MY NAME IS MARCUS, A TENURED TRACK AT NIAMS. IT'S MY PLEASURE TODAY TO INTRODUCE DR. DINSHAW PATEL WHO VISITS US FROM NEW YORK WHERE HE IS THE ABI ROCKEFELLER CHAIR IN EXPERIMENTAL THERAPEUTICS AND A MEMBER IN THE STRUCTURAL BIOLOGY PROGRAM OF THE MEMORIAL SLOAN-KETTERING CANCER CENTER. HE IS ONE OF THE TRUE TITANS IN THE STRUCTURAL BIOLOGY AND HIS LONG AND DISTINGUISHED CAREER INVOLVES MOST PART AROUND THE BIOPHYSICAL DISECTION OF NUKE LAKIC ACID STRUCTURES AND INTERACTIONS. I GOT HIS ENERGY FIRSTHAND IN 2007 DURING MY POSTDOC WITH TOM WHO WAS HIS COLLABORATOR ON A FANTASTIC STRING OF PAPERS THAT FOR THE FIRST TIME REPORTED ON STRUCTURES OF PROTEINS BOUND TO RNA TARGETS AND THESE PROTEINS ARE AT THE CORE OF THE RNA INTERFERENCE PATHWAYS. SO DIN SHAW HAS BEEN STUDYING THIS PATHWAY SINCE THE EMERGENCE IN THE EARLY TWO THOUSANDS AND HIS ELEGANT CRYSTALS OF I COMPONENTS GREATLY CONTRIBUTED TO OUR CURRENT UNDERSTANDING OF SMALL RNA PATHWAYS. HE IS A PIONEER IN NUMEROUS OTHER FIELDS AS WELL AND CONTRIBUTED THE FIRST STRUCTURES OF MULTIPLE RIBOZYMES AND METABOLITE SENSING RNA, RIBOSWITCHES. HE PROVIDED THE STRUCTURAL BASIS FOR EPIGENETIC REGULATION BY HISTONE MARKS AS WELL AS DNA DAMAGE RESOLUTION BYE-BYE PASS POLYMERASES. INVOLVED IN THE DETECTION OF NOVEL INNATE IMMUNE PATHWAY SENSING DNA, AND FINALLY HE ALSO ELUCIDATING THE STRUCTURAL BASIS OF CRISPR-CAS SYSTEMS. A FEW WORDS ABOUT HIS CAREER, HE IS A NATIVE OF MUMBAI AND INDIA WHERE HE ALSO ATTENDED COLLEGE AND OBTAINED HIS BACHELORS IN CHEMISTRY. HE THEN MOVED TO THE U.S. AS A FELLOW AND EARNED A MASTER'S DEGREE FROM CALL TECH FOLLOWED BY PH.D. FROM NEW YORK UNIVERSITY. HE STAYED IN THE AREA AND FIRST DID A POSTDOC AT THE NYU AND MOVED TO THE LEGENDARY BELL LABS IN NEW JERSEY WHERE HE BECAME A IN 1984, HE WAS APPOINTED PROFESSOR OF BIOCHEMISTRY AND BIOPHYSICS AT COLUMBIA AND SINCE 1992 HE IS A MEMBER OF THE STRUCTURAL BIOLOGY PROGRAM. HE IS THE RECIPIENT OF NUMEROUS AWARDS. HE WAS DISTINGUISHED STAFF MEMBER OF BELL LABS AND A DISTINGUISHED ALUM OF NYU, PRESIDENT OF THE HARVEY SOCIETY. HE IS THE RECIPIENT OF THE NIH DIRECTOR'S TRANSFORMATIVE RO1 AND FINALLY HE IS A MEMBER OF THE AMERICAN ASSOCIATION OF ARTS AND SCIENCES AS WELL AS OF THE NATIONAL ACADEMY OF SCIENCES. TODAY HE WILL GIVE US AN OVERVIEW OF MILT PEL ASPECTS OF STRUCTURAL BIOLOGY OF RNA-MEDIATED GENE REGULATION. PLEASE JOIN ME IN WELCOMING DINSHAW PATEL FOR THIS EXCITING LECTURE. [ APPLAUSE ] >> DINSHAW PATEL: THANK YOU VERY MUCH, MARCUS. MY TALK TODAY WILL FOCUS ON OUR WORK CENTERED ON RNA, ITS RECOGNITION, ITS CATALYTIC ACTIVITIES, AND I'LL DO THAT BY DESCRIBING WORK ON RIBOSWITCHES, MAINLY FOCUSED ON RNA ARCHITECTURE AND THE GENERATION OF POCKETS THAT RECOGNIZE SMALL METABOLITES. I'LL TALK ABOUT RIBOZYMES, SHELF-CLEAVING RIBOZYMES THAT CAN CUT THEIR OWN RNA, AND FINALLY, I'LL TALK ABOUT VERY RECENT WORK ON CRISPR-CAS SYSTEM S. SO, LET ME START WITH A BRIEF BACKGROUND ON RIBOSWITCHES. THESE ARE METABOLITE SENSING NON CODING RNA WAS. SHOWN SCHEMATICALLY HERE IN TWO FORMS, THERE IS A SENSING DOMAIN AND THERE IS AN EXPRESSION PLATFORM. METABOLITES ARE RECOGNIZED BY THE SENSING DOMAIN, WHICH UNDERGOES A CONFIRMATIONAL CHANGE, WHICH IS THEN PROPAGATED TO THE ADJACENT EXPRESSION PLATFORM. SO THAT IS SOME MARKER IN THE EXPRESSION PLATFORM, LET'S SAY IS EXPOSED IN ONE STATE ON A METABOLITE-BINDING GETS SEQUESTERED. DEPENDING ON THE CONCENTRATION OF THE METABOLITE, YOU HAVE AN ON OFF SWITCH. TO SHOW YOU AN EXAMPLE, HERE IS A RIBOSWITCH FOR THE METABOLITE TIMING PYROPHOSPHATE. HERE IS ITS SENSING DOMAIN AND HERE IS ITS EXPRESSION PLATFORM. IN THE ABSENCE OF METABOLITE, YOU HAVE THESE PAIRING ALIGNMENTS SHOWN HERE. WHEN THE METABOLITE BINDS, YOU HAVE AN ALTERNATE PAIRING ALIGNMENT. SO IS THAT IN THE FREE STATE, THE ELEMENT IS EXPOSED. IN THE BOUND STATE, IT IS SEQUESTERED. SO TRANSLATIONAL OCCURS IN THE UNBOUND STATE AND INHIBITED IN THE LIGAND-BOUND STATE. WE WOULD LIKE TO STUDY THE STRUCTURE OF THESE RIBOSWITCHES AS WELL AS THEIR LIGAND RECOGNITION POCKETS. GENERALLY THIS IS FEASIBLE FOR THE SENSING DOMAIN BECAUSE WHEN THE LIGAND BINDS, THE STRUCTURE BECOMES COMPACT AND CAN BE STUDIED CRYSTAL GRAPHICALLY. SO THE EMPHASIS IS ON OVERALL RNA ARCHITECTURE. THE BINDING POCKETS, THE LIGAND RECOGNITIONING AND DISCRIMINATION AND SOME OF THE TRANSITIONS THAT ARE MENTIONED AS YOU GO FROM THE FREE TO THE BOUND STATE. SO LET ME JUST SHOW YOU THE INSTRUCT YOU WERE OF THE TIMING PYROPHOSPHATE RIBOSWITCH, BOUND TO THE SENSING DOMAIN. IT CONTAINS A THREE-WAY JUNCTION, WHICH WHEN TIMING PYROPHOSPHATE BINDS, ADOPTS THIS TUNING FORK-LIKE ARCHITECTURE. THERE IS A STEM, A HANDLE, AND TWO PRONGS. YOU HAVE A LOOP HERE INTERACTING WITH THE RECEPTOR HOLDING THESE TWO PRONGS IN ALIGNMENT. THE THREE-WAY JUNCTION IS HERE AND VERY INTERESTINGLY, THE LIGAND, WHICH CONTAINS A RETRO CYCLE AT ONE END, AND A PYROPHOSPHATE AT THE OTHER, BINDS ABOVE THE JUNCTION IN AN EXTENDED FORM WHERE THERE ARE POCKETS IN BOTH HELIXES. IN ONE HELIX, YOU RECOGNIZE THE HETEROCYCLE AND IN THE OTHER HELIX, YOU RECOGNIZE THE PYROPHOSPHATE. SO THIS WAS VERY INTERESTING. HOW DOES AN RNA WHICH IS A POLYPHOSPHATE, RECOGNIZE A LIGAND WHICH ITSELF HAS PHOSPHATES? SO IF YOU BLOW UP THE BINDING POCKET, REMEMBER THERE IS ONE POCKET IN THIS HELIX AND ANOTHER POCKET IN THIS. THIS IS WHAT YOU SEE. HERE IS THE TIMING PYROPHOSPHATE. THE HETEROCYCLE SITS IN ONE POCKET WHERE IT IS SANDWICHED BETWEEN PURINES AND THE EDGES ARE HYDROGEN BONDED. THIS END IS ANCHORED AND THE OTHER END WHERE YOU HAVE THE PYROPHOSPHATE, THE PHOSPHATES ARE COORDINATED TO HYDRATED MAGNESIUM IONS DIRECTLY AND THEN ONE OF THE MAGNESIUM IONS IS IN TURN, COORDINATED TO THE RNA, NOT TO THE PHOSPHATES OF THE RNA BUT TO THE BASE EDGES OF THE RNA. SO RNA CAN FOLD, GENERATE A PAIR OF POCKETS, ANCHOR THE HETEROCYCLIC RING AND ACCOMMODATE A BIPHOSPHATE BY USING MAGNESIUM IONS, HYDRATED MAGNESIUMS, AS RIDGES BETWEEN THE LIGAND AND THE RNA ITSELF. SO THIS WAS QUITE NOVEL. THE DUAL POCKETS, THE EXTENDED STRUCTURE OF THE LIGAND. AND WHAT WE DON'T KNOW IS WHAT THE CONFIRMATION IS IN THE FREE STATE WHETHER THESE HELIXES ARE PARALLEL OR THEY ARE FRAYING, AND ONLY IN THE PRESENCE OF THE LIGAND ARE THEY BROUGHT TOGETHER AND ALIGNED AND PARALLEL. SO WHAT ABOUT POCKETS? HOW SMALL A POCKET CAN ONE GENERATE USING AN RNA BACKBONE? SO HERE I TURN TO ANOTHER RIBOSWITCH, ALL THESE RIBOSWITCHES WERE IDENTIFIED IN THE LAB OF RON BREAKER AT YALE. AND THIS IS A RIBOSWITCH CALLED THE FLUORIDE RIBOSWITCH WHICH IS INVOLVED IN ACTIVATING THE EXPRESSION OF GENES THAT ENCODE FLUORIDE TRANSPORTERS. AND HERE IS THE SENSING DOMAIN OF THIS RIBOSWITCH. IT IS SIMPLE T HAS A STAMINA INTERNAL BUBBLE AND A STEM LOOP AND DANGLING ENDS. THIS RIBOSWITCH BINDS FLUORIDE AND ANINE, WHICH IS STREAMLY SMALL AND ALSO NEGATIVELY CHARGED. SO HOW DOES RNA FORM A VERY SMALL POCKET AND ACCOMMODATE A VERY NEGATIVELY-CHARGED ION? SO AMY IN THE LAB, WHICH SHE SAW THE SENSING DOMAIN OF THE FLUORIDE RIBOSWITCH BOUND TO FLUORIDE AND ANINE, THIS IS THE OVERALL TOPOLOGIY. THE BREAKER LAB THAT IDENTIFIED THIS RIBOSWITCH REALIZED THAT THIS SEGMENT HERE COULD PAIR WITH THIS LOOP SEGMENT TO FORM A PSEUDOKNOT. AND INDEED N OUR STRUCTURE, YOU CAN SEE THE PSEUDOKNOT FORMING A DUPLEX IN PURPLE. AND THE VARIOUS ELEMENTS ARE COLOR-CODED HERE AND IN THE OVERALL STRUCTURE. AND WHAT WE FIND IS THAT THE FLUORIDE ANINE IS POSITION THE IN THE CENTER OF THIS THREE-DIMENSIONAL ARCHITECTURE. SO HOW IS IT ACCOMMODATED IN ITS POCKET? AND HERE WE SEE SOMETHING QUITE REMARKABLE. HERE IS THE FLORIDEANINE. IT'S COORDINATED WHICH IS NEGATIVELY CHARGED, WHICH IS COORDINATED TO THREE MAGNESIUM IONS. AND THEN THE MAGNESIUM IONS IN TURN, ARE COORDINATED TO PHOSPHATE OXYGENS AND WATER MOLECULES WITH THE PHOSPHATE OXYGENS DIRECTED INTO THE INTERIOR OF THE RNA SCAFFOLD. SO YOU HAVE A FLUORIDE ANINE SURROUNDED BY THREE MAGNESIUM IONS AND THE MAGNESIUMS ARE COORDINATED TO PHOSPHATE OXYGENS AND TO WATER MOLECULES. SO NEGATIVE CHARGE SURROUNDED BY POSITIVE CHARGES SURROUNDED BY NEGATIVE CHARGES OF THE PHOSPHATE OXYGENS. AND THE WHOLE CONCEPT IS THAT FLORIDEANINE IS VERY SMALL. YOU CAN NOT FORM A SMALL POCKET SO YOU ENLARGED A POCKET BY NOT ONLY HAVING FLUORIDE BUT THREE DIEVILLANT IONS. SO THE POCKET BECOMES MUCH LARGER AND NOW CAN ACCOMMODATE THE FLORIDEANINE COORDINATED TO THE MAGNESIUMS. NOW THIS RESULT WAS QUITE STRIKING AND WE WERE CONCERNED SO WE LOOKED AT THE PROTEIN WORLD FOR PROTEINS THAT BOUND FLORIDEANINES AND WE FOUND AN EXAMPLE SHOWN HERE WHICH IS COMPARED WITH WHAT I JUST TALKED ABOUT IN THE RNA WORLD. SO IN THE RNA WORLD, YOU HAVE A FLUORIDE SURROUNDED BY THREE DIE VALIANT IONS SURROUNDED BY PHOSPHATE OXYGENS. IN THE PROTEIN WORLD, WE HAVE A FLORIDEAN ION SURROUNDED BY TWO DIVALIANT AND THE PHOSPHATES ARE REPLACED BY CARBOXYLATES. SO NATURE HAS USED SIMILAR PRINCIPLES TO RECOGNIZE FLORIDEANINE AND INWARDLY POINTING EITHER PHOSPHATE OR CABOX LATE GROUPS. NOW WE ARE VERY INTERESTED IN THE CONFIRMATIONAL TRANSITION AS YOU GO FROM THE LIGAND-FREE TO THE LIGAND-BOUND FORM. SO HERE I WANT TO SHOW YOU AN EXAMPLE WHERE WE COULD MONITOR THE SENSING DOMAIN BOTH IN ITS LIGAND-FREE AND ITS LIGAND-BOUND FORMS. AND THIS IS SAY RIBOSWITCH THAT BINDS THE AMINO ACID L GLUTAMINE, AND THE RIBOSWITCH'S POSITION UPSTREAM OF GENES INVOLVED IN NITROGEN METABOLISM. AGAIN, THE TOPOLOGY OF THE SENSING DOMAIN IS VERY SIMPLE. IT CONTAINS THREE STEMS AND AN INTERNAL BUBBLE. NOW TO SOLVE THIS STRUCTURE, WE HAD TO ADD A U1ARNA LOOP BINDED TO THE U1A PROTEIN AND SOLVE THE PROBLEM. BUT FOR THIS SENSING DOMAIN THAT BINDS L GLUTAMINE, WE WERE ABLE TO SOLVE THE STRUCTURE IN THE FREE-FORM. SO YOU HAVE THREE STEMS AND ONCE AGAIN, HAVE YOU THAT TUNING FORK ARCHITECTURE, A HANDLE AND TWO PRONGS. AND WHEN WE LOOK AT THE ENTIRE SCAFFOLD, THERE IS A GGA ELEMENT WHICH IS DISORDERS IN THE FREE-FORM. ONE CAN ASK WHAT STABILIZE THIS HAS FREE-FORM STRUCTURE IN THE ABSENCE OF LIGAND? AND WHAT WE FIND IS THAT THE THREE-WAY JUNCTION ARE A SET OF STACKED BASES STACKING ON THEIR NEIGHBORS. WHEN WE ADD L GLUTAMINE, THE CONFIRMATION CHANGES DRAMATICALLY FROM THE TUNING FORK ARCHITECTURE TO AN L-SHAPED ARCHITECTURE, AND AT THE JUNCTION OF THE L ARCHITECTURE IS WHERE THE L GLUTAMINE BINDS. FURTHER, NOW, THE SEGMENT THAT WAS DISORDERED IN THE FREE STATE, GGA, CAN BE FOLLOWED IN THE BOUND STATE, G22, G23A24, FORMING ONE SIDE OF A POCKET WHERE THE L GLUTAMINE BINDS. IN ADDITION, WE FORM A LONG-RANGE INTERACTION BETWEEN THIS G AND THAT C TO FORM A WATSON CRATE GC PAIR. IT'S AN OPEN POCKET IN WHICH THE L GLUTAMINE BINDS AND AGAIN, THE POCKET IS ENLARGED BY A HYDRATED MAGNESIUM ION. SO IT IS AN OPEN POCKET IN PART ACCOUNTING FOR THE WEAK BINDING AFFINITY. HERE THE IS L GLUTAMINE SITTING IN THE POCKET TOGETHER WITH THE HYDRATED MAGNESIUM ION AND LONG-RANGE GC PAIR. AND WHAT YOU OBSERVED IS ALL THE HETEROATOMS IN L GLUTAMINE ARE INVOLVED IN EITHER DIRECTOR ORDER MEDIATED HYDROGEN BONDS. AND THIS IS IMPORTANT BECAUSE THIS RIBOSWITCH BINDS L GLUTAMINE AND DISCRIMINATES AGAINST ALL OTHER AMINO ACIDS, INCLUDING THEIR D ANALOGUES. SO WHAT WE HAVE OBSERVED FOR THIS RIBOSWITCH FOR L GLUTAMINE IS THAT WE ARE GOING FROM A CONFIRMATION IN ORANGE IN THE FREE STATE, WITH A DISORDERED SEGMENT, TO CONFIRMATION IN BLUE IN THE BROWN STATE, A LARGE CONFIRMATIONAL CHANGE, WITH FORMATION OF THIS LONG-RANGE TERB REPAIR WITH THE BINDING POCKET LOCATED ADJACENT TO IT. THERE ARE QUESTIONS ABOUT THE GLUTAMINE-FREE-FORM. IS IT THE CONFIRMATION WE SEE IN THE CRYSTAL OR ARE THE P2 AND P3 HELIXES ADOPTING OR SAMPLING DIFFERENT CONFIRMATIONS? SO WE WORKED WITH THE LAB AT DUKE UNIVERSITY TO USE MMR, BOTH CHEMICAL SHIFTS AND RESIDUAL COUPLINGS AND ALSO MOLECULE DYNAMIC SIMULATIONS. AND TO SUMMARIZE THAT RESULT THERE, WE CAN EXPLAIN THE RESIDUAL COUPLINGS VERY WELL FOR THE BOUND FORM BUT THE INDICATIONS FOR THE FREE-FORM IS THAT THE FREE-FORM IS SAMPLING A RANGE OF CONFIRMATIONS AND IN THIS WILL RANGE, YOU EITHER HAVE A TUNING FORK OR AN L SHAPE BUT YOU DON'T FORM THAT LONG-RANGE TERB AREA PAIR IN THE PRESENCE OF MAGNESIUM. ONLY WHEN YOU ADD L GLUTAMINE DOES IT TRAP THE BOUND STATE WHICH FORMS THIS LONG-RANGE PAIR THE LAST EXAMPLE I WANT TO SHOW YOU IS TO ADDRESS THE FOLLOWING POINTS: YOU HAVE AN RNA SCAFFOLD, YOU HAVE A NATURAL PETAB LIGHT THAT BINDS SOMEWHERE IN THE SCAFFOLD, CAN WE USE SELECTION METHODS TO IDENTIFY SMALL LIGANDS THAT COULD ALSO TARGET THIS PARTICULAR POCKET? SO MANY YEARS AGO, WE LOOKED AT THIS SIX-STEM JUNCTION RNA, BOUND THE METABOLITE FLAVOR MONONUCLEOTIDE AND SOLVED THE STRUCTURE. AND INTERESTINGLY, DESPITE NO INDICATION OF PSEUDOTWO-FOLD SYMMETRY, YOU HAVE ONE-HALF OF THE RNA RELATED BY SYMMETRY TO THE OTHER HALF, AND THE FMN SITTING IN A JUNCTIONAL POCKET ITSELF IN AN ASYMMETRICAL ORIENTATION RELATIVE TO THESE TWO SEM TREE ELEMENTS. NOW MANY YEARS LATER, A GROUP AT MERCK IDENTIFIED THROUGH SELECTION, A SMALL MOLECULE WHICH THEY CALL RIBOSILL, WHOSE STRUCTURE IS SHOWN HERE. AND IT'S A HIGHLY-SELECTIVE MODULATOR OF BACTERIAL RIBOFLAVIN RIBOSWITCHES BY REPRESSING RIBOSWITCH MEDIATED GENE EXPRESSION AND INHIBITING BACTERIAL CELL GROWTH. SO HERE IS THE FMN SITTING IN ITS POCKET. IT IS SANDWICHED BETWEEN TWO PURINES AND SIDE CHAIN HAS A PHOSPHATE AND A HYDRATED MAGNESIUM ION. AND HERE IS RIBOSILL SITTING IN THE SAME POCKET USING THESE TWO LIGANDS WHICH ARE PLANAR AND SANDWICHED BETWEEN TWO PURINES. THIS THEN ALLOWING STACKING OF THIS HETEROCYCLIC RING. SO THESE POCKETS CAN BE TARGETED BY OTHER MAN MADE LIGANDS. IN THIS CASE, REPRESSING RIBOSWITCH MEDIATED GENE EXPRESSION AND INHIBITING BACTERIAL CELL GROWTH. NOW I WANT TO MOVE ON TO THE SECOND TOPIC WHICH INVOLVES SELF -CLEAVING RIBOZYMES. IN THE LITERATURE, FOR MANY YEARS, THERE HAVE BEEN SHELF-CLEAVING RIBOZYMES WITH NAMES LIKE HAMMERHEAD, AND SATELLITE. AND THEY INVOLVE IN ROLLING CIRCLE BASE REPLICATION OF SATELLITE RNAs. AND FOR LONG TIME NO NEW SHELF-CLEAVING RIBOZYMES WERE IDENTIFIED UNTIL AGAIN THE RONALD BREAKER LAB FOUND A SET OF RIBOZYMES, SMALL RIBOZYMES W NAMES LIKE PISTOL, TWISTER AND HATCHER AND FOR EACH THERE WAS A PARTICULAR POSITION IN THE BACKBONE WHERE CLEAVAGE OCCURRED. PISTOL HERE AND TWISTER HERE AND FOR HATCHER HERE. NOW THE CONCEPT IS WHY DOES CLEAVAGE OCCUR AT A PARTICULAR SITE? AND BASED ON THE EARLIER WORK IF YOU HAVE A DINUCLEOTIDE STEP, WHAT HAPPENS IN NUCLEOLYTIC CLEAVAGE IS TWO-PRIME HIGH DOCKSIL AT THIS POSITION IS SOMEHOW ACTIVATED. IT TARGETED THE PL1 AND NEEDS IN-LINE ALIGNMENT TO DO THAT AND YOU GET PL1 CLEAVAGE. SO YOU NEED SOMETHING TO ACTIVATE THE TWO PRIME HYDROXYL. YOU NEED SOMETHING TO STABILIZE A NEGATIVE CHARGE THAT IS DEVELOPING AT THE CLEAVAGE SITE. AND THE CONCEPT IS THERE IS GENERALLY A GENERAL BASE THAT DOES THE ACTIVATION AND A GENERAL ASSAY INVOLVED IN THE STABILIZATION, AND ONE NEEDS IN-LINE ALIGNMENT. SO THE IMPORTANT POINTS ARE, A SPECIFIC CLEAVAGE SITE, IN-LINE ALIGNMENT, WHAT ARE THE CATALYTIC RESIDUES AND IS THERE A ROLE FOR MAGNESIUM ION? SO WHAT I'M GOING TO TELL YOU TODAY IS THE STRUCTURE OF THE PISTOL RIBOZYME, RIBOZYME CALLED TWISTER SISTER, THAT IS RELATED TO TWISTER, AND WHAT CAN WE LEARN FROM THESE STRUCTURES? SO HERE IS THE PISTOL RIBOZYME. IT'S A VERY SMALL RNA. IT CONTAINS A STEM LOOP, THESE TWO STEMS AND AN INTERNAL BUBBLE, AND FROM THE SEQUENCE THE BRACKETTER LAB WAS ABLE TO SHOW THAT IT MOST LIKELY FORMS A PSEUDOKNOT. SO AIMING IN MY LAB SOLVED THE STRUCTURE OF THE PISTOL RIBOSOME. THE RNAs YOU HAVE TWO STRANDS. THERE IS ONE STRAND HERE AND THEN THERE IS A SECOND STRAND HERE. WE WORK WITH A CHEMISTRY LAB WHO CAN SYNTHESIZE THE RNA AS WELL AS IN CORN 8 ANY ANALOGUES AT WHATEVER POSITION WE REQUIRE. -- INCORPORATE. SHEAR A THREE-DIMENSIONAL STRUCTURE OF THE PISTOL RIBOZYME. YOU WALK THROUGH IT AND START WITH THIS ORANGE STEM, AND THEN YOU HAVE A PSEUDOKNOT IN MAGENTA AND THEN THE GREEN STEM. AND THE REST OF IT IS THEN INVOLVED IN A FOLD-BACK AND AT THE CLEAVAGE SITE, U54G53, THE BASIS INSTEAD OF BEING STACKED, ARE WOULD PLAYED APART. SO WHAT IS OCCURRING AT THE CATALYTIC POCKET? SO FIRST I SHOW YOU THE STRUCTURE ON THE UPPER LEFT. HERE IS G53 AND HERE IS U54. AT THIS SITE, THE CLEAVAGE SITE, THE BASE PAIR BASISES ARE PLAYED APART BUT EQUALLY IMPORTANTLY EACH IS ANCHORED IN PLACE THROUGH STACKING INTERACTIONS OR IN TOTAL ALIGNMENT ON EITHER SIDE. SO NOT ONLY ARE THEY HAVE AD APART BUT ANCHORED IN PLACE. PLAYED APART. SO THE NEXT THING YOU WANT TO ASK IS WHAT ARE THE RESIDUES IN THE VICINITY OF THE G53-U54 STEP? AND WHAT WE FIND IS THAT THERE IS A GUININE WHOSE N1 IS DIRECTED TOWARDS THE TWO PRIME HYDROXYL THAT HAS TO BE ACTIVATED AND THERE IS AN ADD ANINE NEXT TO THE PL1 THAT HAS TO BE BROKEN. SO THROUGH THESE BASES PLAYING A CATALYTIC ROLE IN ACTIVATING THE TWO PRIME HYDROXYL AND STABILIZING THE TRANSITION STATE AS WELL AS AFTER THE PL BOND IS CLEAVED? SO THE MORE SIMPLE THING WE CAN DO IS WE CAN REPLACE THESE NUCLEOTIDES, G40, BY AN AN ANINE AND A32 BY SIGHTA SEEN. NOW THIS IS A TWO 46 STRANDED CONSTRUCT. ONE EAST - TWO-STRANDED CONSTRUCTED. ONE IS A SUBSTRATE STRAND. ADDS MAGNESIUM AND THE SUBSTRATE STRAND GOES DOWN AND AS A RESULT OF CLEAVAGE, THERE ARE TWO NEW STRANDS. SO FOR THE WILDTYPE RNA, FOR THE CRYSTALLOGRAPHY, WE HAD TO REPLACE THE TWO PRIME HYDROXYL OF G53 BY A FRO TON BUT FOR THE WILDTYPE WHICH IS ALL RNA, THE SUBSTRATE STRAND GETS CLEAVED IN THE WILDTYPE SYSTEM. YOU GO WHEN WE MAKE THESE MUTATIONS, GO 40 TO A OR A30 TO C, THERE IS NO CLEAVAGE TELLING US THAT THESE RESIDUES, G40 AND A32 ARE IMPORTANT FOR CLEAVAGE. NOW IF YOU LOOK AT A32, THE ENTRY POSITION OF THE ADDA NINE AND THE TWO PRIME HYDROXYL MAY PLAY A ROLE IN THE CATALYSIS. IF YOU REPLACE THE HYDROXYL BY A PROTON WITH OXYRESIDUE AND DO THE SAME CLEAVAGE ANALYSIS, CLEAVAGE OCCURS BUT AT A SLOWER RATE. AS FAR AS ENTRY POSITION OF THE ADENINE IS CONCERNED, IF YOU REPLACE THE NITROGEN CHEMICALLY BY A CARBON TO MAKE THE C3DA ANALOGUE, YOU COMPLETELY LOSE CLEAVAGE. SUGGESTING THAT THE ENTRY POSITION HAS AN IMPORTANT ROLE IN THE CATALYSIS AND THE TWO PRIME HYDROXYL HAS A ROLE THAT IS LESS IMPORTANT. WE CAN DO ONE MORE EXPERIMENT AND THAT IS TO TRY AND MEASURE THE PKAs OF THESE BASES. BEGANNINE HAS A PK AROUND 12. RNA IS NOT STABLE DURING A PH TITRATION SO WE WERE LIMITED TO TRYING TO ESTIMATE THE MKA OF A32. AND SO THE WAY THIS WAS DONE WAS TO CHEMICALLY INCORPORATE A C13 LABEL SPECIFICALLY AT THE TWO POSITION OF ADENINE SPECIFICALLY FOR ADENINE 32 AND THEN DO AN NMR TITRATION. AND THE PKA WE OBSERVED IS 4.7 WHICH IS ONE PH UNIT ABOVE THE PKA OF ADENINE AND RNA. SO IN THE CONTEXT OF THE RIBOZYME, THE PKA GOES UP BY ABOUT ONE PH UNIT TOWARDS A NEUTRAL PH OF 7. FINALLY, WE WERE INTERESTED IN CLEAVAGE RATE AND TO DO THIS, WE GENERATED A CONSTRUCT CONTAINING TWO STRANDS BUT IN THE SUBSTRATE STRAND IT REPLACED AN ADENINE BY TWO AMINO PURINES. SO IN THE FREE RNAs WILL YOU GET FLUORESCENCE FROM THE TWO AMINO PURINE BUT IF IT'S PART OF A DUPLEX, THAT FLUORESCENCE IS QUENCHED. SO WHEN YOU ADD THE TWO STRANDS IN THE PRESENCE OF MAGNESIUM, YOU GENERATE THE RIBOSWITCH, CLEAVAGE OCCURS AND THE STRANDS BREAK AND ARE SEPARATED. SO YOU START WITH A CERTAIN FLUORESCENCE LEVEL AND IT FORMS THE RIBOSWITCH AND THE TWO AMINO PURINE ATTACKING AND THE FLUORESCENCE GETS QUENCHED AND EVE TIME AS THE STRANDS DISBURSE, THE FLUORESCENCE RECOVERS AND WE OBTAIN A CLEAVAGE RATE OF ONE PER MINUTE, SLOW BUT STILL MEASURABLE. NOW MORE RECENTLY, WE WORKED WITH THE SAME RIBOZYME CALLED TWISTER SISTER. AGAIN IN COLLABORATION WITH THE RONALD LAB, AND FOR THIS RIBOZYME, THERE IS ALSO A STRUCTURE FROM THE DAVID LILI LAB I WILL COMPARE WITH OUR STRUCTURE. SO IF YOU CAN SEE THIS, YOU HAVE A STEM, A BUBBLE, A STEM, TWO LOOPS AND A STEM AND ANOTHER LOOP. AND THE CLEAVAGE SITE OCCURS AT A SPECIFIC SITE IN THIS RIBOZYME AT THIS PARTICULAR STEP. WHEN THE STRUCTURE OF THIS RIBOZYME WAS SOLVED, IT'S OVER ALL ARCHITECTURE IS SHOWN HERE. THERE IS ONE CONTINUOUS HELIX HERE AND A SECOND CONTINUOUS HELIX HERE. AND THE CLEAVAGE SITE ONCE AGAIN CONSISTS OF TWO BASES THAT ARE PLAYED APART. NOW IN ORDER TO ADOPT THIS TOPOLOGY, YOU HAVE LONG-RANGE INTERACTIONS WITHIN THE RNA SCAFFOLD, AND I'M SHOWING YOU ONE OF THEM INVOLVING RESIDUES IN THIS HAPPEN LOOP THAT INTERACT WITH A BASE PAIR HERE TO FORM A TRIPLE G35, FORM A TRIP WELTHIS BASE PAIR AND THEN THERE ARE TWO MORE INTERACTIONS MEDIATED BY DIVALIANT IONS WITH RESIDUES FURTHER AWAY IN THE SEQUENCE, U36 THE ADJACENT BASE, INTERACTS THESE TWO RESIDUES THROUGH COORDINATION VIA HYDRATED MANGANESE YUM IONS. SOCIETY RNA FALSE THROUGH A SERIES OF LONG-RANGE INTERACTIONS TO FORM THIS TOPOLOGY. I APOLOGIZE THAT THE SLIDES ARE NOT VERY CLEAR. WHAT I'M TRYING TO EMPHASIZE HERE IS THAT IN THE RNA SECOND RESTRUCTURE, THERE ARE A LARGE NUMBERS OF RESERVED RESIDUES AND THESE ARE CIRCLEED. THEY ARE ALL OVER IN THE SECOND RESTRUCTURE BUT THEY ARE BROUGHT INTO CLOSE PROXIMITY IN THE TERTIARY STRUCTURE. SO YOU CAN SEE EVEN THOUGH IN THE SECOND RESTRUCTURE THEY ARE FAR AWAY AND HYLEGGED RED AND BROUGHT INTO CLOSE PROXIMITY. YOU HAVE A JUNCTION HERE LINKING THIS RESIDUE AND THAT RESIDUE AND THIS RESIDUE AND THIS RESIDUE, AND AT THE JUNCTION WHAT WE SEE IS THAT THESE ALIGNMENTS ARE BROUGHT INTO CLOSE PROXIMITY AND MEDIATED BY A HYDRATED MAGNESIUM ION AND SUGAR HYDROXYLS ARE HYDROGEN BONDING PHOSPHOLATES ACROSS FROM THEM ON BOTH SIDES. SO MAGNESIUM IS VERY IMPORTANT AS PART OF THE STRUCTURAL COMPONENT HOLDING THE RIBOSWITCH TOGETHER. SO ONCE AGAIN, I'M SORRY YOU CAN'T SEE THESE CLEARLY, BUT THE POINT I'M MAKING HERE IS THAT OUR LAB STUDIED THE RIBOSWITCH SHOWN ON THE TOP LEFT WHERE WE HAVE A FOUR-WAY JUNCTION AND DAVID LILI'S LAB IN DUNDEE STUDIED THE SAME RIBOSWITCH BUT LACKING THE STEM LOOP AND THEREFORE CONTAINING A 3-WAY JUNCTION. IN OUR CASE, AT THE CLEAVAGE SITE, YOU HAVE THE BASES PLAYED APART. C62, A63 IN YELLOW. WHILE IN THE LILI LAB, WITH ONE LESS STEM LOOP, THESE BASES ARE STACKED ON EACH OTHER. AND AS FAR AS THE DETAILS OF THE RESIDUES INVOLVED AROUND THE CLEAVAGE SITE, IN OUR CASE, THE STEP IS C62A63. THE PHOSPHATE GROUP IS COORDINATED BY THE N ONE OF A BEGANNINE AND THROUGH A HYDRATION MAGNESIUM COORDINATED TO THE OTHER PHOSPHATE THROUGH A WATER MOLECULE. IN ADDITION, THERE IS A SECOND HYDRATED MAGNESIUM ION WHOSE WATER MOLECULE IS CLOSE TO THE TWO-PRIME HYDROXYL THAT HAS TO BE ACTIVATED. IN THIS CASE, THE HYDROXYL IS NOT IN LINE RELATIVE TO THE PO BOND. NOR IS IT IN LINE IN THE CASE OF THE DAVID LILI STRUCTURE. WHAT THEY DO SEE HOWEVER, FOR THIS C54-A55 STEP IS ALSO HYDRATED MAGNESIUM WHOSE WATER IS CLOSE TO THE TWO PRIME HYDROXYL. SO TWO DIFFERENT SISTER TWISTER RIBOZYMES ADOPTING GLOBAL VERY SIMILAR FALLS BUT IN THE CATALYTIC POCKET, ADOPTING THE œDISTINCT ALIGNMENTS. WHAT WE HAVE TO REMEMBER IS THAT THESE ARE LEAGUEY PRE-CATALYTIC CONFIRMATIONS AND WE DON'T KNOW WHAT THE TRANSITION STATE LOOKS LIKE. SO FOR INSTANCE IN OUR STRUCTURE, THE ADENINE IS ANCHORED IN PLACE BUT THE CYTOSEEN IS DIRECTED OUTWARDS SO THAT THIS NON-IN LINE CONFIRMATION COULD AS A RESULT OF THE CONFIRMATIONAL CHANGE IN THE TRANSITION STATE, ADOPT AN IN-LINE ALIGNMENT. SO WHAT WE WOULD LIKE TO DO NOW IS TO DO SOMETHING THAT THE LAB HAS DONE IN THE PAST, TO MAKE MIMICS OF THE TRANSITION STATE AND IN THOSE MIMICS SEE HOW THESE ADJACENT RESIDUES ARE ALIGNED AND WHETHER WE HAVE IN-LINE ALIGNMENT OR NOT. AND FOR OUR TWISTER SISTER RIBOZYME, WE COULD AGAIN MUTATE RESIDUES, FOR INSTANCE WE COULD MUTATE C6262 OR ADENINE 63 AT THE CLEAVAGE SITE. C62 ACCELERATES THE CLEAVAGE, THE BASE THAT IS DIRECTED OUTWARD WHEN YOU REPLACE IT BY AN ADENINE, BUT A63 WHEN IT IS REPLACED BY A URACIL, HAS NO CLEAVAGE. G5 WHOSE N1 IS DIRECTED TO THE PHOSPHATE WHEN WE PLACED BY AN ADENINE, AGAIN NO CLEAVAGE AND SO ON. NOW, I WANT TO SWITCH TO THE LAST PORTION OF THE TALK, WHICH DEALS WITH CRISPR-CAS SYSTEMS. SO THIS IS A VERY HOT AREA OF SCIENCE. WHEN A FACE EN RAGES THE HOST, IT INSERTS DOUBLE-STRANDED DNA INTO THE HOST. THAT DNA IS CHOPPED UP BY NUCLEASES AND A PIECE CALLED THE SPACER, IS THEN INSERTED INTO THE CRISPR HOST. AND IN THE CRISPR LOCUS, WE HAVE THESE SPACERS REPRESENTING PAST INVASIONS SEPARATED BY THESE REPEAT ELEMENTS. THE CRISPR DNA, CRISPR-CAS LOCUS DNA IS THEN TRANSCRIBED INTO PRE-KISS PER RNA AND THAT PRECRISPR RN SAMPLE PROCESSED IN TURN INTO MATURE CRISPR RNASs THAT CONTAIN THE SPACER AND THE REPEAT. THE MATURE CRISPR RNAs ARE THEN TAKEN UP INTO PROTEIN EFFECTER COMPLEXES WITH A PROTEINS ARE EITHER SINGLE-SUB UNIT CAS PROTEINS OR MULTISUBUNIT CASCADE PROTEINS. WHEN THE SAME PHAGE NOW INVADES THE HOST AGAIN, IT INSERTS ITS DNA, THAT DNA IS CHOPPED UP, AND IF THE SEQUENCE OF DNA MATCHES THE SPACER RNA, THE EFFECTOR PROTEIN THEN CUTS BOTH STRANDS OF THE DNA. SO FROM A STRUCTURAL BIOLOGY PERSPECTIVE, THE LABS OF JENNIFER AND OTHER IN JAPAN, HAVE LOOKED AT THE SINGLE SUB UNIT CAS PROTEINS, IN THIS CASE, CAS IS 9, AND STUDIED THEIR COMPLEXES BOUND IN THE APOE FORM, FOUND CRISPR RNA AND THEN BOUND TO DOUBLE-STRANDED DNA. WHAT IS IMPORTANT ABOUT THESE KAS PROTEINS IS THAT THEY CONTAIN TWO NUCLEASE DOMAINS, ONE OF THEM CALLED HNH AND THE OTHER ONE CALLED RUVC. SO IN THE SCHEMATIC, WHAT YOU SEE IS THE CAS PROTEIN CONTAINING A RECOGNITION LOBE AND A NUCLEASE LOBE, BOUND TO A CRISPR RNA AND SOMETIMES YOU NEED A TRANSACTIVATING RNA SHOWN IN RED HERE, AND CRISPR WITH TRANSACTIVATING WOULD FORM YOUR BINARR NARY COMPLEX AND THEN DOUBLE-STRANDED DNA WOULD COME IN, A SEGMENT OF THE DNA WOULD PAIR WITH THE CRISPR RNA TO FORM THIS D LOOP, TARGET STRAND PAIRS AND NON TARGET STRAND IS UNPAIRED. AND THEN THE NUCLEASE DOMAINS, HNH, AND THE RUVC, HNH WOULD CUT THE NON-TARGET STRAND AND RUVC WOULD CUT THE TARGET STRAND. NOW YOU NEED TO RECOGNIZE FOREIGN DNA FROM SELF-DNA AND FOREIGN DNAs HAVE A PAN SEGMENT THAT IS RECOGNIZED BY THE CAS PROTEINS. WHAT IS IMPORTANT IN THESE CANONICAL CAS PROTEINS YOU IS NEED TWO NUCLEASES, ONE TO CUT THE TARGET STRAND AND ONE TO CUT THE NON-TARGET STRAND. FURTHER, THE CLEAVAGES ARE BLUNT END. SO THE STRUCTURE BIOLOGISTS HAVE SOLVED THE STRUCTURE OF CAS9 IN THE APOE FORM TOGETHER AS A BINARY COMPLEX WITH THE RNA AND THEN WITH BINARY COMPLEXES INITIALLY CONTAINING THE PAN DUPLEX AND THE TARGET STRAND, AND IN THE LONG-TERM, ACTUALLY DELOOP STRUCTURES. SO THE MECHANISM IS AS FOLLOWS. YOU HAVE APOECAS9, THE CRISPR RNA TOGETHER WITH TRANSACTIVATING RNA BIND, WITHIN THE SPACE OF SEGMENT THERE IS A C DEVELOPMENT THAT IS ACT, THE DNA COMES IN, THE DNA IS CLEANED FROM THE PAN SEQUENCE DO SEE IF IT IS FOREIGN OR HOST. AND IF IT IS FOREIGN, AND IFINGS SEQUENCE MATCHES THAT OR COMPLEMENTARY OF THE CRISPR RNA, YOU START TO FORM A HETERODUPLEX BETWEEN THE CRISPR RNA AND THE DNA AND THE BEGINNING OF A STEM LOOP THAT IS THEN EXPANDED TO FINALLY FORM A CATALYTIC COMPETENT STATE WHERE THE TWO STRANDS ARE CUT, AND IN ALL OF THESE STEPS THERE ARE CONFIRMATIONAL TRANSITIONS THAT FOR CANONICAL CAS IS 9 HAVE BEEN DETERMINED BY STRUCTURAL BIOLOGY. NOW WE GOT INTO THIS AREA LATE AS I SAID, WHEN A NEW FAMILY OF CAS PROTEINS WERE IDENTIFIED AT THE BRODE ANDED THE NIH. AND I'LL START BY TALKING ABOUT TWO OF THEM, CPF1 AND C2C1. THEIR DOMAIN ARCHITECTURES ARE SHOWN HERE. AND THE STRIKING FEATURE OF THESE NEW CAS PROTEINS IS THEY LACK THE DID THE HNH DOMAIN BUT THEY RETAIN THE RU VC DOMAIN. AND WILL FURTHER, THEY HAD A SET OF INTERESTING PROPERTIES THAT DISTINGUISHED THEM FROM KAS PROTEINS. SO FOR INSTANCE, CPF1 REQUIRES CRISPR RNA BUT NOT TRANSACTIVATING RNA, MAKING IT A SIMPLER SYSTEM FOR GENOME EDITING. C2C1 HOWEVER, REQUIRES BOTH CRISPR AND TRANSACTIVATING RNA. I SAID THAT THESE TWO PROTEINS ALSO HAVE ANOTHER FEATURE IN THE CASE OF CPF1 THAT THEY NOT ONLY CUT DOUBLE-STRANDED RNA, BUT THEY ARE ALSO INVOLVED IN THE PROCESSING OF PRECURSOR TO MATURE RNA AND THAT CAME FROM THE MANUAL LAB. ONCE AGAIN, THESE TWO BRO TEENS HAVE A SINGLE RU VC DOMAIN, THEY LACK DNH-HNH DOMAIN YET THEY HAVE TO CUT BOTH STRANDS REMEMBER THE TARGET STRAND AND NON-TARGET STRAND. WHILE CAS9 RESULTS IN BLUNT-END CLEAVAGE, THESE TWO PROTEINS RESULT IN STAGGERED CLEAVAGE OF THE DOUBLE-STRANDED DNA. AND FINALLY, THE PAM SEQUENCES ARE DIFFERENT. IN CANONICAL CAS FINAL, THEY ARE G RICH AND PROXIMAL TO THE CLEAVAGE SITE WHILE IN THESE TWO MOW TEENS, THEY ARE DISTAL TO THE CLEAVAGE SITE. SO WE FELT THAT IT WAS AN OPPORTUNITY TO STUDY THESE NEW CAS PROTEINS WITH VERY DIFFERENT PROPERTIES FROM CANONICAL CAS9. SO I'LL FOCUS ON C2C1, THE SECOND OF THESE TWO PROTEINS. MY LAB WAS ABLE TO GENERATE THE COMPLEX OF C2C1 WITH AN RN. THAT CONTAINS BOTH THE CRISPR RNA AND THE TRANSACTIVATING RNA COVALENTLY LINKED AS A BINARY COMPLEX AND THEN ALSO AS A TERTIARY COMPLEX WITH THE DNA THAT WE HAVE THE PAM CONTAINING DUPLEX, AND THE TARGET STRAND THAT CAN PAIR WITH THE COMPLEMENTARY STRAND ON THE CRISPR RNA. AND THAT STRUCTURE WAS SOLVED LAST YEAR AND IT IS SHOWN HERE. THIS HETERODUPLEX IS IN A CAVITY WITHIN THE OVERALL SCAFFOLD. HERE IS THE PAM DUPLEX, THE CRISPR RNA IS A MAGENTA. THE TRANSACTIVATING RNA IS IN SIGH AM. SO PART OF THE TRANSACTIVATING RNA IS INVOLVED IN PROTEIN RNA RECOGNITION. OTHER ELEMENTS ARE EXPOSED. SO THIS IS THE LARGE COMPLEX. I WANT TO JUST FOCUS ON A SET OF FEATURES. MOST OF THE PROTEIN RNA CONTACTS ARE NON SEQUENCE-SPECIFIC AND I JUST WANT TO SHOW YOU ONE EXAMPLE. SO HERE IS THE HETERODUPLEX INVOLVING RECOGNITION BETWEEN THE CRISPR RNA AND THE TARGET STRAND OF THE DNA AND HERE IS THE ADJACENT PAM-CONTAINING DUPLEX. WHAT YOU SEE IS YOU DON'T HAVE STACKING ALONG ITS ENTIRE LENGTH. THERE IS STACKING HERE. IT IS BROKEN AND THEN YOU HAVE STACKING HERE. SO WHY IS THAT? SO A PORTION OF THIS DUPLEX IS SHOWN UP HERE. A PORTION OF THIS DUPLEX IS SHOWN UP -- DOWN HERE. WHAT YOU SEE IS YOU HAVE A BREAK IN THE CONTINUOUS STACKING BECAUSE AMINO ACID SIDE CHAINS ARE STACKING ON THE TERMINAL PAIRS HERE AND HERE. IN THIS CASE, THERE ARE GLUTAMINES AND FINALLY THE PHOSPHATE LINKING THESE TWO STEMS IS TARGETED BY A SIDE CHAIN AND A BACKBONE OF GLYCIN. SO IF YOU REPLACE THIS ARGININE BY AN ALANIN AND THERE IS A CLEAVAGE ASSAY GENERATING TWO STRANDS, WHAT YOU SEE IS WHEN YOU REPLACE THE ARGININE BY AN ALANIN, THE CLEAVAGE IS ATTENUATED AND WHEN YOU MAKE THE MUCH MORE DISTURBING MODIFICATION GLYCIN TO PROTEIN, THE CLEAVAGE IS COMPLETELY LOST. SO YOU GET NON-SPECIFIC INTERACTIONS BUT INTERACTION SUCH THAT YOU ARE BLAKEING CONTINUOUS STACKING BY STACKING OF THESE SIDE CHAINS ON THE TERMINAL PAIRS. ON THE OTHER HAND, THE KAS PROTEINS HAVE TO RECOGNIZE THE PAM SEQUENCE AND DISTINGUISH BETWEEN FOREIGN AND HOST. SO IN THE CASE OF C2C1, THE PAM SEQUENCE IS AT RICH T HAS TWO AT PAIRS AND A GC PAIR. SO HERE IS ONE OF THE AT PAIRS. HERE IS THE SECONDS ATP AND HERE IS SAY THIRD GC. AND NOW LAW SEE IS INSTEAD OF NON SPECIFIC INTERACTIONS, THE FIRST AT IS RECOGNIZED FROM BOTH THE MAJOR AND MINOR GROOVE. THE SECOND BY FEWER INTERACTIONS AND THE TERMINAL GC, AS SHOWN BEFORE, HAS THESE GLUTAMINE SIDE CHAINS STACKING OVER THE TERMINAL PAIR. SO THE PAM IS THE ONLY SITE WHERE YOU HAVE THESIS SEQUENCE-SPECIFIC RECOGNITIONS. AND SOME OF THE RESIDUES RESULT IN ATTENUATION OF CLEAVAGE. LITTLER LIKE R122 ARE MUTATED TO ALANIN, RESULTING IN TOTAL LOSS OF CLEAVAGE. SO A SINGLE AMINO ACID RECOGNIZING A BASE IS SUFFICIENT WHEN MUTATED TO RESULT IN LOSS OF CLEAVAGE. NOW I HAVEN'T SAID ANYTHING ABOUT THE DNA BEING POSITIONED IN THE CATALYTIC ROUGH C POCKET. AND HERE WE WERE VERY, VERY LUCKY. WHEN WE GENERATED THE COMPLEX OF C2C1 WITH RNA, AND THE DNA CONSTRUCT, WE ADDED ACCESS OF THIS STRAND. SO THE DNA CONSTRUCT CONTAINED THIS STRAND AND THIS STRAND, PAM SEQUENCE IS HERE, HETERODUPLEX FORMATION IS HERE. AND TO OUR SURPRISE, WE FOUND THE EXCESS OF THE STRAND THREADING ITS WAY INTO THE CATALYTIC POCKET. SO HERE SELL RESIDUES 3, 4, 5, 6 AND 7. THE CATALYTIC RESIDUES IN THE ROUGH C POCKET ARE SITTING HERE AND WE SEE AN EXTRA SULPHATE. SO HERE ARE STEPS 4 AND 5 COMING FROM THIS EXCESS RNA STRAND, POSITIONED IN THE CATALYTIC POCKET. IN ORDER TO FORM THE CRYSTALS, WE HAD TO MUTATE ALL THREE ACIDIC RESIDUES ALANIN. WE JUST MODELED THE SIDE CHAINS HERE. THERE IS ALSO A TYROSINE AND THEN AN ARGININE AND ASAREINE FORMING THIS PARTICULAR CATALYTIC POCKET. SINCE WE KNOW THESE RESIDUES, WE CAN MOVEITATE THEM AND THE WILDTYPE REPLACES ANY OF THESE CATALYTIC RESIDUES BY ALANIN, CLEAVAGE IS COMPLETELY LOST. AND FOR THE OTHER RESIDUES, CLEAVAGE IS EITHER LOST OR PREDOMINANTLY LOST. SO IS THIS WAS VERY INTERESTING TO US, THIS EXTRA STRAND WAS POSITIONED IN THE CATALYTIC POCKET AND THERE WAS ALSO A SULPHATE. SO THE LAST TWO BASE PAIRS OF THE HETERODUPLEX ARE SHOWN HERE AND HERE IS THE FIVE-PRIME END OF THE TARGET DNA STRAND AND THIS EXTRA STRAND HAS THE THREE-PRIME END, A SULPHATE IN BETWEEN. SO YOU COULD POSSIBLY LINK THESE. AND THAT IS EXACTLY WHAT WE DID. WE MADE ANOTHER COMPLEX WHERE WE EXTENDED THE TARGET STRAND. AND NOW WHAT YOU SEE IS HERE IS THE HETERODUE PUBLIX AND THE EXTENSION NOW IS FORMING A TURN UP AND IT'S POSITIONING THE PHOSPHATE THAT HAS TO BE CLEAVED IN THE CATALYTIC POCKET. SO HERE IS THE END OF THE DUPLEX, HERE IS THE EXTENSION, AND THE EXTENSION IS INVOLVED A LARGE -- THAT WAS MY WORRY. VOLVES A LARGE NUMBER OF HYDROGEN AND STACKING INTERACTIONS POSITIONING -- IF SOMEBODY COULD GET ME AN UPDATED POINTER, PLEASE. POSITIONING THE CATALYTIC RESIDUES SHOWN IN STARS OPPOSITE THE PHOSPHATE THAT HAS TO BE CLEAVED. SO THAT IS SUMMARIZED IN THIS SLIDE ON THE UPPER LEFT IS THE STRUCTURE CONTAINING THE EXCESS STRAND. ON THE UPPER RIGHT IS THE OVER HANG AND WHAT CAN YOU SEE IS THAT THEY BOTH POSITION IN THE SAME WAY NOW POSITIONING A PARTICULAR PHOSPHATE OPPOSITE THE CLEAVAGE SITE. WHEN WE DO BIOCHEMICAL ASSAYS, AND THE LOWER PANEL WHERE THE TRIANGLES ARE IS WHERE THE CLEAVAGE OCCURS AND FOR THE TARGET STRAND, THE CLEAVAGE THAT WE SEE BIOCHEMICALLY, IS EXACTLY THE CLEAVAGE THAT WE SEE STRUCTURALLY. THANK YOU. SO THE CLEAVE AMAGE SITE IS HERE AND THE EXTRA STRAND AND CLEAVAGE SITE IS HERE AND WE HAVE THE OVER HANG. SO WE SAID OKAY, WHY DON'T WE TRY AND HAVE AN OVER HANG IN THE NON-TARGET STRAND SHOWN ON THE UPPER RIGHT? NOW WE USED THE STRETCH OF Ts BECAUSE WE DIDN'T WANT TO HAVE THE NON-TARGET STRAND PAIR THE TARGET STRAND AND COMPETE WITH PAIRING WITH THE CRISPR STRAND, CRISPR RNA STRAND. WHEN WE SAW THIS STRUCTURE SHOWN ON THE UPPER LEFT, WE COULD SEE A STRETCH OF Ts SITTING IN THE CATALYTIC POCKET BUT BECAUSE THEY WERE ALL Ts, WE COULDN'T SAY WHERE THE CLEAVAGE SITE WAS OCCURRING, JUST SAID THERE IS SAY PHOSPHATE THAT IS POSITIONED IN THE CATALYTIC POCKET. WE TRIED TO INTRODUCE NON-T SEQUENCES BUT UNFORTUNATELY THE CRYSTALS HAVEN'T FORMED. BIOCHEMICALLY, THEY OCCUR ON THE NON TARGET STRAND WHERE THE RED ARROW IS SHOWN. SO WHAT IS VERY INTERESTING ABOUT THIS SYSTEM IS THAT YOU HAVE ONE RUVC POCKET AND IT CLEAVES BOTH THE TARGET AND THE NON-TARGET STRANDS. THAT THE STAGE, WE DON'T KNOW WHAT THE SEQUENTIAL ORDER OF CLEAVAGE IS. NOW, SINCE WE DID THE BINARY COMPLEX AND ALSO THE TURNIARY COMPLEX, WE CAN COMPARE THE TWO STRUCTURES AND ONE INTERESTING FEATURE WAS THE CRISPR RNA IN WHAT WE CALL THE SEED SEGMENT. IN THE BINARY COMPLEX WHERE THERE IS NO DNA, ON THE LEFT YOU SEE RESIDUES 1, 2, 3, 4 AND 5. ON THE CRYSTAL RNA. AND THEN THE REST OF THE CRISPR RNA GRADE IS DISORDERED. IN THE TURNIARY COMPLEX PAIRING WITH THE DNA, YOU HAVE A DUPLEX, YOU CAN FOLLOW THE CRISPR RNA SHOWN ON THE RIGHT BOX PANEL ALONG ITS FULL-LENGTH. BUT WHEN YOU LOOK AT THE FIGURE SHOWN BELOW, IN SILVER IS THE CRISPR RNA AND THE BINARY COMPLEX AND IN MA GENTSA IS THE CRISPR RNA IN THE TERTIARY COMPLEX. WHAT YOU SEE IS A TRYPTOPHAN RESIDUE PACKS AGAIN BASE 5 IN THE BINARY COMPLEX, PREVENTING FURTHER STACKING OF THE CRISPR RNA, BUT IN THE TURNIARY COMPLEX IN MAGENTA, THE TRYPTOPHAN MOVES OUT OF THE WAY AND NOW YOU CAN FOLLOW THE CRISPR RNA ALONG THE FULL-LENGTH. AND FEATURES LIKE THESE WERE ALSO SEEN IN AGERRA NON PROTEINS. THE LAST COMPARISON I WANT TO MAKE IS THAT WE STUDIED NOT ONLY C2C1 ON THE LEFT BUT ALSO CPF1, THE OTHER NEW NUCLEASE. C2C1 HAS TRANSACTIVATING RNA. CPF1 LACKS IT. AND THE CONFIRMATIONAL CHANGE THAT IS WE SEE IS FROM BINARY TO TERNEIARY FOR THESE TWO DOMAINS, ARE MUCH SMALLER FOR C2C1 THAT HAS THE TRANSACTIVATING RNA THAN THE CPF1 THAT LACKS IT. SO TO TRANSACTIVATING RNA MAKES THE OVERALL FOLD MUCH MORE COMPACT RESULTING IN MUCH SMALLER CONFIRMATIONAL CHANGES. I REALLY NEED THE POINTEDDER. IF SOMEBODY CAN HELP OUT. IT'S VERY WEAK. I CAN BARELY SEE IS IT. NOW OF COURSE, PHAGES DO NOT WANT THE CRISPR-CAS SYSTEM TO CLEAVER THE DOUBLE-STRANDED DNA SO THEY EVOLVE PROTEINS AND ANTI-CRISPR PROTEINS THAT TARGET THE CRISPR PATHWAY. AND SEVERAL OF THESE ANTI-CRISPR PROTEINS HAVE NOW BEEN IDENTIFIED AND CAN BE STUDIED STRUCTURALLY. SO, NOW I'M LOOKING AT THE SPY CANONICAL CAS9 TARGETED BY SEVERAL ANTI-CRISPR PROTEINS. AND I WANT TO FOCUS ON ONE OF THEM, ACR2A4. SO WHAT DOES THE ANTI-CRISPR PROTEIN TARGET? DOES IT TARGET APOCAS9? THE BINARY COMPLEX OUTEARNIARY COMPLEX WITH BODGED DOUBLE-STRANDED DNA? IN THESE PULL DOWN EXPERIMENTS, WE CAN SHOW THAT THE INHIBITOR DOES NOT BIND APOECAS9 NOR THE TERTIARY COMPLEX BUT BINDS THE BINARY COMPLEX CONTAINING THE RNA. YOU'RE ABLE TO SOLVE THE CRYSTAL STRUCTURE OF THIS INHIBITOR SHOWN ON THE LEFT SIDE BOTH IN THE RIBBON VIEW AND IN THE SURFACE VIEW. FOLLOWING THAT, WE TOOK SPCSA9, DOMAIN ARCHITECTURE IS SHOWN ON TOP, THE R ARE NA CONTAINS CRISPR RNA AND TRANSACTIVATING RNA AND SOLVED THE STRUCTURE OF THE BINARY COMPLEX BOUND BY THIS. AND IN THE LOWER RIGHT, YOU SEE THE INHIBITOR CIRCLE BINDING TO THE ONE SURFACE OF CAS9 AND THIS WAS DONE IN MY LAB AND IN A LAB IN CHINA. [ CELL PHONE RINGING ] SORRY. EVEN THIS IS WEAK. JUST REPLACE THE BATTERIES ON THE OTHER ONE. SO WHAT YOU SEE IS AN INHIBITOR BINDING TO A SPECIFIC SITE IN THE COMPLEX OF CAS9 BOUND TO LITTLE RNA. AND WHAT IS VERY INTERESTING ABOUT THIS INHIBITOR IS THE BINDING SITE OF THE INHIBITOR IS EXACTLY WHERE THE PAM SEGMENT OF THE DOUBLE-STRANDED DNA TARGET BINDS. SO BASICALLY, THE INHIBITOR IS AINCLUDING THE BINDING SITE FOR RECOGNITION OF THE PAM DOMAIN. BUT AS EQUALLY INTERESTING THERE IS SAY LOOP ON THE INHIBITOR, WHICH PROJECTS OUT AND ENTERS THE CATALYTIC POCKET. WHAT WE ARE SEEING HERE IS THIS LOOP SEGMENT ENTERING AND POSITIONING ITSELF OPPOSITE THE CATALYTIC RESIDUES. SO THIS SEEMS TO HAVE TWO FUNCTIONS. IT OCCLUDES THE PAM SITE AND ALSO INSERTS A LOOP INTO THE CATALYTIC POCKET. SO THUS PREVENTING BOTH DOUBLE-STRANDED DNA BINDING AND EVEN IF THE DOUBLE-STRANDED DNA BOUND, IT WOULD NOT HAVE ACCESS TO THE CATALYTIC POCKET. DOES THE BINDING OF THE INHIBITOR RESULT IN A CONFIRMATIONAL CHANGE IN THE CAS9? WHEN WE GO FROM THE RNA-BINARY COMPLEX AND ADD DOUBLE-STRANDED DNA, THERE ARE VERY LARGE CONFIRMATIONAL CHANGES IN CAS IS FINAL. BUT IF YOU ADD THE INHIBITOR TO THE DNA COMPLEX, THERE IS VERY SMALL MINOR CHANGE LESS SUGGESTING THE ARCHITECTURE OF THE RNA-BOUND CAS9 IS LOCKED IN PLACE AND THE INHIBITOR BINDS IT WITHOUT ADDITIONAL CONFIRMATIONAL CHANGES. THERE IS A FIELD MOVING. WHAT IS THE EXCITING CHALLENGE IN THE FUTURE? THERE WAS A THIRD CAS IDENTIFIED CALLED C2C2. IN THE LAB LATER ON IN ANOTHER LAB IT WAS SHOWN THAT THERE IS NOT ONLY INVOLVED IN TARGET CLEAVAGE, BUT ALSO IN PRECURSOR TO MATURE CRISPR RNA PROCESSING. THIS ENZYME HAS A TOTALLY DIFFERENT ARCHITECTURE. IT HAS THESE ATP DOMAINS, A PAIR OF THEM. THEY ARE BASIC CATALYTIC DOMAINS. AND WHAT IS VERY INTERESTING ABOUT C2C2 IS THAT IT DOES NOT CLEAVE DOUBLE-STRANDED DNA BUT IT CLEAVES RNA. SO NOW WE HAVE A CAS NUCLEASE THAT CLEAVES RNA AND IT CLEAVES IT IN A PROMISCUOUS MANNER. SO THE CLEAVAGE SITES CAN OCCUR IN SINGLE-STRANDED REGIONS AND IN STEM LOOPS. SO A POSTDOC IN MY LAB WHO DID ALL THE WORK IN THE LAB HAS HER OWN LAB AT THE INSTITUTE OF BIOFACE NIX BEIJING AND THIS YEAR, SHE REPORTED ON THE STRUCTURE OF C2C2 BOUND TO MATURE CRISPR RNA. AND AS YOU CAN SEE, THE ARCHITECTURE IS EXTREMELY DIFFERENT FROM CANONICAL CAS PROTEINS. AS FAR AS THE RNA AND THE COMPLEX IS CONCERNED, THEY CAN SEE A STEM LOOP REPEAT BUT THE SPACE ONLY A SMALL SEGMENT OF THE SPACER CAN BE OBSERVED. MOST TESTIFY IS DISORDER MOST OF IT IS DISORDERED IN THE BINARY COMPLEX AND THE TWO DOMAINS ARE THE SEPARATED IN THE SECOND FOLD COMING TOGETHER IN THE TERTIARY FOLD BUT ONE SET OF CATALYTIC RESIDUES HERE AND THE SECOND SET HERE. SO THE CHALLENGE NOW IS TO MEET THE TERTIARY COMPLEXES WITH TARGET RNAs AND SEE IF THEY AND WHAT ARE THE OVERALL CONFIRMATIONAL CHANGES THAT WILL OCCUR AS ONE GOES FROM THE BINARY TO THE TERTIARY COMPLEX. NOW THIS WORK WAS DONE BY VERY TALENTED POSTDOCS, AMY WHO WORKED ON RIBOSWITCHES AND RIBOZYMES AND HAS HER OWN LAB NOW BACK IN CHINA. AND THE CRISPR-CAS WORK WAS DONE BY YANG, WHOSE CURRENTLY IN THE LAB. AND I WANT TO END BY VERY BRIEFLY PRESENTING ONGOING WORK WITH THE LAB AT THE NCI. AND I HAVE TALKED ABOUT CAS PROTEINS AS EFFECT OPPOSITE, SINGLE DOMAIN PROTEINS BUT HERE I'M TALKING ABOUT CASCADE. ANOTHER EFFECTOR WHICH INVOLVES A MULTI-SUB UNIT COMPONENT. SO HERE I'M SHOWING YOU A TYPE I F KAS KATE CADE CONTAINING A SPINE OF SIX, 7.6 SUBUNITS PLUS A 6F SUBUNIT, A 5 AND 8F SUB UNIT AND HERE IS THE CRISPR RNA WITH THE SEED SEGMENT, 5 PRIME END WITH STEM LOOP AT 3 PRIME END. SO THE PROTEIN IS MUCH LARGER. IT'S AMENABLE TO STUDY BY BOTH CRYSTALLOGRAPHY AND CRYO-EM. AND IF YOU'RE INTERESTED IN ANTI-CRISPR PROTEINS THAT TARGET CASCADE, CRYSTAL GRAPHIC EFFORTS DIDN'T WORK AND SO WE TURNED TO WORK ON THE CRYO-EM STRUCTURES. AND TO DATE, WE HAVE LOOKED AT THREE ANTI-CRISPR PROTEINS CALLED 1, 2 AND 10. FOR 1 AND 10, WE HAVE THE X-RAY STRUCTURES OF THE ANTI-CRISPR PROTEINS. AND WORKING WITH ANOTHER LAB, WE HAVE SOLVED IN THIS PARTICULAR VIEW, THE STRUCTURE OF CASCADE BOUND TO TWO ACRF1 ANTI-CRISPR PROTEINS. AND WHAT YOU SEE IS THAT THE ANTI-CRISPR PROTEINS ARE TARGETING THE BINARY COMPLEX OF CASCADE WITH THE CRISPR RNA AT SITES WHERE THE CRISPR RNA WOULD PAIR WITH THE TARGET DNA. SO BASICALLY, ACR ARE F1, WITH THESE TWO BOUND, IS OCCLUDING BINDING OF THE NOVEL EXPANDED DNA AND THEREFORE PREVENTING CLEAVAGE OF THE INVADING PHAGE DOUBLE-STRANDED DNA. WHAT ABOUT 2 AND 10? HERE IS ACRF2 BINDING HERE. HERE IS ACRF10 BINDING HERE. SO 2 AND 10 ARE BINDING AT SITES WHERE THE PAM SEGMENT OF THE DOUBLE-STRANDED DNA WOULD BE POSITIONED ON THE CASCADE SCAFFOLD. SO BOTH INHIBITORS ARE TARGETING THE SAME SITE BUT BECAUSE THESE ARE DIFFERENT PROTEINS, THEY ARE DOING SO IN A DIFFERENT MANNER. SO OVERALL, HERE IS THE CASCADE SCAFFOLD BOUND WITH CRISPR RNA SHOWN IN ACRF1, TWO MOLECULES ARE TARGETING A FACE INVOLVING RECOGNITION OF THE SPACER ELEMENT OF THE RNA WITH THE DOUBLE-STRANDED DNA. SO THEY ARE OCCLUDING BINDING OF DNA AND ACRF2 AND 10 ARE OCCLUDING BINDING OF THE PAM SITE ON THE DNA. THERE ARE MORE INHIBITORS OF THIS FAMILY AND THESE HAVE TO BE STUDIED TO SEE IF THEY BIND BY COMMON PRINCIPLES OR USING DIFFERENT PRINCIPLES. ED AND I ASKED FOR A SLIDE OF HIS LAB. WE HAVE COMPETITION IN THIS AREA. SO HE PUT A LOT OF MEMBERS OF HIS TEAM ON THE PROJECT. AND A PRE-MD STUDENT IN HIS LAB WE HAVE OUR MOST INTERACTIONS WITH. SO I'LL STOP HERE. AND I HOPE I HAVE GIVEN YOU AN IDEA USING RIBOSWITCHES, A FEELING FOR RNA ARCHITECTURE, FOR RNA POCKETS, AND HOW LIGANDS ARE RECOGNIZED IN THESE POCKETS, AND DISCRIMINATED AGAINST, ABOUT CONFIRMATIONAL TRANSATIONS WHEN LIGANDS BIND, AND THEN IN THE CASE OF RIBOZYMES, SMALL RNAs THAT CONTAIN SPECIFIC SITES FOR CLEAVAGE WHERE WE CAN LOOK AT THE ARCHITECTURE OF THOSE SITES, LOOK FOR PLAYED DEPART ALIGNMENTS FOR BASES THAT COULD BE ALIGNED INVOLVED IN ACTIVATION AND STABILIZATION OF THE CLEAVAGE STEP. AND FINALLY, IN THE CRISPR-CAS FIELD, WE HAVE WORKED WITH THESE NEW NUCLEASE THAT IS APPEARED IN THE LAST YEAR OR SO THAT INVOLVES A STAGGERED CLEAVAGE, A SINGLE NUCLEASE POCKET AND STUDY BOTH BINARY AND TERTIARY COMPLEXES AND LEARN SOMETHING ABOUT HOW THE CATALYTIC POSSIBILITIES FUNDS AND HOW THE CLEAVAGE SITES OCCUR AND SO ON. AND FINALLY, WE ARE INTERESTED IN ANTI-CRISPR PROTEINS THAT TARGET BOTH CASCADE AS WELL AS CAS AND WE HAVE USED BOTH X-RAY CRYSTALLOGRAPHY AND CRYO-EM TO ADDRESS THESE ISSUES AND TRY AND UNDERSTAND HOW THESE ANTI-CRISPR PROTEINS FUNCTION TO OCCLUDE DNA AT THE HETERODUPLEX LEVEL OR DO THEY OCCLUDE RECOGNITION OF THE PAM SEQUENCE OR DO THEY OCCLUDE ACCESS TO THE CATALYTIC POCKET? THANK YOU VERY MUCH. >> THERE IS TIME FOR ONE OR TWO QUESTIONS. ELSE WE MOVE TO THE LIBRARY. AND YOU CAN DISCUSS WITH HIM THERE. >> THANK YOU. VERY ELEGANT STUDIES. MY MIND IS USUALLY ON THE APPLICATION OF ALL OF THESE STRUCTURAL AND MECHANISM OF RNA-DNA MOVEMENT OR ARCHITECTURE HAVE YOU NOTICED ANY CHANGES IN ENERGY TRANSFER WHEN YOU SUBSTITUTE ONE MOL DUAL FOR EXAMPLE, IF YOU SUBSTITUTE GLUTAMINE FOR ARGININE AND/OR -- ANOTHER CHALLENGED MOLECULE, WHICH IS OCCUPYING A SPACE AND IT HAS TO BE SOMEWHAT ENERGY-INVOLVED, PLUS HAVE YOU NOTICED -- MAYBE IT'S NOT FAIR TO ASK YOU, BUT HAVE YOU NOTICED WHEN YOU HAVE CONFORMATION AND DECONFIRMATION STATUS OF THE RNA? THAT THERE IS A CHANGE IN ENERGY DYNAMICS? >> LET ME TRY AND ANSWER YOUR QUESTION THE FOLLOWING WAY. THE GLUTAMINE RIBOSWITCH IS VERY SPECIFIC FOR GLUTAMINE. YOU CAN NOT REPLACE IT BY ANY OTHER AMINO ACID. YOU CAN'T EVEN REPLACE BY L GLUTAMATE. SO ONE INTERESTING QUESTION IS, HAVE YOU THE STRUCTURE OF THE GLUTAMINE RIBOSWITCH. CAN YOU MAKE CHANGES EITHER IN THE POCKET OR FAR FROM THE POCKET SO THAT THE GLUTAMINE RIBOSWITCH WOULD BIND GLUTAMTE? WHICH HAS A CHARGE? AND THAT IS NOT STRAIGHTFORWARD BECAUSE WORK WE HAVE DONE EARLIER TELLS US THAT THE CHANGES THAT OCCUR NOT ONLY OCCUR IN THE CATALYTIC POCKET, OR BINDING POCKET, BUT ALSO FAR AWAY FROM THE BINDING POCKET. SO WE LOOKED AT A PURINE RIBOSWITCH WITH AND WITHOUT A SUGAR ATTACHED TO IT. AND THE CHANGES WERE NOT ONLY IN THE CATALYTIC POCKET BUT THEY WERE ALSO FAR FROM THE CATALYTIC POCKET SO IT IS VERY DIFFICULT TO TRY AND DAIC A STRUCTURE AND RELEVEL THE POCKET TO BIND THE CLOSE ANALOGUE. IT'S VERY DIFFICULT. >> WHAT ABOUT IN THE PRESENCE OF YOUR INHIBITORS? >> YOU'RE TALKING OF THE ANTI-CRISPR 1234. >> RIGHT. >> THAT WORK IS VERY NEW. AND SINCE THEY ARE PROTEINS, WE COULD LOOK AT THE INTERFACE AND REPLACE AMINO ACID CHANGES TO CHANGE THE ELECTROSTATICS AND SEE WHAT THE CONSEQUENCES ARE. AND IT IS ALL VERY NEW AND WE HAVEN'T DONE ANYTHING ALONG THOSE LINES. >> THANK YOU. >> SO LET'S MEET IN THE LIBRARY. [ APPLAUSE ]