCRATCH MODELING OF THE 763 00:30:31,394 --> 00:30:32,795 STRUCTURES. 764 00:30:32,795 --> 00:30:35,297 DISAISH ON WE DIDN'T HAVE -- SO 765 00:30:35,297 --> 00:30:37,299 WE DIDN'T HAVE TO DO SCRATCH 766 00:30:37,299 --> 00:30:42,338 MODELING OF THE STRUCTURES. 767 00:30:42,338 --> 00:30:52,481 THANKS. 768 00:30:56,786 --> 00:30:58,054 >> (AWAY FROM MICROPHONE). 769 00:30:58,054 --> 00:31:01,690 >> SO YEAH, TR AT THAT POINT WE 770 00:31:01,690 --> 00:31:03,325 DIDN'T HAVE ANY STRUCTURE OF THE 771 00:31:03,325 --> 00:31:06,362 COMPLEX OF RAS BOUND TO BRAF. 772 00:31:06,362 --> 00:31:08,230 I THINK LIKE THIS YEAR THERE WAS 773 00:31:08,230 --> 00:31:10,332 A RELEASE OF A STRUCTURE, BUT 774 00:31:10,332 --> 00:31:14,170 LOW RESOLUTION OF RAS BINDING TO 775 00:31:14,170 --> 00:31:14,370 BRAF. 776 00:31:14,370 --> 00:31:16,005 BUT AT THAT POINT, THERE WAS NOT 777 00:31:16,005 --> 00:31:17,873 A STRUCTURE OF THE COMPLEX, SO 778 00:31:17,873 --> 00:31:22,611 BASICALLY WHAT WE USED IS THAT F 779 00:31:22,611 --> 00:31:27,783 WE LOOK AT HERE, THERE WAS KNOWN 780 00:31:27,783 --> 00:31:30,386 INTERACTION OF THE RAS WITH JUST 781 00:31:30,386 --> 00:31:34,990 THE R AF BINDING DOMAIN OF THE 782 00:31:34,990 --> 00:31:36,959 PROTEIN WITH RAS AND THERE WAS A 783 00:31:36,959 --> 00:31:39,061 CHRYSALIS STRUCTURE OF THIS TWO 784 00:31:39,061 --> 00:31:40,463 DOMAIN BINDING BUT THAT WAS NOT 785 00:31:40,463 --> 00:31:42,865 IN COMPLEX OF THE FULL LENGTH 786 00:31:42,865 --> 00:31:43,532 BRAF. 787 00:31:43,532 --> 00:31:46,035 JUST RAF BINDING DOMAIN YOU BIND 788 00:31:46,035 --> 00:31:47,136 WITH THE RAS. 789 00:31:47,136 --> 00:31:48,804 WE USE THAT HAD TO KIND OF 790 00:31:48,804 --> 00:31:51,107 SUPERIMPOSE ON OUR COMPLEX. 791 00:31:51,107 --> 00:31:52,641 THAT'S HOW WE MODEL WHAT WILL 792 00:31:52,641 --> 00:31:57,913 HAPPEN WHEN THIS INTERACTION IS 793 00:31:57,913 --> 00:32:08,124 ESTABLISHED. 794 00:32:12,561 --> 00:32:22,938 >> (AWAY FROM MICROPHONE). 795 00:32:29,912 --> 00:32:31,313 DO YOU HAVE ANY THOUGHTS ON 796 00:32:31,313 --> 00:32:34,717 WHETHER OR HOW THIS METHOD COULD 797 00:32:34,717 --> 00:32:37,219 BE USED, OR -- 798 00:32:37,219 --> 00:32:40,289 >> YEAH, I THINK THEY'RE 799 00:32:40,289 --> 00:32:41,390 DEFINITELY VERY COMPLEMENTARY TO 800 00:32:41,390 --> 00:32:42,158 EACH OTHER. 801 00:32:42,158 --> 00:32:44,660 WHEN YOU LOOK AT SPECIFICALLY IN 802 00:32:44,660 --> 00:32:46,962 OUR LAB, WE WORK A LOT WITH 803 00:32:46,962 --> 00:32:47,396 KINASES. 804 00:32:47,396 --> 00:32:50,466 SO IF YOU LOOK AT ALL THE 805 00:32:50,466 --> 00:32:52,034 DEPOSIT, LIKE STRUCTURE FOR 806 00:32:52,034 --> 00:32:53,869 INSTANCE IN ALPHAFOLD, THERE ARE 807 00:32:53,869 --> 00:32:56,071 ALL MODELS OF KINASE ACTIVE 808 00:32:56,071 --> 00:32:56,472 STRUCTURES. 809 00:32:56,472 --> 00:32:57,473 SO IF YOU GO ON THERE AND YOU 810 00:32:57,473 --> 00:33:00,309 LOOK AT THEM, YOU CAN HAVE A 811 00:33:00,309 --> 00:33:02,278 STARTING POINT FOR WHAT YOU WANT 812 00:33:02,278 --> 00:33:04,380 TO INVESTIGATE IN YOUR PROTEINS, 813 00:33:04,380 --> 00:33:08,317 BUT WE HAVE SEEN HOW THESE 814 00:33:08,317 --> 00:33:17,426 ACTIVE CONFORMATIONS THAT ARE 815 00:33:17,426 --> 00:33:18,861 DEPOSITS, THEY DON'T -- WHEN YOU 816 00:33:18,861 --> 00:33:20,162 DON'T HAVE ANY OTHER 817 00:33:20,162 --> 00:33:21,363 INFORMATION, THEY ARE A GOOD 818 00:33:21,363 --> 00:33:22,364 STARTING POINT FOR YOU TO KIND 819 00:33:22,364 --> 00:33:24,233 OF FORMULATE YOUR HYPOTHESIS AND 820 00:33:24,233 --> 00:33:26,635 START DOING YOUR EXPERIMENTS. 821 00:33:26,635 --> 00:33:29,238 BUT DEFINITELY I FEEL LIKE WHEN 822 00:33:29,238 --> 00:33:33,209 YOU DO THE STRUCTURE 823 00:33:33,209 --> 00:33:35,144 DETERMINATION, YOU CAN GET LIKE 824 00:33:35,144 --> 00:33:36,378 REALLY INSILENT OF HOW THE 825 00:33:36,378 --> 00:33:38,214 PROTEIN IS REALLY LIKE WORKING. 826 00:33:38,214 --> 00:33:39,748 SO SOMETIMES THEY WILL AGREE, 827 00:33:39,748 --> 00:33:42,518 SOMETIME THEY WON'T. 828 00:33:42,518 --> 00:33:47,423 YEAH. 829 00:33:47,423 --> 00:33:52,061 THANKS. 830 00:33:52,061 --> 00:33:58,968 >> (AWAY FROM MICROPHONE). 831 00:33:58,968 --> 00:34:01,203 >> SO IF YOU LOOK AT JUST THE 832 00:34:01,203 --> 00:34:03,305 KINASE ACTIVITY, THEY ARE VERY 833 00:34:03,305 --> 00:34:04,840 SIMILAR IN TERMS OF THE 834 00:34:04,840 --> 00:34:06,008 SUBSTRATES THAT THEY 835 00:34:06,008 --> 00:34:07,676 PHOSPHORYLATE ARE ALSO VERY 836 00:34:07,676 --> 00:34:08,110 SIMILAR. 837 00:34:08,110 --> 00:34:09,211 THE SEQUENCE IDENTITY THEY HAIR 838 00:34:09,211 --> 00:34:12,615 IS ALSO VERY HIGH, BUT IN 839 00:34:12,615 --> 00:34:15,050 DISEASE STATES, YOU CAN SEE 840 00:34:15,050 --> 00:34:16,552 DIFFERENT KIND OF -- THERE IS A 841 00:34:16,552 --> 00:34:18,220 DIFFERENT FUNCTION, YOU CAN SEE 842 00:34:18,220 --> 00:34:20,289 IN SOME TYPE OF CANCERS, THERE 843 00:34:20,289 --> 00:34:22,458 ARE NO MUTATIONS IN BRAF AND 844 00:34:22,458 --> 00:34:24,426 OTHER ONES THERE ARE NO 845 00:34:24,426 --> 00:34:26,028 MUTATIONS IN CRAF. 846 00:34:26,028 --> 00:34:27,162 THAT'S WHY WE ARE TRYING TO 847 00:34:27,162 --> 00:34:28,697 UNDERSTAND THE STRUCTURE IF 848 00:34:28,697 --> 00:34:32,001 THESE PROTEINS DIFFER IN THE 849 00:34:32,001 --> 00:34:33,202 FORMATION SPECIFICALLY OF THESE 850 00:34:33,202 --> 00:34:35,271 OUTER INHIBITORY COMPLEX ASK 851 00:34:35,271 --> 00:34:36,171 ACTIVE COME PLEKS. 852 00:34:36,171 --> 00:34:37,940 RIGHT NOW WE FINISHED THIS WHICH 853 00:34:37,940 --> 00:34:39,208 WAS THE STRUCTURE OF THE BRAF 854 00:34:39,208 --> 00:34:41,076 AND THEN WE MOVE IT TO TRY TO 855 00:34:41,076 --> 00:34:43,379 STUDY THE CRAF PROTEINS, AND WE 856 00:34:43,379 --> 00:34:45,014 HAVEN'T BEEN ABLE TO SOLVE YET 857 00:34:45,014 --> 00:34:47,416 THE STRUCTURES, BUT WE HAVE SOME 858 00:34:47,416 --> 00:34:49,184 PRELIMINARY DATA THAT INDICATES 859 00:34:49,184 --> 00:34:51,153 THAT THIS OUTER INHIBITORY STATE 860 00:34:51,153 --> 00:34:51,887 IS DIFFERENT. 861 00:34:51,887 --> 00:34:52,788 THE MAIN DIFFERENCE WE ARE 862 00:34:52,788 --> 00:34:55,491 SEEING SO FAR IS THAT THE CRT 863 00:34:55,491 --> 00:34:56,925 DOMAIN THE ONE I SHOWED IN 864 00:34:56,925 --> 00:34:58,160 VIOLET THAT IS IN THE ERNT OF 865 00:34:58,160 --> 00:35:00,029 THE STATE IN THE BRAF MIGHT NOT 866 00:35:00,029 --> 00:35:02,665 BE IN THE SAME POSITION IN THE 867 00:35:02,665 --> 00:35:02,865 CRAF. 868 00:35:02,865 --> 00:35:05,067 IF THAT IS THE CASE, IT MAY 869 00:35:05,067 --> 00:35:07,269 INDICATE THAT A CRAF MAY HAVE A 870 00:35:07,269 --> 00:35:09,004 MORE ACTIVE STATE BECAUSE IT HAS 871 00:35:09,004 --> 00:35:13,842 A LESS OUTER STATE THAN BRA -- 872 00:35:13,842 --> 00:35:15,911 BECAUSE IT HAS ALPHA HELIX STATE 873 00:35:15,911 --> 00:35:17,646 OF BRAF BUT THAT'S SOMETHING WE 874 00:35:17,646 --> 00:35:20,249 ARE LOOKING AT NOW. 875 00:35:20,249 --> 00:35:30,693 >> (AWAY FROM MICROPHONE). 876 00:35:32,428 --> 00:35:34,830 >> SO BASICALLY, WHEN YOU 877 00:35:34,830 --> 00:35:37,333 ANALYZE THE DATA, BASICALLY THE 878 00:35:37,333 --> 00:35:40,002 PROCESS OF RECONSTRUCTING IS 879 00:35:40,002 --> 00:35:43,372 LIKE AVERAGING, DOIRCHT THE 880 00:35:43,372 --> 00:35:44,606 CLASSES -- DIFFERENT 2D CLASSES. 881 00:35:44,606 --> 00:35:46,008 SO IF YOU HAVE SOME PORTIONS OF 882 00:35:46,008 --> 00:35:48,844 THE PROTEIN, THIS IS A VERY 883 00:35:48,844 --> 00:35:49,712 SIMPLIFIED OF THE PROCESS BUT 884 00:35:49,712 --> 00:35:50,679 BASICALLY WHAT YOU USE. 885 00:35:50,679 --> 00:35:52,348 SO IF YOU HAVE THE PROTEINS THAT 886 00:35:52,348 --> 00:35:54,450 ARE VERY FLEXIBLE YOU CAN REALLY 887 00:35:54,450 --> 00:35:55,184 POPULATE ONE STATE. 888 00:35:55,184 --> 00:35:56,618 SO IT'S KIND OF LIKE YOU HAVE 889 00:35:56,618 --> 00:35:57,820 SOMETHING HERE, SOMETHING HERE, 890 00:35:57,820 --> 00:35:59,254 SOMETHING HERE. 891 00:35:59,254 --> 00:36:01,223 SO THE AVERAGE THAT YOU GET IS 892 00:36:01,223 --> 00:36:05,594 NOT VERY WELL DEFINED BECAUSE IT 893 00:36:05,594 --> 00:36:09,198 IS ACTUALLY EVIDENT MOST 894 00:36:09,198 --> 00:36:10,632 CONFORMATIONS SO IT MAKES 895 00:36:10,632 --> 00:36:14,570 STRUCTURE TO HAVE LESS 896 00:36:14,570 --> 00:36:14,870 RESOLUTION. 897 00:36:14,870 --> 00:36:16,638 >> (AWAY FROM MICROPHONE). 898 00:36:16,638 --> 00:36:18,674 >> YES. 899 00:36:18,674 --> 00:36:22,144 >> SO ALL OF THIS IS DMURG 900 00:36:22,144 --> 00:36:23,445 TWO-DIMENSIONAL -- IS OCCURRING 901 00:36:23,445 --> 00:36:25,047 IN TWO-DIMENSIONAL. 902 00:36:25,047 --> 00:36:26,815 YOU WERE SAYING THE N TERMINAL 903 00:36:26,815 --> 00:36:30,119 IS THAT BECAUSE THEY INTERACT 904 00:36:30,119 --> 00:36:30,352 WITH -- 905 00:36:30,352 --> 00:36:32,221 >> WHEN WE SAW THE STRUCTURES 906 00:36:32,221 --> 00:36:33,122 THEY WERE IN SOLUTION AND WE 907 00:36:33,122 --> 00:36:35,324 DON'T HAVE ANY MEMBRANE PRESENT, 908 00:36:35,324 --> 00:36:38,127 BUT OBVIOUSLY WHAT WE'RE SUSPECT 909 00:36:38,127 --> 00:36:40,763 SG THAT ALL OF THIS PROCESS 910 00:36:40,763 --> 00:36:42,498 HAPPENS AT THE MEMBRANE BECAUSE 911 00:36:42,498 --> 00:36:43,932 RAS IS LOCALIZED IN THE 912 00:36:43,932 --> 00:36:44,199 MEMBRANE. 913 00:36:44,199 --> 00:36:45,868 SO THERE IS ACTUALLY SOME 914 00:36:45,868 --> 00:36:48,604 EVIDENCE THAT BRAF ITSELF MAY BE 915 00:36:48,604 --> 00:36:49,671 INTERACTING WITH THE MEMBRANE BY 916 00:36:49,671 --> 00:36:52,341 THE CRD DOMAIN. 917 00:36:52,341 --> 00:36:55,844 BUT IN THE CYTOSOL, THE CRT WAS 918 00:36:55,844 --> 00:36:58,147 COMPLETELY OCCLUDED BY THE 919 00:36:58,147 --> 00:36:59,348 14-3-3 AND INTERACTIONS WITH THE 920 00:36:59,348 --> 00:37:01,417 KINASE DOMAIN. 921 00:37:01,417 --> 00:37:02,851 SO IT IS EXPECTED AT SOME POINT 922 00:37:02,851 --> 00:37:05,053 DURING THE ACTIVATION THAT THE 923 00:37:05,053 --> 00:37:07,322 CRD DOMAIN BECOMES EXPOSED AND 924 00:37:07,322 --> 00:37:09,625 ENTER ABLGHTS ALSO WITH THE 925 00:37:09,625 --> 00:37:10,392 MEMBRANE -- INTERACTS ALSO WITH 926 00:37:10,392 --> 00:37:11,393 THE MEMBRANE BECAUSE THERE IS 927 00:37:11,393 --> 00:37:12,928 SOME EVIDENCE OF THAT HAPPENING. 928 00:37:12,928 --> 00:37:14,430 SO I GUESS YES TO FULLY 929 00:37:14,430 --> 00:37:17,199 UNDERSTAND THIS ACTIVATION 930 00:37:17,199 --> 00:37:18,267 MECHANISM, IT WOULD PROBABLY BE 931 00:37:18,267 --> 00:37:19,935 VERY IMPORTANT TO TRY TO 932 00:37:19,935 --> 00:37:21,770 UNDERSTAND HOW THIS HAPPENS IN 933 00:37:21,770 --> 00:37:24,406 THE CONTEXT OF SOME KIND OF 934 00:37:24,406 --> 00:37:28,010 MEMBRANE, SO PROBABLY SOME 935 00:37:28,010 --> 00:37:29,545 LIPIDS, AS A LOT OF ACTIVE 936 00:37:29,545 --> 00:37:39,922 RESEARCH IN THAT AREA. 937 00:37:43,492 --> 00:37:44,827 THANK YOU VERY MUCH. 938 00:37:44,827 --> 00:37:55,003 [APPLAUSE] 939 00:38:01,910 --> 00:38:05,814 [THE EVENT CONCLUDED AT 940 00:38:05,814 --> 00:38:16,091 11:42 A.M. EST]