>> WELCOME TO THE BIOWULF SEMINAR SERIES. OUR SPEAKER IS SAGAR CHITTORI FROM THE LAB OF CELLULAR BIOLOGY AT NCI. SAGAR GRADUATEDED FROM THE INDIAN INSTITUTE OF SCIENCE AND CAME TO THE NIH AS POST DOC IN 2012 WHERE HE WAS WORKED WITH MARK MAYER'S GROUP. THREE YEARS AGO HE MOVED TO NCI AND TODAY HE'S GOING TO SPEAK ABOUT CryO-EM STUDIES OF GLUTAMATE RECEPTORS IN NUCLEOSOMES. >> THANK YOU. THE BIOWULF TEAM FOR THE INVITATION. I'M PLEASED TO PRESENT MY WORK HERE TODAY. SO CryO-EM FIELD IS REALLY EXPANDING SO I THOUGHT I WOULD START WITH SOME NUMBERS AND SO WHAT I'M SHOWING HERE IS A CHART WHICH HAVE -- IT SHOWS HOW MANY MAPS ARE RELEASED BY THE -- YOU SEE THE NUMBERS ARE ACTUALLY INCREASING A LOT, IN THE LAST FEW YEARS. YOU SEE A LOT OF MAPS NOW BEING RELEASED DEPOSITED IN THE BANK AND WHEN YOU LOOK AT THIS -- MOST AFT MAPS ARE -- PARTICLE, THE ONE I DID ON GLUTAMATE RECEPTORS AND NUCLEOSOMES ARE ACTUALLY COMES FROM SINGLE PARTICLE PROTEINS. CryO-EM IS NOW ACTUALLY GIVING RESOLUTIONS VERY CLOSE TO NEAR RESOLUTIONS, THE RESOLUTION IS STILL ABOUT -- STILL LOWER THAN -- YOU HAVE STRUCTURES BEING DEPOSITED CLOSE TO -- EVERY YEAR. THEN SINCE YOU HAVE HIGHEST RESOLUTION MAPS, THAT MEANS THAT A LOT OF MODELS NOW DEPOSITED IN THE PROTEIN DATA BANK, SO THIS IS -- HERE IS A GRAPH THAT SHOWS NUMBER OF STRUCTURES WHICH ARE AVAILABLE IN PROTEIN DATA BANK THAT WERE DERIVED FROM THE -- SO ORANGE COLOR HERE SHOWS NUMBER OF DEPOSITED AND THE BLUE COLOR SHOWS HERE THE TOTAL AVAILABLE IN THE EM DATA BANK. THE 2018 IS STILL GROWING AND IT WILL GO UP FURTHER AS WE MOVE FORWARD IN THIS YEAR. SO HERE IS MY OUTLINE FOR TODAY. I WILL BE GIVING A ONE SLIDE BRIEF OVERALL ABOUT PARTICLES SINGLE PARTICLE CryO-EM. AND THEN I WILL TOUCH UPON THE WORK WE DID ON GLUTAMATE RECEPTOR, I WILL HAVE A BRIEF BACKGROUND THEN PRESENT THE MEAN STRUCTURAL RESULTS FROM THESE STUDIES BUT THESE STUDY WAS DONE IN COUPLE OF YEARS AGO AND THE FIRST -- IN FACT THE FIRST WE STARTED THIS IN AROUND 2012 AND SO THIS IS MORE -- THIS WORK WAS DONE PREVIOUS TO THE WORK WE DID ON NUCLEOSOMES WHICH WAS DONE RECENTLY SO I WILL BE TOUCHING AND PROVIDING MORE DETAILS ON WHAT WE DID ON NUCLEOSOME COMPLEX. AND THIS STRUCTURE WE ALSO BUILD STRUCTURE OF THE -- SO WHEN WE WERE DOING THIS COMPLEX STRUCTURE WE DID NOT HAVE ANY STRUCTURE PERMISSION FOR THE COMPLEX PART WHICH IS THE KINETOCHORE PROTEIN. SO WE USE THE CENP-N MAP. WE ALSO HAD VALIDATION OF THE MODEL USING SOME PERSONAL DATA THAT WE DID IN COLLABORATION IN THE LAB AT NCI. SO LOOK AT THE CryO-EM WORK YOU CAN DIVIDE THAT INTO PRE-AND POST MICROSCOPIC SESSION. SO THE PRE-MICROSCOPIC SESSION WILL INCLUDE PROFESSIONAL -- THIS IS YOU ALREADY HAVE PROTEIN IDENTIFIED AND IS READY TO BE USED TO MAKE GRADES. SO HERE I'M SHOWING A GRID, YOU APPLY YOUR PROTEIN MOLECULE ON THIS SOLUTION OF -- SOLUTION WHICH MOLECULES AND THEN YOU BLOCK THIS USING A PIECE OF PAPER AND IMMEDIATELY YOU DO A -- LIQUID NITROGEN TEMPERATURE AND THIS CAN BE STORED IN LIQUID NITROGEN FOR A LONG TIME SO YOU CAN MAKE THESE GRIDS AHEAD OF TIME AND THEN TAKE THIS GRID WHENEVER YOU HAVE THE SCOPE TIME AVAILABLE AND LOOK AT THE MICROGRAPHS. SO THIS IS LIKE A SCHEMATIC EXAMPLE OF A MICROGRAPH, HOW A MICROGRAPH LOOKS LIKE SO IF YOU'RE LUCKY YOU WILL SEE FROM TEEN MOLECULES RANDOM ILY ORIENTED. YOU'RE SEEING IS THE PROJECTIONS OF THOSE BIOMOLECULES. SO NEXT STEP IS TO USE COMPUTATIONAL POWER TO TRY TO SELECT PARTICLES WHICH LOOK ALIKE AND SOLVE THEM INTO DIFFERENT GROUPS DEPENDING HOW THEY LOOK. WHEN YOU COMBINE THAT INFORMATION AND THEN AGAIN USE COMPUTATIONAL PROCESSING TO GET THROUGH THE DENSITY OF THE MAP. I WOULD LIKE TO EMPHASIZE HERE THIS IS ALL POSSIBLE BECAUSE WE DO HAVE VERY HIGH COMPUTING CLUSTER HERE. THE SISTER SIGNAL IS PRETTY LOW EM IMAGES, IT REQUIRES THAT WE SHOULD HAVE VERY GOOD CLUSTER AND ALSO A VERY GOOD SOFTWARE SUPPORT SO THERE ARE A LOT OF PROGRAMS WHICH ARE COMING -- WHICH HAVE DEVELOPING ON ALMOST ON DAILY BASIS SO WE ALSO HAVE TO KEEP OURSELVES UPDATED WITH THE DIFFERENT PROGRAMS THAT ONE CAN USE. I WOULD LIKE TO THANK BIOWULF FOR MAINTAINING THESE ASPECTS OF OUR WORK. SO I'LL COME TO NOW THE WORK WHICH WE DID ON GLUTAMATE RECEPTOR AND I WILL SHOW EXAMPLE OF HOW WE USE EM DENSITY, ANALYSIS TO DERIVE FUNCTIONAL INFORMATION ABILITY THE GLUTAMATE RECEPTOR. HERE IS A LITTLE BIT OF BACKGROUND ON GLUTAMATE RECEPTORS. THESE ARE LIGAND CHANNELS THAT ARE PRESENT AT SYNAPSES IN THE BRAIN. AND THEY BIND TO THE GLUTAMATE WHICH IS THE MOST ABUNDANT EXCITATORY NEUROTRANSMITTER AND THEN YOU LOOK AT THE HUMAN GENOME YOU WILL FIND THERE ARE ABOUT 18 GENES FOR THE GLUTAMATE RECEPTOR THAT CAN BE BROADLY CLASSIFIED INTO SEVEN GENE FAMILIES BUT BASICALLY THEY ARE ON BROAD LEVEL BASICALLY DIVIDING TO TWO FAMILY CALLED NMDA AND NON-GLUTAMATE RECEPTORS SO THE IMPACT ON DATA RECEPTOR PART OF THE RECEPTOR MORE CLOSELY RELATED IN TERMS OF STRUCTURE AND FUNCTION. SO IN MY TALK I WILL BE TALKING ABOUT THE RECEPTOR FROM AMPA FAMILY AS WELL AS ONE RECEPTOR FROM THE FAMILY CALLED IGlRSTHE GLU BELONGS TO HAVE A VERY COMMON ARCHITECTURE SO YOU HAVE A DOMAIN FOLLOWED BY LIGAND BINDING DOMAIN WHICH CHANNEL THAT'S OPENING. BUT IF YOU LOOK AT -- IF YOU FOLLOW THE POLYPEPTIDE CHAIN HERE YOU WILL NOTICE IT THE DOMAIN IS CONTINUOUS SO THE POLYPEPTIDE FINISHES THE DOMAIN AND THEN IT GOES AND FORMS THE ONE DOMAIN SUBDOMAIN OF THE LIGAND BINDING LAYER AND THEN IT ACTUALLY GOES TO THE TRANSMEMBRANE DOMAIN AND FORMS THESE THREE HELICES AND GOES BACK AGAIN TO FORM THE LUMEN OF LIGAND BINDING LAYER AND ULTIMATELY BACK TO THE -- SO THIS DOMAIN IS CONTINUOUS WHILE YOU'RE -- HAS THE POLYPEPTIDE MOVE BACK AND FORTH AND IT IS IMPORTANT BECAUSE IT IS -- AMPA 3 IS THE HELIX WHICH SURROUNDS THE ION CHANNEL PORE AND HAS THIS MOTIF AT THE END OF THE HELIX. WHICH IS INVOLVED IN THE GATING OF THE CHANNEL. SO BEFORE WE STARTED THIS STRUCTURAL WORK ON GLUTAMATE RECEPTOR THERE WERE A LOT OF STRUCTURES WHICH WERE AVAILABLE FOR THE ISOLATED DOMAIN AS WELL AS ISOLATED LIGAND BINDING DOMAINS BUT THE FIRST STARE FOR THE RECEPTOR COMES FROM -- LAB IN 2009. SO THOUGH THERE WAS A LOT OF INFORMATION ON THE ISOLATED DOMAIN THE RECEPTOR SHOWED THAT HOW THIS DOMAINS ARE ARRANGED IN THE CONTEXT OF THE DEBT TROUGHMERIC GLUTAMATE RECEPTOR AND ALSO INTERESTINGLY AS YOU WILL SEE THERE IS A DIME HE WERE OF DIMERRING LAYER AND DIMER OF DIMER IN THE LIGAND BINDING DOMAIN SO HERE THE TWO DIMERS ARE ARRANGED, AND THESE ARE ARRANGED BUT LOOKING CAREFULLY YOU REALIZE THE DIMER COMPOSITION IS DIFFERENT SO HERE COMES DIMER LBD LAYER THERE IS A CHANGE, IT ACTUALLY NOW FORMS DIMER WITH A D. SO THERE IS A SUBUNIT CROSS OVER AS YOU CAN SEE THAT SUBUNIT HERE ACTUALLY MOVES FROM MOVES OVER FROM THE GREEN AND MAKESES IT APPEAR WITH THE BLUE ONE IN THE LBD LAYER SO IT WAS INTERESTING AND COMPLETELY UNANTICIPATED BEFORE THE RECEPTOR WAS AVAILABLE. ANOTHER INTERESTING THING IN THIS STRUCTURE WAS THAT IT FORMS TWO FOLD SO LIKE A DIMER AND DIMER AND RELATED BY TWO POLE SO IF YOU MOVE BY ONE DEGREE YOU GENERATE THE SAME STRUCTURE AGAIN. THAT'S THE SAME THING FOR THE LIGAND BINDING DOMAIN BUT WHEN YOU COME TO THE TRANSMEMBRANE DOMAIN IT WAS ARRANGED AS SYMMETRY INSTEAD OF TWO FOLD SYMMETRY THAT WAS SHOWN IN THE TERMINAL AS WELL AS LIE GAPPED BINDING DOMAIN. SO -- LIGAND BINDING DOMAIN SO IT REINFORCED THE LINK WHICH CONNECTS THESE LAYERS ALSO PLAYING A CRUCIAL ROLE IN SYMMETRY TRANSITION OR WHETHER IT IS IN SUBUNIT CROSS OVER. SO I'M CUTTING SHORT HERE THE BIOCHEMISTRY PART, I SPENT -- I DID BIOCHEMISTRY ON THIS BUT THOUGHT IT BE MORE APPROPRIATE TO DO STRUCTURE WORK THAN BIOCHEMISTRY SO I'M JUST BRIEFLY SAYING HOW WE PRODUCE THIS RECEPTOR SO I DID THIS BIOCHEMISTRY WORK WITH MARK MAYER IN BUILDING 35. SO WE USED VIRUS MEDIATED PROTEIN EXPRESSION IN INSECT CELL LINES. AND THEN WE USED THIS EXPRESS PROTEIN AND SOLUBILIZE USING -- AND THEN USED AFFINITY CHROME -- CHROMATOGRAPHY WHICH WAS STILL USING DIFFERENT LIGANDS SO IF YOU WANT TO STUDY A WE USE ANTIBODIES TO MAKE SURE FAMILIES CLOSE AND IS HOMOGENOUS, THAT KIND OF SAMPLE WENT FOR THE CryO-EM STUDY. SO I JUST PULLED UP A FIGURE FROM THE PAPER THAT WAS PUBLISHED, SO IT WAS THE FIRST PAPER PUBLISHED FROM COLLABORATION THAT WAS FORMED BETWEEN MARK MAYER'S LAB IN BUILDING 35 AND THE LAB IN BUILDING 50 AND I WAS NOT INVOLVED IN THIS PROJECT. THIS SHOWS HERE IS THE ANALYSIS TO SHOW THE PURITY OF THE PROTEIN SAMPLE, THE PURIFIED PROTEIN SAMPLE WAS HIGH LIE PURE OF. WE HAVE A SET ANALYSIS TO SHOW THAT WHEN YOU INCUBATE THE LIGAND, INCUBATE THE PROTEIN WITH THE DIFFERENT LIGANDS, IT REMAINS COMPLETELY HOMOGENOUS AND GOOD ENOUGH FOR THE STRUCTURE STUDIES SO I DID NOT GO INTO DETAILS OF THIS PARTICULAR WORK BUT I WANT TO JUST ADD THAT SO THIS WAS A CRYOELECTRON CHROMATOGRAPH STUDY. THIS STUDY WE WERE ABLE TO RESOLVE RESTING OF THE GLUTAMATE 2 RECEPTOR AT ABOUT 20-ANGSTROM SOLUTION. SO NOW I'LL COME TO THE PART WHERE I GOT INVOLVED FROM -- SO I WAS WORKING WITH MARK, DOING BIOCHEMISTRY AND THERE WAS THE LAB DOING THE PROCESSING PART OF THIS PROJECT. SO WHAT WE DID IN THIS STRUCTURE ANALYSIS, IS THE IDEA WAS TO USE SINGLE PARTICLE TO GET HIGH RESOLUTION STRUCTURES OF THE RECEPTOR. SO WHAT I'M USING HERE IS THE -- WHAT WE ARE USING HERE IS THE GLUA 2 AMPA RECEPTOR SUBTYPE, WHEN YOU ADD ANTIBODIES IT WILL CLOSE THE CHANNEL. AND WE COULD DISSOLVE RECEPTOR AT ABOUT I DON'T KNOW WHAT ALL RESOLUTION ANGSTROM. AND THE MOLECULE INTERPRETATION WAS DONE FOR THIS EM DENSITY MAP IT WILL INTERPRETATION WAS DONE USING HIGH RESOLUTION STRUCTURE WHICH WAS BINDING IF IN THE LAB PREVIOUSLY IN TWINE. -- 2009. SIMILARLY ATD DIMERS, TWO COPIES AND ONE COPY OF DOMAIN INTO THE EM DENSITY MAP, ANGSTROM RESOLUTION. SO WHAT WE SAW THE ANTAGONIST BOUND CLOSE VERY SIMILAR TO THE STRUCTURE THAT WAS ALSO OBTAINED BY CRYSTAL STRUCTURE IN A SIMILAR CONDITION. THERE WERE MINOR CHANGES BUT THE CHANGES WERE MOSTLY REFLECTIVE OF THE FACT THAT WE USED ALMOST NEARLY RELATIVELY NEAR WILD TYPE CONSTRUCT COMPARED TO THE CONSTRUCT THAT WAS USED FOR CRYSTALLOGRAPHY AND HAD TO BE HIGHLY LYNN YEAR FOR FACILITATING THE SOLUTION OF THE PROTEIN. SO NEXT, WE HAVE THIS RESTING AT 10-ANGSTROM THEN WE WANT TO CAPTURE THAT GLUTAMATE RECEPTOR. SO HOW DO WE ACHIEVE THAT? WHEN YOU ACTIVATE RECEPTOR WITH AGONIST WHICH IS A GLUTAMATE, AND IT BINDS TO GLUTAMATE LIGAND BINDING DOMAIN BUT THE BINDING THAT OPEN THE CHANNEL OPENING HAPPENS FOR A FEW MILLISECONDS AND THEN ULTIMATELY THE CHANNEL WILL DESENSITIZE. WE ALSO ADDED ALLOSTERIC MODELING WHICH CAN STABILIZE -- PREVENT ENTRY INTO THE DESENSITIZED STATE SO THE OPEN STATE WILL BE THERE FOR LONGER TIME AND WE WILL HAVE -- WE WILL PROBABLY HAVE SUFFICIENT TIME TO BE ABLE TO CAPTURE THIS TO BE ABLE TO FREEZE THIS SAMPLE AND TAKE IMAGES. SO WE WERE ABLE TO DISSOLVE THIS EM DENSITY MAP AT 12-ANGSTROM, RESOLUTION AND THEN WE FITTED ATD DIMERS FROM THE CRYSTAL STRUCTURE AND THEN LBD DIMERS FROM A STRUCTURE THAT WAS GLUTAMATE ONLY FOR LIGAND BINDING DOMAIN ONLY IN PRESENCE OF GLUTAMATE SO YOU CAN SEE THE DIFFERENCE HERE THAT WHEN YOU HAVE -- WHEN YOU HAVE ANTIBODIES , THIS FLAP HERE IS OPEN, BUT WHEN YOU ADD GLUTAMATE THE FLAP HERE CLOSES. SO GLUTAMATE BINDING ALSO LEADS TO THIS LBD CLAM SHELL CLOSURE. SO WHAT HAPPENS OVERALL GOING FROM RESTIN STATE TO THE ACTIVE STATE? WHAT I HAVE HERE IS A MR, I HAVE TO ONE WAS RESTING STATE, STARTING AT A POINT AND THEN WE HAVE AN END DATA POINT THE ACTIVATED STATE. AND THE STEPS IN BETWEEN ARE NOT REALISTIC BUT IT GIVES YOU A SENSE OF THE CHANGES THAT ARE HAPPENING GOING FROM RESTING TO ACTIVATED STATE. SO I WILL REPLAY THIS. FIRST THING YOU WILL SEE ASK THERE IS A PARTICLE INTERACTION OF RECEPTOR, SO THE RECEPTOR WAS LONGER AND WHEN IT ACTIVATES IT BECOMES SHORTER. THEN THERE IS ALSO A RATION IN LBD LAYER, WHY WOULD THE LTD DOMAIN IS -- BEHAVES AS RIGID BODY. WE SAW THIS DIFFERENT CHANGES OVERALL GOING FROM RESTING STATE TO ACTIVE STATE SO THERE WAS A VERTICAL CONTRACTION, THERE IS ALSO ROTATION AND ROTATION IN LBD AND THERE IS ALSO A SEPARATION -- THERE IS ALSO SEPARATION OF LOWER GLOBES, IF YOU LOOK AT THIS -- HELICES, NOW COMING APART. AND THEN NEXT WE TRIED TO GET A STRUCTURE FOR THE DESENSITIZED STATE FOR THE GLUTAMATE RECEPTOR BUT WHAT WE FOUND THAT IT WAS CONFIRMATIONALLY HETEROGENEOUS AND THOUGH WE COULD GET CLASSES OUT OF ONE SAMPLE AND RESOLVE AT 25-ANGSTROM BUT IT WAS NOT POSSIBLE TO GET MORE INFORMATION THAN THAT. SO WHAT WE DID NEXT WAS TO SWITCH TO SUBTYPE OF GLUTAMATE RECEPTOR WHICH IS THE KINASE RECEPTOR SUBTYPE. SO WE KNOW FROM THE ELECTROPHYSIOLOGICAL ANALYSIS AND ALSO FROM THE CHROMATOGRAPH STUDY I DIP PRESENT IN DETAIL BUT IN THE RECEPTOR THE DESENSITIZED STATE IS MORE STABLE COMPARED TO THE DESENSETIVE STATE OF THE GLUTE MAY RECEPTOR SO WE THOUGHT IT WAS A PROMISING CANDIDATE FOR THE STUDIES. SO WE STABILIZED THE DESENSITIZED STATE USING AN AGONIST SO BECAUSE THERE IS NO ALLOSTERIC MODEL WHICH CAN PREVENT THE -- WHAT HAPPENS IS THAT IN PRESENCE OF AGONIST, ALMOST ALL THE RECEPTOR HAS GONE INTO A DESENSITIZED CONFIRMATION AND WE RESOLVE THIS CONFIRMATION AT HIGHEST RESOLUTION FOR THAT PARTICULAR STUDY WHICH WAS AT ABOUT 7.6-ANGSTROM. THIS WAS AROUND 2014. SO WE ALSO INTERPRETED THIS MAP USING TWO DIMERS THAT WERE TAKEN FOR -- THAT WERE TAKEN FROM THE STRUCTURES OF ISOLATED ATDs AND THEN WE ALSO ADDED FOUR LBDs TO FIT INTO LIGAND BINDING LAYER. SO INTERESTINGLY, THIS WAS ALSO -- WE ALSO BROUGHT FROM THE CRYOELECTRON CHOUGH MAT GRAPH BUT WE HAD -- CHROMATOGRAPH, THERE IS A LARGE CHANGE IN LIGAND BINDING LAYER, THE LIGAND BINDING LAYER WAS BEHAVING LIKE TO FOLD SO DIMERS WERE TWO FOLD BUT WHEN YOU GET TO THE DESENSITIZED STATE THIS LIGAND, THERE IS APERTURE OF THE DIMER -- RUPTURE OF DIMER ASSEMBLY AND THEN SYMMETRY. SO IT WILL BE MORE -- IT WILL BE MORE EASIER TO SEE IN THIS, SO WHAT I'M SHOWING HERE IS JUST A LBD LAYER ASSEMBLY HERE, REMOVED ATD WHICH ARE MORE OR LESS RIGID, BEHAVES LIKE A RIGID BODY DURING THE TRANSITION SO WE DID NOT HAVE INFORMATION FOR TRANSMEMBRANE DOMAIN, I'M SHOWING THE LIGAND BINDING FOR SUBUNITS THAT COARSE RESPONDS TO LIGAND BINDING DOMAIN OF THIS RECEPTOR. SO IF YOU REMEMBER THAT IN THE STIFF STATE THERE WAS PARTICULAR INTERACTION SO RECEPTOR GOES BACK. AND THEN IN THIS YOU WILL BE ABLE TO SEE THERE'S A LOT OF -- THERE IS A CLOCKWISE ROTATION IN TWO OF THE SUBUNIT OF THE RECEPTOR AND WHICH LEADS TO THIS RUPTURE OF LBD ASSEMBLY AND TRANSITION FROM THE TWO FOLD TO SYMMETRY FOR LBD LAYER. SO THE QUESTION WE HAD WAS SO THERE WAS A RUPTURE IN THE LBD DIMER ASSEMBLY SO WE WERE BASICALLY MAKING SOME INTERACTIONS AND WE ALSO KNEW THAT THIS IS ALSO THE STATE WHICH IS HIGHLY STABLE. SO WHAT WAS HAPPENING? WERE THERE INTERESTING INTERACTIONS STABILIZING THIS CONFIRMATION. SO WE TOOK THIS DESENSITIZED STATE AND IN THE SECOND PHASE OF STUDY WHICH WAS DONE MORE RECENTLY, WE WERE ABLE TO RESOLVE THIS RECEPTOR AT 3.8-ANGSTROM RESOLUTION AND WE COULD BUILD THE MODEL STARTING -- WE DID NOT HAVE TO DO ANY FITTING OF UNKNOWN CRYSTAL STRUCTURE SO HERE IN THIS STRUCTURE WE CAN SEE LTD, WE CAN CLEARLY SEE LBD BUT ALSO WE WERE ABLE TO SEE THE TRANSMEMBRANE DOMAIN AND FIT THE HELICES HERE. NOT ONLY THE TRANSMEMBRANE DOMAIN, THIS PROTEIN HAS A LOT OF GLYCO SOLUTION INSIDE, WE COULD SEE DENSITY FOR THE MOLECULES WHICH WERE VASCULAR GLYCO SOLUTION SIDE. BUT IMPORTANTLY BIOLOGICALLY WE WERE NOW ABLE TO LOOK AT LBD LAYER AGAIN AND ACTUALLY FOUND LOT OF COMPENSATING TRACTION THAT WERE FORMED OR UNIQUE TO THIS DESENSITIZED STATE AND ARE NOT PRESENT IN THE RESTING OR EVACUATED STATE SO THIS SHOWS THAT THEIR INTERACTION THAT FORMS WHEN THE RECEPTOR GOES FROM ACTIVE STATE TO DESENSITIZED STATE. AND THERE WERE ALSO INTERACTIONS, NEW INTERACTIONS FORMED BETWEEN LTD AND LBD LAYER. SO THIS ATD INTERESTING STATE, NEGLIGIBLE INTERACTION BETWEEN ATD AND LBD LAYER BUT WE FOUND THERE WERE SOME RESIDUS THAT NOW INTERACT WHEN RECEPTOR GOES INTO DESENSITIZED STATE. SO THESE TWO SITE OF INTERACTION NOW COMPENSATES FOR THE BREAKING LBD DIMER ASSEMBLY AND STABILIZES THE RECEPTOR. SO I WILL FINISH THIS PART OF THE -- I'M FINISHING THIS PART OF THE TALK HERE BUT IF THERE ARE ANY QUESTIONS ON THE GLUTAMATE RECEPTOR, I'LL BE HAPPY THE ANSWER BEFORE I MOVE ON TO THE NUCLEOSOMES. (OFF MIC) >> YEAH. SO EVACUATED STATE WE GOT WAS AT 12-ANGSTROM SO WE REALLY DIDN'T SEE THE TRANSMEMBRANE HELICES IN THAT PARTICULAR STUDY. SO IT'S VERY DIFFICULT TO SPECULATE ON WHAT EXACTLY IS HAPPENING IN THE TRANSMEMBRANE DOMAIN BUT WHAT WE KNOW FROM THE STUDY THAT -- SO THE -- WHICH COVER IT IS ION CHANNEL CORE IS CONNECTED TO LIGAND BINDING DOMAIN AND WE SEE A LOT OF MOVEMENT IN THE GLUTAMATE LIGAND BINDING DOMAIN. THIS MOVEMENT ALSO PULLS THE M 3, THAT CAUSES UNBINDING OF THE HELIX AND PROBABLY THE CHANNEL OPENING. THAT'S INFORMATION THAT IS DERIVED FROM WHAT WE UNDERSTAND FROM THE LIGAND BINDING REARRANGEMENT AND THE LIGAND BINDING DOMAIN. (OFF MIC) YOU MEAN IN THE NMDA RECEPTOR WITHOUT ANY LIGAND? (OFF MIC) >> YOU AT LEAST -- WHAT I CAN SAY THAT LIGANDS BINDING CAN MAKE IT FAVORABLE AND THERE MAYBE SOME CHANGES THAT CAN HAPPEN BY ITSELF BUT I'M NOT SURE. (OFF MIC) L >> SO I DON'T RECALL, I DON'T REMEMBER ANY INTERACTION THAT COMES FROM THE GLYCOSOLUTION SIDE BUT YOU RAISE A NICE POINT. THERE IS A GLYCOSLATION SIDE CLOSE TO THIS PLACE BUT -- EXACTLY. SO THIS IS WHAT YOU SEE THE GLYCOSYLATION. IT'S CLOSE TO THE LBD LAYER BUT I DON'T EXACTLY RECALL IF THERE WAS ANY BIOLOGICAL -- (OFF MIC) SO NOW I'LL MOVE TO THE STUDY WHICH WE DID RECENTLY IN COLLABORATION WITH ALEX KELLY'S LAB IN BUILDING 37 THERE ALSO PART OF NCI. SO THIS IS -- IN THIS STUDY WE DISSOLVE STRUCTURE OF THE NUCLEOSOME IN COMPLEX WITH PROTEIN CENP-N. SO WHILE I NOTICE THAT IN LAST TWO TALK ALSO HAVE NUCLEOSOME AND I ALSO SAW THAT THE COVER PAGE OF -- ALSO HAVE NUCLEOSOMES SO IT SEEMS LIKE A VERY POPULAR VALUABLE THING I GUESS. AND I MAY NOT HAVE INTRODUCED THEM ABOUT THE KNEW CASH FLOWSOME BUT WHAT I -- NUCLEOSOME BUT WHAT I SHOW YOU ON THE LEFT IS UNPACKING OF DNA. HERE IS A LINEAR DNA AND THE BASIC UNIT OF PACKING DNA IS NUCLEOSOMES WHERE DNA IS AROUND THE HISTONE AND YOU CAN GO ON DIFFERENT LEVELS OF PACKING, AND IF YOU COME TO THE META PHASE CHROMOSOME THE PACKING ACTUALLY CHANGES THE LENS, SO IF YOU HAVE ONE NMDA IT CAN REDUCE THE LENGTH BY 10,000 FOLD WHEN IT GOES TO CHROMOSOMES SO THIS PATTERN IS REALLY COMPLEX BUT OF COURSE YOU KNOW THIS ALSO HAS TO BE DYNAMIC AND DIVERSABLE BECAUSE SOME OF THE PROCESSES LIKE TRANSCRIPTION AND TRANSLATION WOULD LIKE TO ACCESS THIS DNA DURING THIS PROCESS SO THIS IS ACTUALLY PRETTY REVERSIBLE BUT IT'S AIDS MAZING HOW -- AMAZING HOW DNA CAN BE ACTUALLY CONDENSED INTO VERY, VERY, VERY SMALL CELL. SO THE FIRST RESOLUTION STRUCTURE OF NUCLEOSOME BECAME AVAILABLE IN 1997, THIS WAS ACTUALLY BY -- WHO HAS BEEN DOING A LOT OF WORK ON NUCLEOSOMES AND IT SHOWS THAT HOW THE DNA IS WRAPPED AROUND, SO THERE WERE ABOUT 146 BASE PAIR OF DNA AND IT COVERS -- MAKES ABOUT 1.65 TURNS AROUND HISTONE SO STARTING FROM HERE YOU MAKE A FULL TURN AND 1.65, SO .65 TURN TO GET HERE BEFORE IT GOES TO -- BEFORE IT GOES OUT AND STARTS TO CONNECT WITH NUCLEOSOME. AFTER THAT THERE WERE OTHER STRUCTURES BUT A STRUCTURE INTERESTING STRUCTURE IN THE CONTEXT OF TODAY'S SEMINAR CAME OUT IN 2011 WHERE THEY EXCHANGE ONE OF THE HISTONE S-3 WITH ANOTHER HISTONE LIKE PROTEIN CALLED CYTOCHROME PROTEIN A. THE SIGNIFICANCE OF THIS EXCHANGE IS THAT SEN TOEMERIC NUCLEOSOME IS FORMED AT SENT MERES AND IT HELPS ATTACHMENT OF MICROTUBULES TO THE -- IT IS IMPORTANT DURING CELL DIVISION AND APPROPRIATE OF THE CHROMOSOMES. IF YOU ZOOM ON THIS SMALLER PART YOU WILL SEE THAT THERE ARE ACTUALLY A LOT OF PROTEINS WHICH ARE INVOLVED IN THIS INTERACTION. SO KIN STOW -- KINETOCHORE IS A DYNAMIC ASSEMBLY YOU SEE A BUNCH OF KNEW COLLIESOMES AND -- NUCLEOSOMES CONTAIN S-3. YOU SEE SOME PROTEINS FROM KINETOCHORE WHICH DIRECTLY RECOGNIZE DIRECTLY RECOGNIZE OUT OF THIS BUNCH OF MIX OF NUCLEOSOMES SO CENP-N IS ONE OF THOSE PROTEINS. WE WANTED TO STUDY HOW THAT HAPPEN. SO WE STARTED WITH OBJECT RESOLVING THE STRUCTURE OF CENTROMERIC NUCLEOSOME IN COMPLEX WITH THE KINETOCHORE PROTEINS TO UNDERSTAND THE STRUCTURE BASIS OF THIS INTERACTION AND WHY S-3 IS NOT RECOGNIZED WHILE IT CAN RECOGNIZE CENP-N IN THE MIXTURE. SO AGAIN, I'M SHORTENING THE BIOCHEMISTRY PART HERE BUT I WOULD LIKE TO EMPHASIZE THAT YOU DEFINE CENP-N WAS PRETTY CHALLENGING, THAT IS MOSTLY BECAUSE WHEN PROTEIN IS FORMING PART OF COMPLEX, IT BASICALLY NEEDS A LOT OF PARTNER TO BE STABILIZED. IT CREATES A PROBLEM WHEN YOU TRY TO ISOLATE AND TRY TO SOLUBILIZE ISOLATED DOMAIN, SO IT WAS VERY DIFFICULT, MY POST DOC FROM MY LAB MANAGED TO GET THIS INSOLUBLE FRACTION WITH A CONSTRUCT WHERE HE HAD ATTACHED BINDING PROTEIN TO THE CENP-N. SO KNEW CASH -- NUCLEOSOME IS STANDARD, YOU MIX THEM TOGETHER AND THEN DO A GEL FILTRATION TO PURIFY THE COMPLEX. YOU ALSO GET DNA FREE DNA AND SO IT'S STILL NOT A VERY CLEAN SAMPLE BUT IT WAS STILL HAVE A LOT OF FREE DNA AND THINGS WHICH WOULD NOT HAVE BEEN IN THE COMPLEX FORM BUT ONCE YOU DO GEL PREPARATION YOU CAN SELECT FOR FRACTIONS WHICH HAS MOSTLY YOUR COMPLEX AND THEN THEY ALSO DID A LOT OF BIOPHYSICAL ANALYSIS TO MAKE SURE THE COMPLEX WAS FORM AND IT WAS PRETTY HOMOGENOUS. AND THEN THIS COMPLEX WENT FOR THE CryO-EM ANALYSIS. SO WE DID WE STARTED THIS WITH NEGATIVE STAIN SCREENING WHICH I'M NOT SHOWING HERE BUT IT TELLS YOU THAT'S THE QUALITATIVE THING TO MAKE SURE THAT YOU HAVE THE RIGHT SAMPLE AND IT'S NOT DISSOCIATING AND YOU CAN ACTUALLY GET SOME INFORMATION. SO THEN WE COLLECTED A HIGH RESOLUTION CryO-EM DATA SET SO THIS IS ONE OF THE MICROGRAPH THAT I'M SHOWING FROM THE DATA SET WE COLLECTED TO DISSOLVE THE STRUCTURE OF THIS COMPLEX. SO HERE ARE HIGHLIGHTED SOME OF THE NUCLEOSOME PARTICLES BUT IT WAS VERY DIFFICULT TO SAY WHETHER THEY HAVE THE COMPLEX HERE OR NOT BECAUSE ULTIMATELY EVEN AFTER THIS OPTIMIZING IT WAS NOT EASY TO GET MICROGRAPH BECAUSE THIS NUCLEOSOME HAVE BEEN ON TRACK WITH EACH OTHER, INVOLVED IN STANDARD PACKING SO YOU ALWAYS TEND TO SEE A LOT OF REMEDIATIONS ON THE MICROGRAPH. WE WERE ULTIMATELY ABLE TO SELECT THIS PARTICLE AND THEN OF COURSE YOU CAN ALIGN AND THEN SORT THEM INTO GROUPS, DIFFERENT ORIENTATION. THIS GOES AND GIVES YOU A MAP WHICH WAS RESOLVED AT 3.9-ANGSTROM RESOLUTION, THAT SOUNDS LOW BUT IT WAS IN FACT ONE OF THE TOP MOST RESOLUTION FOR THE NUCLEOSOME COMPLEXES AT THAT TIME. SO WE HAVE -- I HAVE COLORED THIS IN THREE -- I HAVE USED THREE DIFFERENT COLORS HERE SO THE GRAY PART SHOWS THE CENTROMERIC PART SO YOU HAVE HISTOTONES AND DNA HERE AND THE ORANGE REPRESENTS THE CENP-N AND AS I MENTIONED EARLIER, WE LAB USED NBP TO SOLVE, WE ALSO SOLVED FOR BLOOD AND DENSITY PROTEIN COMPLEX. SO FAR SO GOOD, AND I HAVE REMOVED THE BINDING DENSITY HERE BECAUSE THAT WAS PRETTY WEAK AND IT MAKES SENSE JUST TO SHOW THESE TWO HERE. AND SO THE STRUCTURE OF SEN TOEMERIC NUCLEOSOME WAS DISSOLVED AND THERE'S KNEW COLLIESOME STRUCTURE -- NUCLEOSOMES SO WE COULD USE DIFFERENT CRESTAL STRUCTURES -- CRYSTAL STRUCTURES AVAILABLE AND COMBINE THEM TOGETHER TO GET A COMPLEX TO STRUCTURE OF THE SENTMERIC CORE. SO WE HAVE ARTIFICIALLY MOVED THE ORANGE PART TO SHOW HOW THE CENTROMERIC NUCLEOSOME LOOKS LIKE BUT THE SAME COMPLEX. SO WHAT IS REMAINING AT THAT TIME IS HOW TO BUILD THIS ORANGE PART. AND WHEN WE LOOKED AT DATABASE PROTEIN DATA BANK THERE WAS NO KNOWN STRUCTURE FOR CENP-N. SO I'M SEEING HERE 1 TO 86, CENP-N IS ABOUT 339 NANOMETERS BUT FOR THE CONSTRUCT FOR THE WHO STUDY THE COMPLEX WE ONLY USED RESOLUTION ABOUT 286 BECAUSE THIS WAS REGION WHICH WAS SHOWN TO BE IMPORTANT FOR INTERACTION WITH THE CENTROMERIC NUCLEOSOME CORE AND WE DIDN'T WANT TO DO ANYTHING NOT NEED FOR THIS COMPLEX FORMATION TO GET MORE STRUCTURALLY HOMOGENOUS PREPARATION. AND THEN WHEN CENP-N STRUCTURE WE ALSO TRIED HOMOLOGY MODELING BUT ALL OF THESE LED TO FAILURE MAINLY BECAUSE THERE WAS ACTUALLY NO HOMOLOGOUS AVAILABLE IN THE PROTEIN DATA BANK. SO THIS IS -- WHAT YOU DO, YOU TAKE YOUR SEQUENCE OF YOUR PROTEIN AND SITE IT AGAINST THE PROTEIN DATA BANK, IT GIVE US A HIT BUT ONLY SHORT SEGMENT WHICH MASKED WITH ONE OF THE MICROBACTERIA PROTEIN IN THE PROTEIN DOMAIN. SO SHORT ANSWER HERE IS THAT THERE IS NO STRUCTURE AVAILABLE, THERE IS NOT EVEN HOMOLOGOUS STRUCTURE AVAILABLE FOR CENP-N THAT CAN BE USED TO BUILD A MODEL AND THEN INTERPRET THIS DENSITY. SO WHAT WE DO? SO WE -- THE ONLY STRUCTURAL INFORMATION WE HAD WAS FROM THE DENSITY MAP AND YOU COULD -- WE COULD SEE THAT IF YOU LOOK CAREFULLY THAT WE CAN DIVIDE THIS EM DENSITY MAP INTO TWO LOBES. LOOKS LIKE ONE LOBE HERE AND ONE SUBDOMAIN HERE SO LOOKED LIKE IT'S FORMING TWO SUBDOMAINS AND WE COULD VISUALIZE SOME OF THE STRUCTURES OF THIS LOOK LIKE A LOT OF HELICES GOING HERE, PROBABLY A BETA SHEATHE HERE AND THAT'S SOME VERY FUZZY DON'T KNOW WHAT IT IS N. I ALSO FOLLOWED THE SIZE. SO WE WERE SUPPOSED TO HAVE ABOUT 286 RESIDUES BUT IT DIDN'T LOOK LIKE THAT IT CAN ACTUALLY JUSTIFY 286 RESIDUES IN THIS MASS DENSITY SO WE FOUND THERE ARE PROBABLY SOME REGIONS WHICH ARE NOT -- PROBABLY FLEXIBLE AND ARE NOT SEEN IN THE DENSITY MAP. SO NEXT THING I DID MADE SOME PREDICTIONS. ON THE LEFT HERE IS THE PREDICTION OF DISORDERNESS OF THIS SEQUENCE. SO YOU JUST DID THE SEQUENCE, THERE'S NO OTHER INFORMATION NEEDED HERE. AND SO ON THE Y AXIS PROBABLY ANYTHING WHICH GOES BEYOND THE RED LINE IS LIKELY TO BE DISORDER, STRUCTURALLY NOT STABLE SO I COULD SEE THAT THE REGION OF THE C TERMINUS HAS LOT OF FLEXIBILITY. SO IT'S POSSIBLE THAT WHAT WE ARE SEEING IS MOSTLY COMES FROM FIRST MAYBE 200 RESIDUES AND THE REST OF THE REGION IS PROBABLY FLOPPING AROUND. THEN I MADE ANOTHER PREDICTION USING A TRACK DOME SERVER, IT GIVES YOU, IT CAN LOOK AT THE SEQUENCE AND THEN DOES AN ALGORITHM CALCULATION AND GIVES YOU SOME NUMBERS AND PREDICTS THAT IT COULD ACTUALLY BE FORMING PRE-SUBDOMAINS SO THE FIRST SUBDOMAIN WOULD BE ENDS TERMINUS DOMAIN OF RESIDUE ABOUT 1 TO 78 THEN YOU COULD HAVE A SUBDOMAIN OF RESIDUES ABOUT 80 RESIDUES AND THEN REST OF THE REGION AT C TERMINAL WAS FORMING A SUBDOMAIN. BUT THIS IS BASICALLY JUST THE SUBDOMAIN INFORMATION, IT DOESN'T SAY THAT IT WILL BE STRUCTURE AS WELL. SO I HAD TWO INFORMATIONS HERE THAT I COULD ROUGHLY DIVIDE MY SEQUENCE INTO SUBDOMAINS AND I KNOW C TERMINAL IS LIKELY VERY DISORDERED HERE. SO BASICALLY WHAT I WAS TRYING TO DO IS INITIALLY WE WERE PREDICTING STRUCTURE OF WHOLE PROTEIN BUT NOW I'M TRYING TO BREAK DOWN THIS PROBLEM INTO SMALLER SEGMENTS SO THAT THE PROGRAM, THE 3-D PREDICTION PROGRAM WILL HAVE A BETTER CHANCE TO PREDICT STRUCTURE OF THIS SUBDOMAIN INSTEAD OF WHOLE PROTEIN. SO WHAT I HAVE SHOWN HERE IS THE PREDICTION HERE BECAUSE -- THE SUBDOMAIN PREDICTION SHOWED 80 RESIDUES IN THE END TERMINUS. I WANT TO TAKE THIS WITH A PUNCH OF SALT AND MAKE SURE I'M JUST NOT LOOKING FOR ONE SEGMENT, TRYING TO MAKE SURE THAT I HAVE MADE TEMPLATES WHICH STARTS FROM RESIDUE 60 AND THEN STARTED DIVIDING THE SEQUENCE AND STARTED ADDING THIS SECONDARY STRUCTURES. SO I GOT A LOT OF SEQUENCE, I MADE A LOT OF TEMPLATES AND THEN I USED THIS MANY SEQUENCE TEMPLATES AND PRE-DIMER PREDICTION TO SEE OKAY, YOU NEED STRUCTURES FOR THIS ONE, AND IF YOU CAN CALCULATE. SO I DID SIMILAR THING FOR CENTRAL DOMAIN AS WELL AS C TERMINAL DOMAIN SO I MADE CONSTRUCT WHICH RANGE FROM RESIDUE 75 TO 215 FOR THE C TERMINUS CENTER DOMAIN AND THEN I ALSO MADE A LOT OF TEMPLATES FOR THE C TERMINAL DOMAIN, STARTING FROM RESIDUE 150 TO 286. I IMMEDIATELY REALIZED FROM THE STRUCTURE STARTED COTOM IN, THAT C TERMINUS IS INDEED VERY FLEXIBLE. SO IT'S LIKELY WE ARE NOT SEEING THAT IN THE EM DENSITY MAP SO I -- AFTERWARDS I FOCUS ON END TERMINAL AND CENTRAL DOMAIN STRUCTURE OF THE END TERMINAL AND CENTRAL DOMAIN. SO IT LOOKED GOOD AND SO I'M ONLY SHOWING AN EXAMPLE OF SOMETHING THAT ACTUALLY ULTIMATELY WEPT INTO THE -- WENT INTO THE INTERPRETATION OF THE END DENSITY MAP SO THIS WAS END TERMINAL TEMPLATE OF RESIDUE 1 TO 81 OF CENP-N AND I GOT THIS MODEL FROM A SERVER. SO THIS IS ONE OF THE SER VERY I USE, I GOT QUITE A FEW SERVER AND I WAS GETTING MODELS BUT EVEN FROM THE SAME SERVER, YOU COULD GUT MODELS WHICH LOOKS SLIGHTLY DIFFERENT. SO THEY DON'T GIVE YOU ONE EXAM ANSWER THIS IS YOUR STRUCTURE, THAT'S IT, DONE. BUT IT ACTUALLY GAVE YOU STRUCTURES WHICH WERE LIGHTLY DIFFERENT AND -- SLIGHTLY DIFFERENT. THIS END TERMINAL DOMAIN WAS MOSTLY ALPHA HELICAL AND EASIER TO PREDICT COMPARED TO THE CENTRAL DOMAIN. BUT EVEN IN THIS IT WAS NOT STABLE ANSWER THAT YOU WOULD HAVE A STRUCTURE THAT CAN INTERPRET YOUR EM DENSITY MAP. SO WHAT I WAS DOING AFTER THAT IS TAKE THIS AND START PUTTING INTO THE EM DENSITY MAP. SO HERE I'M SHOWING THE FINER SELECTED MODELS FOR THE END TERMINAL AS WELL AS THE -- SO I DID THE SAME THING FOR THE CENTRAL DOMAIN AND I STARTED FITTING THEM INTO THE EM DENSITY MAP AND ULTIMATELY I COULD GET TWO COMBINATIONS WHICH SEEM TO FIT ADEQUATELY INTO THE ION DENSITY MAP. SO THIS IS THE ENDS TERMINAL REGION RESIDUE 1 TO 18, THIS IS THE CENTRAL DOMAIN RESIDUES ABOUT 1 -- STARTING FROM 101 TO 185 RESIDUES. SO I -- ONCE YOU PUT BOTH OF THESE WE COULD ACCOUNT FOR MOST OF THE DENSITY WHICH WAS PRESENT. SO WE REALIZE IT IS ONLY ABOUT 200 RESIDUES THAT ARE VISIBLE IN THE ION DENSITY MAP. SO THIS MOVIE HERE IS TO SHOW THE FITTING, SO I TOOK THIS INITIAL MAP AND DID BACK AND FORTH MANUAL REBUILDING AND REAL SPACE REFINEMENT IN PHOENIX AND TO GET TO THIS FINAL MODEL. HERE JUST TO SHOW IT SEEMED LIKE A REAL NICE FIT AND WAS JUSTIFYING THE DENSITY VERY WELL. SO I HAVE THIS ONE, THIS PORTION IS THE CENTER DOMAIN AND THE PORTION HERE IS THE ALPHA HELICAL, FIVE HELICES THAT RESPOND TO END TERMINAL DOMAIN OF THIS PROTEIN. SO USING THAT CENP-N STRUCTURE WE ALREADY HAD THE NUCLEOSOME CORE WE WERE ABLE TO GENERATE A STRUCTURAL MODEL FOR THE WHOLE COMPLEX. THAT'S HOW THE WHOLE COMPLEX LOOKED. SO THE CENP-N BINDS TO ONE KERNAL OF THIS PROTEIN AND MAKES A LOT OF INTERACTION WITH DNA AND SOME INTERACTION WITH SOME OF THE -- LOOKS LIKE MAKING SOME INTERACTION WITH THE HISTONE SHOWN IN THE CENP-N MODEL HERE. SO THIS IS THE ONE WHICH IS CENP WHICH REPLACES THE PROTEIN, I WILL EXPLAIN LATER THAT WHY CENP-N IS IMPORTANT FOR INTERACTION AND WHY SENSE OF CENP-N AND PRESENCE OF S-3 PRESENCE OF S-3 DOES NOT INTERACT SPECIFICALLY TO THE NUCLEOSOME CORE. SO THIS IS A SUMMARY SLIDE OF ALL THE MODELING PARTS. SO WE HAD A -- THIS IS THE DENSITY MAP AND WE COULD FIT ALL THE HISTONES WE ULTIMATELY WERE ABLE TO ALSO FIT MODELS FOR THE CENP-N BUT OF COURSE WE COULD ONLY FIT ABOUT 185 RESIDUES AND BUT THE PROTEIN HAD ABOUT 286 RESIDUES. WE FEEL THAT RESIDUES ARE C TERMINUS OF PROTEIN FOR INTERACTION OF CENP-N WITH NUCLEOSOME CORE. BUT IF YOU LOOK AT THE QUALITY OF THIS MAP IS -- YOU COULD SEE SIDE CHAINS FOR ALL THE HISTONES BUT DNA RESOLVE AT LOW RESOLUTION COMPARED TO HISTONE AND THEN WHEN YOU LOOK AT THE CENP-N PART IT IS RESOLVED, IT'S THE MOST -- LESS RESOLVED PORTION OF THIS EM DENSITY MAP AND WE COULD ONLY BUILD A -- WE HAD TO TRUNCATE THE SIDE CHANGES BECAUSE THERE WAS NO DENSITY TO FIT THE SIDE CHAINS SO WHAT WE DID UP FOR THE MODELING PART, I REMOVED THE SIDE CHANGES AND WE ONLY HAD THE MAIN CHAIN GOING INTO THE SEQUENCE GOING INTO THE EM DENSITY MAP. SO IT LOOKED LIKE THAT WE HAVE A MODEL, WE HAD A MODEL AND NOW WE WANTED TO MAKE SURE THAT IT CAN ACTUALLY BE TESTED AS WELL. BECAUSE WE HAD NO CLUE ABOUT THE RIGHT MODEL FOR THE DENSITY BECAUSE WE COULD MAKE MISTAKES IN THE SEQUENCE, WE COULD PUT HELICES BUT WE WON'T KNOW IF IT HAS THE RIGHT SEQUENCE FOR THIS AS WELL. SO WHAT WE DID, WE USED THIS MODEL TO PREDICT THE RESIDUES WHICH ARE AT THE INTERFACE AND WE SAW THAT THERE ARE A LOT OF POSITIVELY CHARGED RESIDUES THAT SEEMS TO FACE THE NEGATIVELY CHARGED PHOSPHATE OF THE DNA. LOOKS LIKE THERE IS A STABILIZING INTERACTION WITHIN POSITIVE CHARGE OF THE CENP-N WITH THE NEGATIVE CHARGE OF THE DNA HERE SO WHAT WE DID NEXT IS MUTATE THESE RESIDUES SO THIS WORK WAS DONE IN THE BAI LAB, THE BIOCHEMISTRY WAS DONE THERE. WE ARE MUTATING THE RESIDUES AND LOOKING AT THE CENP-N STILL INTACT WITH THE NUCLEOSOME CORE. SO HERE IS A MOBILITY SHIFT ASSAY. SO THIS IS WHEN YOU JURISRUN THE NUCLEOSOME, THIS IS WHEN YOU ONE THE NUCLEOSOME IN PRESENCE OF CENP-N, WILD TYPE SO THERE IS NO MUTATION HERE. SO WHAT HAPPENS WHEN CENP-N BINDS TO NUCLEOSOME? THERE IS A SHIFT BECAUSE KNOW BUILT IS NOW RETARDED SO IT -- MOBILITY IS NOW RETARDED. SO THERE'S A BINDING OF CENP-N, THE PEAK WILL GO UP, BECAUSE THE BINDING IS MAKING THE SPEED GO SLOWER. BUT IF THE MUTATION IS AFFECTING YOU WILL SEE A LOT OF -- YOU WILL SEE NUCLEOSOME COMING DOWN BECAUSE THE CENP-N IS NOT BINDING TO THE NUCLEOSOME CORE AND IT'S NOT PREVENTING ITS MOTION SO IT BEHAVES MORE LIKE YOU SEE IT MAY HAVES LIKE A FREE -- BEHAVES LIKE A FREE NUCLEOSOME. SO WE SAW THAT IN ALL THESE CASES THE RESIDUES WHICH WE IS PROBABLY INTERACTING WITH THE DNA SEEM TO ACTUALLY INTERACT WITH THE DNA AND AFFECTED THE MOBILITY OF THE CENTROMERIC NUCLEOSOME CORE. BUT WE ALSO WANTED TO MAKE SURE THAT IT'S NOT AN ARTIFACT SO WHAT WE ALSO DID IS WE MUTATED RESIDUES POSITIVELY CHARGED FROM OTHER PART OF THE CENP-N. WHICH IS BASICALLY NOT -- WHICH SHOULD NOT BE ACCORDING TO MODEL NOT BE INTERACTING WITH THE DNA AND WE SEE THAT IN ALL THOSE CASES, THE CENP-N INTERACT WITH THE DNA SO THE MOBILITY IS RETARDING IN ALL THOSE CASES. SO IF YOU MUTATE A POSITIVELY CHARGED RESIDUE WHICH IS NOT IMPACTING WITH DNA, YOU DON'T SEE ANY CHANGE IN THE MOMENT BUT IF YOU INTERACT ANY OF THESE RESIDUE YOU SEE THAT THERE IS A LOT OF FREE NUCLEOSOME THAT COMES DOWN BECAUSE THERE IS NO INTERACTION, CENP-N IS NOT HOLDING THE NUCLEOSOME ANY MORE. WE ALSO VERIFIED THIS IN COLLABORATION WITH ALEX KELLY IN THE IN VIVO SYSTEM AND SAW WHEN YOU MUTATE THE RESIDUE IT AFFECTS THE CENTROMERIC LOCALIZATION OF CENP-N. SO CENP-N BECAUSE THERE IS NO -- BECAUSE THE CENP-N IS NOT MUTATED IT DOES NOT BIND AND DOES NOT STAY AT THE CENTROMERES BECAUSE THERE'S NO INTERACTION, THE INTERACTION IS AFFECTED WITH THE CENTROMERIC NUCLEOSOME. SO IN THIS INTERACTION WITH DNA -- HOW DOES IT MAKE A SPACE FOR THE CENTROMERIC NUCLEOSOME? DNA IS ALSO PRESENT WHETHER YOU TAKE A STAIN NUCLEOSOME OR CENTROMERIC NUCLEOSOME, DNA IS PRESENT IN BOTH. IT WAS KNOWN THE LITERATURE IF YOU HAVE A CENTROMERIC PROTEIN WHICH HAVE TWO RESIDUES IN ADDITION WHEN YOU COMPARED THOSE -- WHEN YOU COMPARE THE SEQUENCE OF S-3 WITH CENP, THEY HAVE TWO RESIDUES ARGININE AND GLYCENE THAT FORMS TO MAKE A LOOP INTERACTING WITH HELIX 1 OF CENP-N. SO WHEN YOU HAVE S-3 MISSING THOSE TWO RESIDUE THIS INTERACTION IS LOST AND THAT IS WHAT WE OBSERVED THAT THIS ARGININE IS NOW INTERACTING WITH THE GLUTAMATE PRESENT ON THE HELIX ONE OF CENP-N SO WHEN YOU MUTATE THIS GLUTAMATE, YOU SEE THAT THE BINDING IS HAPPENING, THAT IS PROBABLY BECAUSE IT CAN STILL BIND TO THE DNA BUT THE BINDING IS NO MORE SPECIFIC, YOU SEE MORE BANDS SO WHAT HAPPENS NOW INSTEAD OF BINDING TO JUST THIS PART OF THE COMPLEX, IT CAN ACTUALLY NOW BIND NON-SPECIFICALLY TO ANY MUTAGEN, WHEREVER THE DNA -- WHENEVER THE POSITIVE RESIDUES CHARGE AND FORM INTERACTIONS WITH THE NEGATIVE PHOSPHATES SO IT MAKES IT NON-SPECIFIC AND UNPRODUCTIVE BINDING. SO OKAY, SO FAR WE HAVE THE MODEL AND WE WERE ABLE TO DO INTERPRET DENSITY MAP AND WE ALSO -- THE MODEL MUTATION THAT I HAVE SHOWN IS PRETTY LESS COMPARED TO WHAT WE DID FOR THE STUDY. WE MADE LOT OF MUTATION TO MAKE SURE THAT THE MODEL ACTUALLY IS CONSISTENT 100% WITH THE FUNCTIONAL DATA. BUT ULTIMATELY IT WAS STILL A MODEL THAT WAS RESOLVED AT LIKE FIVISH ANGSTROM AND WAS BUILT KIND OF ON THE EM DENSITY MAP. SO INTERESTINGLY WHEN WE -- AFTER WE PUBLISHED OUR PAPER, WITHIN SIX DAYS THERE WAS ANOTHER PAPER FROM IN COLLABORATION WITH OTHER LABS THEY PUBLISHED A CryO-EM DENSITY MAP FOR THE SAME STRUCTURE. SO THEY ALSO HAD NUCLEOSOMES CENP-N COMPLEX BUT THEY ALSO IN THE SAME STUDY THEY ALSO RESOLVE STRUCTURE OF CENP-N USING CRYSTALLOGRAPHY SO WE HAVE A STRUCTURE THAT WAS REMINDED AT HIGH RESOLUTION, USING CRYSTALLOGRAPHY AND WE COULD GET A CHANCE TO COMPARE THIS WITH WHAT WE DERIVE, MODEL WE DERIVE ON ION DENSITY MAP. WHEN I SUPERIMPOSE THESE TWO STRUCTURES, THEY SEEM LIKE A REALLY NICE FIT. THIS IS TO SHOW THAT THERE SEEMS TO BE THE MAP SEQUENCE BY SEQUENCE SO LOOK LIKE THAT ONE COULD ACTUALLY DERIVE MODELS FROM EM DENSITY MAP AND LOW RESOLUTION BUT OF COURSE YOU NEED TO BE VERY CAREFUL ABOUT THIS, YOU NEED TO -- BEFORE WE ACTUALLY SUBMITTED THIS WE DID A LOT OF FUNCTIONAL VALIDATION TO MAKE SURE -- WE STAND A CHANCE TO GET A CORRECT MODEL HERE. SO WITH THIS I WOULD LIKE TO THANK THE TEAM. I WOULD LIKE TO THANK WHO GAVE ME THE NUCLEOSOME PROJECT AND AT THAT TIME I HAD NO EXPERIENCE ON EM PROCESSING AND HE SUPPORTED A LOT ME DOING THIS PROJECT. AND I DID A LOT OF WORK IN THE LAB GLUTAMATE RECEPTOR BIOCHEMISTRY. FOR TIMING WE ALSO COLLABORATED WITH ONE OF THE GLUTAMATE RECEPTOR PROJECT AND WE HAD EXTENSIVE COLLABORATION WITH YAVIN AND ALEX KELLY'S LAB FOR THE EM NUCLEOSOME PROJECT. I WOULD LIKE TO THANK SENTENCER FOR MOLECULAR MYCROPSCOPY FOR THE DATA -- MICROSCOPY FOR PRESENTING THE DATA WE PRESENT HERE AND COMPUTING RESOURCES. THANK YOU FOR YOUR PATIENCE AND LISTENING. [APPLAUSE] I WOULD BE HAPPY TO ANSWER ANY QUESTIONS. YES. (OFF MIC) >> YES. I USE THE LAB. I HAVE BEEN USING THE LAB FOR ALMOST EVERYTHING. I THINK IT'S LIKE WHEN YOU BECOME GOOD AT ONE. (OFF MIC) >> I HAVE DONE LIKE WHEN I WAS ACTUALLY LEARNING I TRIED IMAN IS THE SECOND MOST, I HAVE NOT REALLY SOLVED THE REAL STRUCTURE. ED ALWAYS STICK TO THE LAB. UH-HUH. (OFF MIC) >> SO FOR THE SEQUENCE CHEMISTRY, ACTUALLY I STARTED LIKE THAT, I HAVE A LOT OF CRYSTALLOGRAPHY EXPERIENCE SO I CAN ACTUALLY BUILD THINGS INTO THE DENSITY MAP. HOW DO YOU GET THE SEQUENCE REGISTRY? I COULD BUILD HELICES, I KNOW YOU CAN BUILD THEM THAT'S SOMETHING I DID. SO I WAS DOING INITIALLY BEFORE I DID ALL THIS, IS TO BUILD SOME HELICES AND NOW USE THE STRUCTURE WHICH IS LIKE WE DON'T HAVE ANY SEQUENCE, TO SEE IF I CAN ACTUALLY PREDICT STRUCTURE HOMOLOGOUS TO THIS BECAUSE PREDICTING A STRUCTURE FROM SEQUENCE IS TOUGH AND THE FORCE -- SO I WAS HOPING TO PICK UP SOMETHING BUT IT DIDN'T REALLY CAME OUT WELL BUT I COULD BUILD HELICES. (OFF MIC) >> CENP-N. YEAH. (OFF MIC) >> UH-HUH. (OFF MIC) >> NO, NO, NO. SO IT HAS A VERY SIMILAR FOLD TO THAT OF CENP-N AND IT SUPERIMPOSES FOR MOST OF THE STRUCTURE, EXCEPT LOW VISION AND SOME AGENT. IT'S A VERY HIGHLY SUPERIMPOSABLE, IT HAS A VERY SIMILAR CORE. SO IN FACT I DIDN'T SHOW THAT DATA BUT IF YOU CAN PUT JUST THAT LOOP ARGININE RESIDUES ON S-3 AND IT STARTS TO INTERACT WITH THE CENP-N. THAT'S THE ONLY LOOP THAT IS DIFFERENT IN THE CONTEXT OF BINDING TO CENP-N. (OFF MIC) >> SO THIS WAS -- SO THE DNA SEQUENCE WAS NOT SYMMETRY, YOU LOSE RESOLUTION BECAUSE OF THAT. IT'S EASY TO BILLION DOLLARS IN A SENSE, YOU REALLY WON'T SEE YOUR BASES, AT THIS RESOLUTION. SO YOU DON'T KNOW IF YOU ARE -- YOU WON'T BE ABLE TO BUILD DE NOVO BUT YOU CAN FIT THE DNA SEQUENCE, BUT YOU WON'T BE ABLE TO TELL WHICH BASE IS WHERE. IT'S NOT EASY BUT BECAUSE THE STRUCTURES ARE THE SAME DNA SEQUENCE WE USE FOR OUR STUDY, WE COULD JUST USE THAT AS A VISIBILITY PROTEIN. (OFF MIC) >> YEAH. (OFF MIC) >> CENP-N? THAT WAS -- THAT WAS AT ABOUT 4.5-ANGSTROM, TO 5-ANGSTROM. I CAN SHOW YOU THAT. SO THE RESOLUTION WAS NOT VERY HIGH FOR THAT REGION. (OFF MIC) >> FOR THE CRYSTAL STRUCTURE? THE CRYSTAL STRUCTURE, IF I REMEMBER CORRECTLY, IT WAS AT ABOUT TWO ANGSTROM BUT THE CRYSTAL STRUCTURE WAS ONLY FOR THE CENP-N PART, THEY ALSO HAD AN EM DENSITY MAP AND THEY USE THE CENP-N CRYSTAL STRUCTURE FORFEITING. UH-HUH. (OFF MIC) >> SO I THINK RIGHT NOW, I WAS READING SOME PAPERS AND I THINK THAT'S WHY I WAS INSPIRED TO DO THIS BREAKDOWN. SO I SEE THAT PROGRAMSES THAT CAN DO A MEANINGFUL RECONSTRUCTION OF ABOUT LIKE 100, IF YOU GO BEYOND COMPLEXES, IT WAS EASY TO GET STRUCTURE FOR THE END TERMINAL DOMAIN, THEY'RE EASY TO PREDICT BUT I HAD VERY LITTLE TIME GETTING A MODEL FOR THE CENTRAL DOMAIN. THAT IS FOR TWO REASON, BECAUSE IT WAS DIFFICULT TO PREDICT BECAUSE THEY WERE LIKE BETA SHEATHES AND UNLESS THE END TERMINAL DOMAIN COMES THERE'S NO SUPPORT FOR THE BETA SHEATHE. SO IF YOU BREAKDOWN SEPARATELY THERE'S NO SUPPORT SO MOST OF THE PROGRAM FAILS TO RECOGNIZE THEY'RE MISSING A PROTEIN, STRUCTURE OF THE PROTEIN HERE SO WHAT IT WILL DO, IT WAS JUST PUTTING ONE HELIX FROM SOMEWHERE ELSE TO SUPPORT THAT BETA STRAND SO MOST ARE GETTING FROM THE -- SO THAT HAVE TO DO THIS KIND OF THING I HAVE TO DO MANUALLY, LIKE MAKE SOME DECISIONS RIGHT THERE. BUT I HAVE SEEN THAT IF YOU HAVE LIKE SHORT PROTEIN AND ALPHA HELICAL YOU CAN GET SOME SORT OF -- BUT YOU NEED TO HAVE SOME VALIDATION AGAIN, BECAUSE EVEN FOR THE END TERMINAL I GOT MODELS WHICH WERE COMPLETELY DIFFERENT. (OFF MIC) >> THAT'S SOMETHING -- THAT IS SOMETHING I DID AND I COULD SEE THAT I HAD BETA CONFIDENCE ON THOSE MODELS COMPARED TO SOMETHING WHICH WAS LARGER THAN THAT. BUT I HAVE ALSO VERY LIMITED EXPERIENCE, I'M NOT DOING MODELING AT THAT LEVEL FOR SO LONG. UH-HUH. (OFF MIC) >> EXCUSE ME, I DIDN'T GET -- >> SO MBP IN THE ION DENSITY MAP, DOES NOT SEEM TO MAKE ANY INTERACTION WITH EITHER WITH CENP-N, IT IS LINKED TO THE CENP-N BUT IT DOESN'T FORM AN INTERFACE WITH CENP-N OR WITH THE NUCLEOSOME CORE. SO IT'S NOT AFFECTING HOW THE NUCLEOSOME COMPLEX WOULD LOOK LIKE IN THE CENP-N, SO IN FACT WE DID SOME FUNCTIONAL VALIDATION FOR THAT. SO WE DID WE LOOKED AT THE BINDINGS AFFINITIES FOR CENP-N, WITH AND WITHOUT NBP SO YOU CAN PURIFY CENP-N WITHOUT BUT IT WAS NOT GOOD ENOUGH FOR THE STRUCTURAL STUDIES. KEEP IT STABLE FOR A WRONG TIME BUT IF YOU LOOK AT THE BINDING CONSTANT THEY WERE PRETTY SIMILAR WHETHER YOU HAVE MBP OR DO NOT HAVE MPB. (OFF MIC) >> SO THAT WORK WAS DONE IN VAVIN'S LAB SO GINGIN, A POST DOC IN THE LAB CAN ANSWER THIS MORE IF I CAN CHECK WITH HIM BUT WHAT I HEARD FROM THEM IS JUST NOT STABLE, THEY WERE VERY RELUCTANT, WE WERE RELUCTANT TO DO THIS ASSAYS IN SENSE OF MBP BECAUSE MOST OF THE PROTEIN WILL JUST FALL OUT OF THE SOLUTION. SO IT WAS -- I CAN SAY IN LATER STUDIES IT WAS VERY DIFFICULT TO KEEP THE PROTEINS IN SOLUTION. SO YOU PURIFY IMAGE YOU DO ASSAYS I GUESS BUT YOU CANNOT KEEP IT FOR LONG. (OFF MIC) >> OKAY. THANK YOU. [APPLAUSE]