IT'S MY PLEASURE TO INTRODUCE TODAY'S SPEAKER FOR THE MIDER LECTURE, BURROUGHS MIDER LECTURE, ESTABLISHED IN 1968, RECOGNIZING AN NIH INTRAMURAL SCIENTIST, TODAY'S LECTURER, SRIRAM SUBRAMANIAM, IS HIGHLY DESERVED OF THIS LECTURESHIP, A REAL LEADER, A REAL FORCE IN THE TOPIC TODAY OF CRYO-EM ELECTRON MICROSCOPY. I GOT HIS UNDERGRADUATE TRAINING IN INDIA IN ENGINEERING AND SCIENCE, AND Ph.D. IN FISCAL CHEMISTRY AT STANFORD, FOLLOWED BY POSTDOCTORAL FELLOWSHIP IN CHEMISTRY AND BIOLOGY AT M.I.T. HE IS SENIOR INVESTIGATOR AT NCI, THE FOUNDING DIRECTOR OF THE MOLECULAR MICROSCOPY CENTER, AS WELL AS THE NATIONAL CRYO-EM FACILITY, NATIONAL LAB AT FREDERICK. HOLDS VISITING APPOINTMENTS AT HOPKINS AND UNIVERSITY OF MARYLAND, AND TITLE TODAY IS THE CRYO-EM REVOLUTION, THIS HAS BEEN A REVOLUTION, THIS TECHNOLOGY HAS REALLY HAD GREAT IMPLICATION FROM BASIC SCIENCE, MEDICINAL CHEMISTRY, A BRIDGING TECHNOLOGY THAT'S HAD AN AMAZING IMPACT. IF THIS IS A REVOLUTION, SRIRAM IS THE LENIN OF IT, THE EARLY THOUGHT LEADER AND VERY PERSON IMPORTANT, I EXPECT A FASCINATING TALK. LET'S WELCOME OUR SPEAKER. [APPLAUSE] >> THANK YOU VERY MUCH, NED. THANK YOU, MICHAEL. AND MANY OTHERS AT THE NIH THAT INVITED ME TO GIVE THIS LECTURE. WE CAN HAVE THE FIRST SLIDE PLEASE. YEAH, I'D LIKE TO ACTUALLY TELL YOU ABOUT THE WORK IN MY LAB IN THE CRYO-EM AND RELATED FIELDS, AND WHEN I CAME TO THE NIH I HAVE TO SAY IT WAS WITH A LOT OF TRUST THAT I WOULD DO SOMETHING RELATED TO ELECTRON MICROSCOPY AND MEMBRANE PROTEINS, AND EVENTUALLY THAT FREEDOM TO DO THAT SOMETHING REALLY HELPED US WORK IN MANY EARS THAT WE FOUND VERY INTERESTING, AND IMPORTANT. CRY CRYO-EM, MANY THE LIGHTS COULD GO DOWN AS WE DISCUSS THIS. IT'S NOW A WELL-KNOWN WORD. IT WAS LABELED AS METHOD OF THE YEAR BY NATURE METHODS AND THERE'S BEEN DRAMATIC GROWTH IN THE FIELD. BUT I WANTED TO POINT OUT THAT THIS IS ACTUALLY NOT REALLY A NEW METHOD. CERTAINLY ITS IMPACT IS SEEN AS BEING REVOLUTIONARY, AND IT IS, BUT IF YOU GO BACK TO A DECADE AGO, AS EARLY AS 2008, THERE WAS STRUCTURE AT NEAR ATOMIC RESOLUTION OF VIRUSES PUBLISHED BY A PIONEER IN THIS AREA, OVER THE YEARS MANY STRUCTURES AT PROGRESSIVELY HIGHER RESOLUTIONS, YOU CAN SEE ANYTIME CHARTS, NOT JUST TO OBTAIN HIGH RESOLUTION STRUCTURES BUT A VARIETY OF DIFFERENT RESOLUTIONS RANGING FROM NEAR ATOMIC ALL THE WAY TO MEDIUM WHERE USEFUL BIOLOGY COULD BE LEARNED. I'D LIKE TO PRESENT A HISTORICAL LECTURE HOW I AND MY COLLEAGUES CAME TO THE FIELD, HOW WE START AND HOW WE CUT A PATH TO BE ABLE TO EXPLORE METHODS TO LEARN ABOUT -- TO DEVELOP MECHANISTIC INSIGHTS INTO BIOLOGICALLY COMPLEX SYSTEMS AND LARGER ASSEMBLIES. MANY YEARS AGO WHEN I FIRST WAS AT THE NIH, I DIDN'T ACTUALLY QUITE KNOW EXACTLY WHAT TO DO. DONNY BLISS, WHO WAS WORKING IN MEDICAL ILLUSTRATION AT THAT TIME, HELPED ME PUT TOGETHER THIS SLIDE, WHICH WAS A WAY TO THINK ABOUT WHERE THE IMAGING GAPS LAY IN VIROLOGY AND MEDICINE. THE SLIDE HAS THE CLICHƒED MOLECULES TO MAN PIECE COVERED FROM THE LEFT HAND WHERE WE THINK OF MOLECULES TO INDIVIDUALS WHO WE DON'T THINK OF IN TERMS OF THE NUMBER BUT NEVERTHELESS GIVES YOU A FEELING FOR SIZE, ABOUT 27 ORDERS OF MAGNITUDE. AND WHAT'S CLEAR LOOKING AT THIS, X-RAY METHODS HAVE BEEN AND CONTINUE TO BE VERY POWERFUL, THE TWIN ENDS OF THE SPECTRUM, AND CONVENTIONAL MICROSCOPY, LIKE AND ELECTRON, HAS CONTRIBUTED A GREAT DEAL TO UNDERSTANDING THE MIDDLE OF THE SPECTRUM BUT THE POINT THAT GUIDED THE WORK IN THE LAB WAS RECOGNITION OF GAPS. THE ONE THAT INTERESTED US THE MOST WAS WHERE GAPS ENTER AROUND THE SIZE OF A VIRUS, AND PARTICULARLY DYNAMIC ASSEMBLIES THAT COULD NOT BE EASILY ADDRESSED BY X-RAY CRYSTALLOGRAPHY BECAUSE YOU HAVE TO CAPTURE A PARTICULAR STATE, ALSO WHERE EVEN SUPER RESOLUTION LIKE MICROSCOPIC METHODS MIGHT NOT LET YOU GET AT THE CHEMICAL QUESTIONS WE WERE INTERESTED THE FOCUS IN THE LAB HAS BEEN IN BRIDGING THE GAP, BY WAY OF PERMUTATION WHAT DREW ME, HERE IS A VIDEO SHOT OF A HUMAN NEUTROPHIL, EXTENSIVELY CHASING A BACTERIUM, TAKEN NEARLY 60 YEARS AGO. AND THIS IS A 20 KILOBYTE, WHAT'S FASCINATING, WHILE YOU CAN MAKE AN ADDITIVE OF WHAT MIGHT BE GOING ON, SUCH AS THE NEUTROPHIL CHASING THE BACTERIUM, ONE MIGHT WIN, THERE'S A LOT OF CHEMISTRY GOING ON HERE, CHEMO TAX IS, GRADIENTS, GENES BEING TURNED ON AND OFF, REARRANGEMENTS OF PROTEINS. ULTIMATELY WE'D LIKE TO BE ABLE TO PREDICT THE OUTCOME OF DYNAMIC PROCESSES LIKE THIS, LONG-TERM VISION, BUT IN THE NEAR TERM AN IMPORTANT THING IS TO BE ABLE TO UNDERSTAND ALL THE PIECES THAT ARE INVOLVED IN DYNAMIC PROCESSES SUCH AS THIS. TO THIS END, WE SET OUT AMBITIOUS PROGRAM TO BRIDGE THIS IMAGING GAP. IT WAS CLEAR FROM EARLY DAYS WE NEEDED TOOLS TO GO AFTER ENTITIES ACROSS THE SPECTRUM FROM PROTEINS TYPICALLY WITH SIZES IN THE RANGE OF 10 TO 20 NANOMETERS TO VIRUSES THAT COULD NEVER BE CRYSTALLIZED TO HOLD BACTERIAL CELLS WHERE EACH REEL CELL WOULD BE DIFFERENT FROM ITS TISSUE, NO SIMPLE METHODS WOULD LET YOU DETERMINE ENTITIES. IF I WERE WRITING AN R01 GRANT TO DETERMINE ATOMIC STRUCTURE OF A BACTERIAL CELL IT WOULD NOT BE MET WELL BUT I COULD PRESENT IDEAS LIKE THAT IN THE INTRAMURAL PROGRAM WHICH LET US TAKE RISKY STEPS TO ADDRESSING THESE PROBLEMS. WORK IN THE LAB WAS GEARED TOWARDS FINDING TOOLS THAT WOULD HELP US ADDRESS THESE GAPS BY TACKLING INDIVIDUALLY STRUCTURAL ASPECTS OF EACH FROM PROTEINS, VIRUSES AND CELLS TO TISSUE. SO TODAY I'D LIKE TO ESSENTIALLY PROVIDE AN OUTLINE OF THIS WORK. I'D LIKE TO BEGIN WITH A VERY BRIEF INTRODUCTION TO ELECTRON CRYSTALLOGRAPHY WHICH IS WHERE THE ORIGINS OF CRYO-EM REVOLUTION LIE, I'D LIKE TO TOUCH ON SOME EARLY WORK WE DID ON BACTERIAL AND VIRUS STRUCTURES, WHERE WE DEVELOPED AND USED METHODS IN CRYOELECTRON TOMOGRAPHY, INCREASINGLY BEING SEEN TO BE IMPORTANT TO STUDY LARGE AND COMPLEX ASSEMBLIES. I'D LIKE TO TOUCH ON SOMETHING THAT'S SLIGHTLY OFFER TOPIC FROM CRYO-EM BUT NEVERTHELESS IMPORTANT IN THE SCHEME OF THINGS. HOW WE'RE USING SCANNING ELECTRON MICROSCOPIC TO LOOK AT TISSUE, IN THE NEXT PORTION, THE PRIMARY FOCUS, I'D LIKE TO TALK ON NEW DEVELOPMENTS IN CRYO-EM AND WE'RE USING IT IN THE FIELD ACROSS THE WORLD, USING IT TO GET AFTER STRUCTURES THAT WERE PREVIOUSLY INTRACTABLE, AND TO LEARN MECHANISM OF HOW ASSEMBLIES WORK. FINALLY A PERSPECTIVE ON WHERE THIS MIGHT BE HEADED, AND HOW NIH MIGHT PLAY A ROLE IN DISSEMINATING THIS TECHNOLOGY. BY WAY OF INTRODUCTION TO ELECTRON MICROSCOPY, TRANSMISSION, HERE ARE TWO IN WHICH WE COLLECT INFORMATION IN AC ELECTRON MICROSCOPE, ON THE LEFT, THERE'S A SENSE, SOURCE OF RADIATION, SPECIMEN, WE COLLECT INFORMATION IN DIFFRACTION PLANE OR IMAGING PLANE. IF YOU COLLECT IMAGES OF INDIVIDUAL MOLECULES, IN DIFFERENT ORIENTATIONS, YOU COLLECT MANY THOUSANDS, TENS OF THOUSANDS OF THESE IMAGES, THESE PROJECTION IMAGES, 2D IMAGES CAN BE COMBINED TO GENERATE A 3D PICTURE, AND THAT'S THE APPROACH USED IN CRYO-EM. ALTERNATIVE APPROACH WHICH I'LL TOUCH ON AS WELL TO USE TOMOGRAPHY WHERE THE IDEA IS SIMILAR TO THAT USED IN COMPUTER AXIAL TOMOGRAPHY, YOU COLLECT 3D IMAGES FROM A SINGLE OBJECT BY VARYING ORIENTATION OF THE SPECIMEN, USE OF ELECTRON BEAM, THAT PRINCIPLE OF TOMOGRAPHY IS POWERFUL IN DISCERNING STRUCTURES OF LARGE ENTITIES LIKE WHOLE CELLS AND VIRUSES. THE ORIGINS AS I MENTIONED BEFORE IN TERMS OF USING ELECTRON MICROSCOPES TO GET AT PROTEIN STRUCTURES GOES BACK TO CAMBRIDGE WHERE THEY DEMONSTRATED IN THE MID-'70s ONE COULD USE ELECTRON MICROSCOPES, ELECTRON DIFFRACTION TO DETERMINE STRUCTURES OF PROTEINS, WINNING THE NOBEL PRIZE LAST YEAR WITH FRANK. MOLECULES PRESENT IN 2D CRYSTALS COULD DETERMINE ATOMIC MODEL FOR BACTERIA, REPORTED IN 1990 AFTER MANY YEARS OF WORK. WHEN I JOINED HIM AT THE END OF THE 1990s BEFORE COMING TO THE NIH I WORKED WITH HIM DEMONSTRATING WE COULD INDEED GET INFORMATION IN DIFFRACTION SPACE THAT WENT TO HIGH RESOLUTION, LED US TO DETERMINE THE STRUCTURE OF THIS MOLECULE BACTERIADOPSIN, UNDERSTANDING IN WHAT HAPPENS TO THE PROTEIN WHEN IT IS IN THE ACTIVE TRANSLOCATING OF PROTONS, TRANSPORTERS THIS IS AN IMPORTANT BIOLOGICAL QUESTION. THIS IS REALLY THE STARTING POINT FOR MY OWN INTEREST IN USING ELECTRONS TO DERIVE PROTEIN STRUCTURE AND ONCE AT THE NIH WE BEGAN TO APPLY THIS TO OTHER TRANSPORTERS AND IN WORK WE DID WITH PETER MALONEY WHO WAS AT HOPKINS AT THE TIME, WE BEGAN TO APPLY THIS TO HEELIC TRANSPORTERS, WHAT YOU SEE ON THE LEFT-HAND SIDE, TYPICAL TUBULAR TWO DIMENSIONAL CRYSTAL, IF YOU ZOOM IN WITH THE ELECTRON MICROSCOPE A COLLECTION OF MOLECULES IN TWO DIMENSION, USING IMAGE PROCESSING TOOLS DETERMINE 3D STRUCTURES AND IN 2002 WE DEMONSTRATED THAT WE COULD ACTUALLY OBTAIN STRUCTURES OF THIS PROTEIN AT LOW RESOLUTION BUT IN COMBINATION WITH WORK THAT RON TABAK HAD DONE, DETERMINE MECHANISTIC, AND THIS MODE OF ACTION SEEMS TO BE CONSERVED IN TRANSPORTERS IN THIS FAMILY. THE BUSINESS OF MAKING 2D CRYSTALS WAS A TEDIOUS PROCEDURE, WE BEGAN TO MOVE TOWARDS TRYING TO INVESTIGATE STRUCTURES OF THESE MEMBRANE PROTEINS IN CELLS, AND BOB WEISS HELPED US GET STARTED IN CHEMO TAXIC RECEPTORS, IN A VARIETY OF BACTERIAL CELLS, COAL OH BACK TORE, E. COLI, WE COULD VISUALIZE WHAT YOU SEE IN THE LEFT-HAND SITE, CENTER OF THE CELL, TWO DIMENSIONAL RECEPTORS, CHEMO RECEPTOR ASSEMBLIES WITH SIGNALING COMPONENTS, BECAUSE OF THE CONSIDERABLE WORK IN THIS FIELD IN ADDITION TO CRYSTALOGRAPHIC, WE FELT INSPIRED TO USE THIS AS A PARADIGM TO LEARN SOMETHING ABOUT THE ORGANIZATION OF RESEPTEMBERORIES IN INTACT CELLS WHERE WE USED SOME METHODS BUT APPLIED THEM TO WHOLE CELLS AND DERIVED MESOSCALE ORGANIZATION AND ASSEMBLIES OF INTACT CELLS AND MANY YEARS AGO THIS WAS THE FIRST EFFORT, VERY FIRST EFFORTS TO DETERMINE STRUCTURES OF MEMBRANE PROTEINS INSITU WHERE WE COULD ADD LIGANDS AND LOOK AT CHANGES, THE ORGANIZATION OF THE ASSEMBLY AND INDIVIDUAL PROTEIN STRUCTURES. THIS HAD THE VALUE THAT IT LED US TAKE ON AT THAT TIME WHAT WAS A CONSIDERABLY MORE CHALLENGING PROBLEM, TO LOOK AT ALL THE OTHER PIECES. THIS IS ONE EXAMPLE OF WORK LOOKING AT CELLS WHERE WE'RE NOW WALKING INTO A SINGLE DELALIBRIO CELL, THE LEVEL ADEQUATE, EACH OF THE SINGLE DOTS ARE ESSENTIALLY RIBOSOMES, WE CANNOT PROVE THEY ARE BUT THEY ARE CONSISTENT WITH THE RIBOSOMES. THE FACT THAT YOU CAN COUNT ENTITIES LIKE THIS IS A POWERFUL WAY TO INTRIINDICATE THE ARCHITECTURE. WE THEN TACKLED A PROBLEM THAT WAS IMPORTANT AT THE NIH, CERTAINLY WAS SUPPORTED VERY MUCH BY A PROGRAM THAT MICHAEL GOTTESMAN RAN. IT WAS SOURCE OF TECHNOLOGY SUPPORT BUT THEY SOON IN MY LAB WE BECAME ATTACHED TO WORKING ON THIS CHALLENGING PROBLEM AND THE CENTRAL FOCUS OF WHAT WE DID WAS TO TAKE ON WHAT SEEMED AT THAT TIME AN INTRACTABLE PROBLEM, WHICH WAS TO DETERMINE THE STRUCTURES OF PROTEINS ON THE SURFACE OF VIRUSES CAPABLE OF INFECTING CELLS BUT WHERE EVERY SINGLE VIRION WAS DIFFERENTLY SHAPED, DIFFERENT STRUCTURES, DIFFERENT NUMBERS OF PROTEINS IN THE BOX ON THE RIGHT-HAND SIDE. SO THE PROBLEM WE WANTED TO SOLVE WAS USE TOMOGRAPHY TO CUT A PATH TO BEING ABLE TO DO STRUCTURES BY COMBINING THE METHODS OF AVERAGING IN 3D WITH TOMOGRAPHY, WE WERE NOT THE FIRST TO THINK ABOUT THIS, EARLIER WORK HAD LED THE PATH TO THIS, AND KEN TAYLOR'S GROUP AT FLORIDA STATE HAD GONE IN THIS DIRECTION, AND ONCE WE HAD AN IDEA WHAT WE NEEDED TO DO WE BEGAN TO ACTUALLY CARRY OUT ELECTRON TOMOGRAPHY, PLANT GROWTH SPECIMENS OF VIRUSES, IN THIS WORK AND MUCH OF THE HIV WORK WE WERE REALLY ENORMOUSLY HELPED BY A VERY, VERY PRODUCTIVE COLLABORATION WITH JEFF LIFSON AT FREDERICK AND COLLEAGUES WHO TAUGHT ME A LOT ABOUT HIV AND THE IMPORTANT PROBLEMS. AND THE GOAL OF THIS WORK WAS TO GO FROM SUSPENSION OF HIV VIRUSES IN THE TUBE TO A MAP OF WHAT THE VIRUS LOOKED LIKE IN 3D, AND THIS IS THE UNIT OF DATA THAT TYPICALLY COMES OUT OF A SINGLE TOMOGRAM. THE STRATEGY WE DEVELOPED, WAS TO TAKE TOMOGRAM LIKE THIS AND EXTRACT THE REGIONS CORRESPONDING TO TRIMERIC PROTEINS, ALIGNING THEM AND CLASSIFYING THEM WE THEN DERIVED THREE DIMENSIONAL STRUCTURE OF THE HIV ENVELOPE TRIMER IN 2008, THE FIRST GLIMPSE INTO WHAT IT LOOKED LIKE ON THE SURFACE OF THE VIRUS AND CAME ALIVE BECAUSE WE HAD ALREADY VERY POWERFUL STRUCTURE FROM PETERWANG, IN COMBINATION WITH CD4 AND ANTIBODIES, POWERFUL INFORMATION FOR US TO POSITION MOLECULES INTO THE DENSITY MAP. THIS IS AN OCCASION FOR ME TO THANK BIOWULF AND THE STAFF, WITHOUT THEIR HELP COULD NOT DO WORK IN THE LAST DECADE. THE INFORMATION IN THE TOMOGRAMS WAS USEFUL, LOW RESOLUTION. BECAUSE THE DENSITY MAPS WERE HIGH RESOLUTION TO POSITION STRUCTURES OF INDIVIDUAL COMPLEXES, IT LED US TO DEVELOP MODELS FOR WHAT HAPPENED AT THE SURFACE OF THESE VIRUSES AND IN PARTICULAR STEPS THAT TOOK PLACE IN THE COURSE OF INFECTION. OUR INTEREST ALL ALONG WAS TO BE OBSERVERS, AT THE SITE OF ENTRY BETWEEN THE VIRUS AND T CELL, AND BY BRINGING ESSENTIALLY PIECES OF THE CELL TO THE VIRUS WE COULD THEN BEGIN TO VISUALIZE CHANGES SUCH AS THE RESULT OF CD4 BINDING AND IN THE COURSE OF THE LAST DECADE HAVE DONE HUNDRED OR SO STRUCTURES OF GLIOPROTEINS, HIV, EBOLA, INFLUENZA, AND THAT'S LED TO A POWERFUL DATABASE OF UNDERSTANDING THE DYNAMICS AND ANTIGENICITY OF THESE MOLECULES ON THE SURFACE. MECHANISTICALLY WE WERE VERY MUCH -- WE LEARNED A LOT CERTAINLY FROM BEING ABLE TO DEVELOP METHODS TO UNDERSTAND THE HETEROGENEITY. AND THIS IS A VERY BROAD PROBLEM IN THE SENSE VIRUSES, ENVELOPE VIRUSES LIKE HIV, INFLUENZA OR EBOLA DISPLAY DIFFERENT COMPLEXES ON THE SURFACE, AND THE INTEREST IN THESE INFECTIOUS VIRUSES DRAWS METHODS IN THE LAB BEING ABLE TO NOT JUST DESCRIBE STRUCTURES BUT ALSO EDUCATE OURSELVES ON THE PLASTICITY OF THESE ENVELOPE GLYCOPROTEINS TO UNDERSTAND WHICH CONFIRMATIONS WHICH BIND WHICH ANTIBOIES LESS OR MORE TIGHTLY. THIS EARLY WORK LED TO MORE RECENT WORK ON INFLUENZA VIRUS AND IN A VERY POWERFUL COLLABORATION WITH PETER PALIS AND KRAMER, EXTENDING METHODS TO TRY AND UNDERSTAND HOW WE CAN CONTRIBUTE TO THE DEVELOPMENT OF UNIVERSAL VACCINE FOR INFLUENZA, BOTH IN TERMS OF THE HA CYCLE AND NEURO AMINODASE TETRIMER. WE'VE BEEN WORKING WITH JUDY WHITE ON EBOLA. THE ZMapp HAS BEEN VERY EFFECTIVE. OUR CONTRIBUTION HAS BEEN TO TRY AND UNDERSTAND WHERE THESE DIFFERENT ANTIBODIES BIND ON THE GLYCOPROTEIN TO DISCERN HOW DIFFERENCES MIGHT TRANSLATE INTO THE WAY IN WHICH VIRUSES ENTER AND INFECT CELLS. SO FAR I'VE SPOKEN A LITTLE BIT ABOUT ELECTRON CRYSTALLOGRAPHY AND THE CONTRIBUTION OF TOMOGRAPHY TO BACTERIAL AND VIRUS STRUCTURES. BUT I WANT TO DIGRESS TO TALK ABOUT THE DIRECTION WE TOOK SEVERAL YEARS AGO TO BE ABLE TO ADAPT ELECTRON-BASED IMAGING METHODS TO LOOK AT MUCH LARGER THINGS. ONE OF THE CHALLENGES WITH TRANSMISSION ELECTRON MICROSCOPY IS YOU CANNOT LOOK ALL THE WAY THROUGH A LARGE MAMMALIAN CELL OR COLLECTION OF CELLS. SIMPLY BECAUSE GESTATIONAL ELECTRO MICROSCOPES DO NOT REALLY LET YOU LOOK AT THINGS MUCH THICKER THAN A MICRON. AND CELLS, MAMMALIAN CELLS IT BE TENS OF MICRON, TISSUE IS THICK. I BECAME INTERESTED IN DEVELOPING METHODS TO LOOK AT THESE CELLS, BUT TO GO BEYOND WHAT WE WERE ABLE TO DO A DECADE AGO, TO TAKE TEN SECTIONS WHICH IS A METHOD JOSH PALADI DEVELOPED MANY DECADES AGO SO ONE CAN TAKE THIS CANONICAL PICTURE OF A LYMPHOCYTE MAKING CONTACT WITH DENDRITIC CELL, IF YOU WANT TO KNOW WHAT WAS AT THE JUNCTION BETWEEN THE TWO CELLS YOU WOULD THEN TAKE A SECTION, HERE IS AN IMAGE IN MY LAB, AND THIS JUNCTION WHICH IS CALLED THE VIROLOGICAL SYNAPSE THAT TOM HOLT AND QUENTIN DISCOVERED AND CHARACTERIZED IN GREAT DETAIL HAS A LOT OF INFORMATION ON CELL-CELL CONTACT. IN OUR CASE IT HAS INFORMATION ON HOW VIRUSES TAKEN UP BY DENDRITIC CELL OR ANTIGEN PRESENTING CELLS GET TRANSMITTED TO T CELLS. THIS IS IN HIV BIOLOGY A CRITICAL PROBLEM WHERE WE LIKE TO UNDERSTAND EARLY ON HOW THESE VIRUSES GET TO T CELLS AND INFECT THEM. AND THIS, TO GET AT THIS, I HAD THE OCCASION TO KNOW FROM THE WORK IN THE SEMI CONDUCT YEAR FIELD, ION BEAMS WOULD LET YOU CUT INTO VERY LARGE AND CERTAINLY METALLIC OBJECTS, SEMI CONDUCTOR OBJECTS, IN A COLLABORATION WITH FEI COMPANY THAT MAKES MICROSCOPES FOR SEMI CONDUCTOR WORLD WE USED THEM FOR BIOLOGY. THE WAY THEY WORK IN ADDITION TO HAVING A SCANNING ELECTRON BEAM TO IMAGE THE SURFACE OF OBJECTS SUCH AS CELLS OR TISSUE OR SILICON CHIPS FOR THAT MATTER, THEY HAVE AN ADDITIONAL FOCUS ION BEAM, MADE OF GALLEON, ESSENTIALLY WHEN FOCUSED ON THE SURFACE REMOVAL MATERIAL AND IN A VERY FIRST EXPERIMENT WE DEMONSTRATED USING THE DIVIDED YEAST CELLS, YOU COULD FOCUS GALLIAN ION BEAMS, COLLECT IMAGES AND COMBINE TO 3D IMAGE, HUGELY EXCITING, WE COULD GET PICTURES LIKE THIS, BUT A DECADE AGO THIS WAS NOT VERY EXCITING BECAUSE AT THAT TIME YOU COULD ALREADY GET TO RESOLUTIONS LIKE WITH CONFOCAL MICROSCOPY. PERHAPS 200 NANOMETERS, 150 BUT NOW BETWEEN 5 TO 10 NANOMETERS, IN 3D, IN THE Z DIRECTION, THAT OF COURSE IS A VERY DIFFERENT LEVEL OF INFORMATION. BUT THE DEVELOPMENT OF THE TECHNOLOGY WAS EVIDENCE FOR THE HIV WORK WE WERE DOING. ONE PROBLEM IN PARTICULAR THAT WE REALLY WANTED TO UNDERSTAND WAS THE NATURE OF TRANSMISSION OF VIRUSES FROM A DENDRITIC TO T CELL, USING THESE METHODS WERE ABLE TO IMAGE IN 3D CONTACT ZONES BETWEEN HIV, BETWEEN DENDRITIC TO T CELLS, RECOGNIZE WHAT LOOK LIKE DENDRITES ON THE LEFT ARE NOT DENDRITES BUT HUGE WALLS OF MEMBRANES THAT COME OUT OF DENDRITIC CELLS AND BIOLOGICAL SYNAPSE ITSELF IS ACTUALLY NESTLED DEEP, DEEP IN THIS CONTACT ZONE. AND THIS LED TO THE PICTURE THAT SOME OF YOU MAY RECALL, THIS IS USED IN THE RESEARCH FESTIVAL A COUPLE YEARS AGO. OUR IMAGE OF WHAT DENDRITIC CELLS LOOKED LIKE EVOLVED QUICKLY TO BE ABLE TO BE LOOK AT THEM IN TERMS OF HUGE SHEETS OF MEMBRANE COMING OUT, SURPRISES BECAUSE WE WERE THEN ABLE TO SHOW NOT ONLY WAS THIS CONTACT ZONE COVERED BY SHEETS OF MEMBRANE BUT THE PARADIGM WE HAD IN OUR MIND FOR HOW VIRUSES WERE TRANSMITTED TO CELLS WAS VERY DIFFERENT THAN WHAT YOU SEE IN THIS IMAGE, IS THAT AS WE LOOKED IN DETAIL AT THE SYNAPSE WE REALIZED THAT THE T CELLS THAT WERE BEING INFECTED ACTUALLY WERE APPEARING TO INFECT THEMSELVES, REACHING INTO DENDRITIC CELLS, CONTROVERSIAL AT THAT TIME BECAUSE THE IDEA SOMEHOW CELLS UNINFECTED WOULD SOMEHOW PLAY A ROLE IN THEIR INFECTION WAS NOT RECEIVED WITHOUT CRITICISM, BUT WE DID PUBLISH. ULTIMATELY SHOWED THIS WAS A COMMON MECHANISM. THIS IS SYNAPSE BETWEEN TWO CELLS, THE CELL IN CENTER INFECTED, FLESH COLOR IS UNINFECTED, WE CAN SEE WHERE THE VIRUSES ARE. YET AGAIN YOU CAN SEE IT APPEARS THE CELLS NOT INFECTED SEEM TO MAKE TRACKS TO THE CELL THAT'S INFECTED, WORK I WANT TO ACKNOWLEDGE DONE BY GAVIN MURPHY WHO PIONEERED THE DEVELOPMENT OF THESE METHODS IN MY LAB AND UNFORTUNATELY PASSED AWAY LAST YEAR. YET ANOTHER EXAMPLE OF THE USE OF THESE METHODS TO UNRAVEL MECHANISM OF HIV TRANSFER CAME FROM APPLICATION TO ASTROCYTES, FETAL ASTROCYTES, DERIVED FROM HOPKINS AT THAT TIME, CULTURED IN THE LAB, AND WHEN YOU BEGAN TO LOOK AT SYNAPSES BETWEEN INFECTED T CELLS AND FETAL ASTROCYTES AT FIRST IT WAS DISAPPOINTING BECAUSE IT LOOKED LIKE THERE WAS NOTHING OF HAPPENING AT THE JUNCTION BETWEEN THE CELLS, YOU CAN SEE THE CONTACT ZONE SEEMS RELATIVELY UNOCCUPIED, BUT WHEN YOU LOOKED AT THE SAME TIME IN 3D, IT BECAME CLEAR THAT IN THIS CONTACT ZONE WHICH DEVELOPED OVER THE COURSE OF A FEW HOURS THE ASTROCYTES WHICH ARE UNINFECTED ARE SENDING OUT EXTENSIONS TO THE HIV TO SERVE ON THEM, AND EVENTUALLY INFECTED THE ASTROCYTE, SO THE IDEA UNINFECTED CELLS PLAYED THIS ACTIVE ROLE IN BEING INFECTED IS NOW I THINK MORE ACCEPTED THAN IT WAS AT THAT TIME. AND BUT THESE INSIGHTS CAME FROM APPLICATION OF FOCUS TIME BEAM TECHNOLOGY. ONE OF THE REASONS WE STARTED TO DO THIS WAS OF COURSE THE APPLICATION TO CANCER, AND FOR MANY YEARS WORKED ON MELANOMA CELLS TO TRY AND UNDERSTAND EXACTLY WHAT IT IS WE CAN LEARN FROM THE ARCHITECTURE OF THE CELLS. HERE IS THE EXAMPLE OF MELANOMA CELL WORK STARTED IN THE LAB, AND WE CONTINUED TO WORK IN THIS AREA ACTIVELY TO TRY AND UNDERSTAND WHAT DIFFERENCES MIGHT BE BETWEEN NORMAL AND TRANSFORMED CELLS BUT ONE OF THESE, THE GOOD THINGS ABOUT WORKING IN CELLS AND BEING AT THE NIH WAS WE BEGAN TO DEVELOP VERY USEFUL COLLABORATIONS AND ONE OF THEM WAS NHLBI LED TO WORK PUBLISHED A COUPLE YEARS AGO THAT BRIAN IN THE LAB AND LISA IN MY LAB, WHERE WE USED THESE METHODS TO VISUALIZE THE MUSCLE MITOCHONDRIA POWER GRID AND SOLVE HOW ELECTRICITY IS CONDUCTED, LEADING TO VISUALIZATION OF SINGLE MITOCHONDRIA ON THE LEFT-HAND SIDE DON'T LOOK LIKE WHAT YOU FIND IN TEXT BOOKS. THE FACT THAT WE HAVE NOW A RICH SET OF DEEP INSIGHT INTO HOW MITOCHONDRIA MIGHT BE ORGANIZED IN 3D. COLORS IN THE LOWER ANIMATION CORRESPOND TO A SINGLE MITOCHONDRIAM, AT THESE JUNCTIONS. THIS WAS UNEXPECTED. CAME FROM THE APPLICATION OF THESE TECHNOLOGIES, TWO IMPORTANT PROBLEMS. THE ONE THAT WE'RE NOW VERY MUCH INTERESTED IN, THE BEGINNING, JUST LAUNCHED WITH WITH THE INSTITUTE OF AGING WITH LOUIE, TO USE THESE METHODS TO APPLY THIS TO A VERY LARGE COHORT OF PATIENTS WHOM THEY STUDY, TRYING TO DESCRIBE AND DEVELOP GENERAL METRICS FOR AGING. FROM OUR POINT OF VIEW, WE'D LIKE TO CONTRIBUTE TO BE ABLE TO BRING STRUCTURAL BIOLOGY TO THE PROBLEM AND DEVELOP METHODS THAT MIGHT DEFINE WHAT IT IS THAT CHANGES WHEN PEOPLE AGE, AND WHAT DIFFERENCES MIGHT BE CAPTURED BETWEEN PEOPLE OF THE SAME AGE, BY DIFFERENT LEVELS OF WELLNESS, WORK BEING DONE BY BRIAN CAFFREY, LISA AND MARTHA, AND LUIGI' LAB. BRIAN HAS DEVELOPED HIGH THROUGHPUT METHODS TO GO TO YOU LARGE SECTIONS FROM HEALTHY VOLUNTEERS, SEGMENTED TO IDENTIFY MITOCHONDRIA. THIS IS NEW INFORMATION. WE WANT TO KNOW HOW THESE NETWORKS OF MITOCHONDRIA ARE CONNECTED, WE CAN ACTUALLY GO IN COMPUTATIONALLY AND EXTRACT INDIVIDUAL PIECES, WHAT YOU SEE ARE THE SINGLE CONNECTED NETWORK, PROBABLY HAS MANY MITOCHONDRIA IN IT, BUT THIS IS INFORMATION WE THINK MIGHT BEGIN TO LAY THE FOUNDATIONS FOR DEFINING WHAT TO LOOK FOR IN THIS AGING COHORT. THESE ARE EXAMPLES HOW TECHNOLOGY FOR IMAGING FOR TISSUE IMAGING REALLY IS LIKELY TO BE USEFUL LOOKING INTO THE FUTURE. SO THE REST OF MY TALK I'D LIKE TO FOCUS ON CRYO-EM. AND WHERE WE ACTUALLY -- HOW WE BENEFITED IN MANY RESPECTS FROM WORKING ON DIFFERENT PROBLEMS, TO BE ABLE TO TAKE ADVANTAGE OF WHAT HAS REALLY CHANGEED IN THE LAST SEVERAL YEARS. TO SET THE STAGEF YOU LOOK AT WHAT PEOPLE STUDIED USING CRYO-EM, AS I MENTIONED BEFORE IT'S NOT A NEW THING. A DECADE OR SO AGO, THE TRADITIONAL CRYO-EM TARGETS WERE LARGE ENTITIES. VIRUSES, HEP-V, A PROTEIN AT 7 AXON RESOLUTION. FROM THE STUDY OF LARGE ENTITIES LIKE RIBOSOMES THE IDEA THAT WE LOOK AT GARDEN VARIETY EXAMPLES OF CELLULAR PROTEINS, ENZYMES, AND THE FACT THAT THEY ARE ACCESSIBLE TO CRYO-EM AND MIGHT PROVIDE INFORMATION THAT IS BEYOND WHAT YOU MIGHT GET CRYSTALLOGRAPHY IS EXCITEMENT IN THE FIELD. IN OUR LAB WE WORKED ON A NUMBER OF THESE EXAMPLES, AT THE RIGHT END OF THE SPECTRUM. I'LL QUICKLY GO OVER EXAMPLES. FOR THOSE THAT HAVEN'T DONE CRYO-EM BEFORE, ON THE FACE IT LOOKS EASY. HERE IS ANIMATION OF WHAT A TYPICAL PERSON MIGHT DO WHEN CARRYING THIS OUT. IT STARTS WITH A DROPLET DELIVERED ON AN EM GRID 3 MILLIMETERS ACROSS, BLOTTED WITH NUMBER 1 FILTER PAPER THAT MOST LABS HAVE. AND AT THAT POINT AT LEAST 10 FILM OF LIQUID THAT CONTAINS PROTEIN OF INTEREST. WHEN THAT'S PLUNGED FROZEN, COOLED, SAMPLE VITRIFIED, PRESERVES THE SAMPLE, NEAR PHYSIOLOGICAL. IT STAYS AT LIQUID NITROGEN TEMPERATURES, DELIVERED TO THE MICROSCOPE AS THE ONE SHOWN HERE. ONCE IN, THE SAMPLE CAN BE IMAGED, SUBSEQUENT STEPS NOW LARGELY AUTOMATED, THE UNIT OF INFORMATION THAT EMERGES IS PROJECTION IMAGE LIKE THIS WHICH I MENTIONED AT THE VERY BEGINNING, EACH ONE OF THESE IS A PROJECTION IMAGE OF THE SINGLE PROTEIN MOLECULE, WE COLLECT THOUSANDS OF IMAGES LIKE THIS, THEY CAN BE CUT UP, PROCESSED, EVENTUALLY A FEW STEPS LATER OUTCOMES, DENSITY MAP, 3D MODEL OF THE PROTEIN OF INTEREST, AT THE RESOLUTION OF DATA AND OPERATOR ABLE TO GET. SO THIS WORKFLOW IS NOT REALLY NEW. THIS IS WHAT WE AND MANY OTHERS IN THE FIELD HAVE BEEN DOING FOR A WHILE. SO A DECK EIGHT OR SO AGO WHEN WE BEGAN TO WORK IN CRYO-EM OR FOCUS WAS ON METABOLIC PATHWAYS. OUR INTEREST WAS TO TAKE THIS METABOLIC PATHWAY SHOT WHICH THE BIOCHEMISTS OF PREVIOUS GENERATION STUDIED AND HAVE IT COME ALIVE IN 3D. WE STUDIED. WE KNOW WHAT ENZYMES ARE. BUT WHAT IF WE COULD FIND WHAT THEY LOOK LIKE IN 3D? THIS REMAINS AN AMBITION FOR US TO PULL TOGETHER THIS WHOLE METABOLIC PATHWAY SHOT INTO 3D. AND WE BEGAN TO LOOK AT METABOLITES, AND THERE WERE SOME EARLY SUCCESSES, THAT LED TO A VISIT FROM THE LEADERSHIP AT THE NIH. MICHAEL GOTTESMAN, ELISA, STONY CLIFFORD AND OTHERS, LOOKING AT IN THE SLIDE THE WORK OF JACQUELINE MILL AT THAT TIME WHEREAS A RESULT OF STUDYING THESE DEHYDROGENASE COMPOUNDS, HOW IT MIGHT BE SYNTHESIZED AND ANNULAR GAP MIGHT BE USED TO SHUTTLE TWO CARBONS FROM PYRO VATE INTO THE CORE WHERE IT MET UP AT ACETYLTRANSFERASE ENZYME, MANY YEARS OF WORK, AND THE HOPE WE COULD DO THIS WITH SMALLER MOLECULES WAS REALLY A DREAM BUT NEVER REALIZED UNTIL DEVELOPMENTS IN THE FIELD LED TO BETTER DETECTORS, FOR US IN THE LAB KEY EVENTS WERE TWO PAPERS FROM UCSF, THAT SHOWED YOU COULD GET STRUCTURES AT NEAR ATOMIC RESOLUTION OF MEMBRANE PROTEINS AND LARGE COMPLEXES WITH NEW FAMILY OF DETECTORS, KNOWN AS THE K-2 DETECTORS DEVELOPED BY GATAN. OUR INTEREST WAS TO USE THESE DETECTORS AND PUSH FURTHER, IN THE EM FIELD THREE AXOMS IS CONSIDERED LIKE THE SUN BARRIER, WORK THAT ALLEN MERCK IN THE LAB PIONEERED LED TO PUSH THESE TO COLLECT PROGRESSIVELY HIGH RESOLUTION, IN WORK HE AND ALBERTO AND SUZIE DID LED TO FOR US CERTAINLY A BREAKTHROUGH AT THAT TIME, DEMONSTRATE YOU COULD USE CRYOELECTRICRON MICROSCOPY APPROACHING TWO ANGSTROMS, ASIDE FROM THE FACT WE HAD HIGH RESOLUTION THE FACT WE COULD VISUALIZE CHAINS OF WATER MOLECULES IN SAMPLE OF EMBEDDED VITREOUS, NOW YOU LOOK AT THE CHEMISTRY OF HOW THESE THINGS WORK. ANOTHER HIGHLIGHT OF THIS WORK WAS THE STRUCTURE WE DETERMINED IF A SMALL MOLECULE BOUND TO A DRUG SHEET AND WE COULD LOOK AT WATER MOLECULES AT THE JUNCTION, THESE WERE EXCITING TIMES. ONCE WE GOT THIS TO THIS STAGE,& IT BECAME CLEAR WE NEEDED TO DEMONSTRATE APPLICATION TO VARIETY OF EXAMPLES, VARIETY OF SYSTEMS, WE FOCUSED ON APPLYING THIS TO THREE CLASSES, ONE METABOLIC ENZYMES OF INTEREST TO THE CANCER INSTITUTE, SECOND MEMBRANE PROTEIN AND WENT AFTER THESE AND COULD NOT HAVE DONE THIS WITHOUT EXTREMELY PRODUCTIVE COLLABORATIONS WE HAD BOTH ON CAMPUS AND OFF CAMPUS, THE WORK WITH HYDROGENASE LET TO THIS, GLUTAMATE HYDROGENASE COULD BE RESOLVED AT HIGH RESOLUTION. IN THE 330 KILODALTON ENZYME, VISUALIZE THE CORE AT HIGH RESOLUTION, DESPITE FLEXIBILITY ON THE PERIPHERY, THESE A FEATURE MANY PROTEINS WILL HAVE AND THIS TODAY THIS IS ONE OF THE HIGHEST RESOLUTIONS IF NOT THE HIGHEST RESOLUTION STRUCTURE REPORTED BY SINGLE PARTICLE CRYO-EM. WE APPLIED THIS TO OTHER DEHYDROGENASES BUT I WANT I WANT TO MENTION IS WORK WE DID WITH MUTANT IDH, IMPLICATED IN CANCERS, AND IN PATIENTS THAT HAVE A SINGLE POINT MUTANT IN IDH1, 132, RELATED TO CYSTEINE OR OTHER RESIDUES, THERE'S A CHANGE IN METABOLISM, INSTEAD OF PRODUCING ALPHA KETA, IT PRODUCES AN ONCOMETABOLITE. WORK AT NCATS AND MANY OTHER ENTITIES FOCUSED ON FINDING MOLECULES THAT TARGET MUTANT BUT NOT WILDTYPE FORM. IN A PRODUCTIVE COLLABORATION WITH NCATS, WE WERE ABLE TO LOOK AT THE COMPLEX OF THE MOLECULE THAT THEY HAD DEVELOPED AT NCATS BUT COULD NOT GET X-RAY STRUCTURES WITH MUTANT IDH1, AT THAT TIME THIS WAS THE SMALLEST PROTEIN FOR WHICH NEAR ATOMIC RESOLUTION CRYO-EM STRUCTURES COULD BE DETERMINED. THE FACT WE COULD LOCATE THIS MOLECULE OPENED THE DOOR TO THE NEXT STEP WHICH IS TO UNDERSTAND WHY IT MIGHT NOT HAVE CRYSTALLIZED SO EASILY AND WHAT YOU SEE HERE IS THAT THE CONSEQUENCE OF BINDING OF THIS MOLECULE LEADS TO PROTEIN CONFIRMATION CHANGE AND ESSENTIALLY CRYO-EM STRUCTURE NOW BECOMES AN ASSAY FOR BINDING OF THIS MOLECULE, IN ADDITION WE CAN NOW COMPARE THE LOCATION OF THE BINDING SITE TO OTHERS THAT HAVE BEEN REPORTED, ONE FROM GSK, ONE FROM SANOFI, A VERY EXCITING AREA. SECOND EXAMPLE, WORK ON P 97, LED IN THE LAB BY SUJI, WHO I WANT TO ACKNOWLEDGE, SADLY HE PASSED AWAY LAST YEAR FROM CLL, ACTUALLY WAS HERE AT THE NIH CLINICAL CENTER FOR MANY OF HIS LAST MONTHS. HIS WORK LED TO THE STRUCTURAL BIOLOGY OF P 97 IN THE APP BOUND FORM, MORE IMPORTANTLY AT THAT TIME THE FIRST EXAMPLE WHERE WE COULD RESOLVE SMALL MOLECULE BOUND AT NEAR ATOMIC RESOLUTION PUBLISHED IN "SCIENCE" IN 2016, AND FOR THE FIRST TIME WE COULD THEN SHOW THAT USING CRYO-EM WE COULD DERIVE LIGAND PLOTS LIKE THIS WHICH WE WERE USED TO FROM X-RAY CRYSTALLOGRAPHY LOOKING AT INTERACTION OF EACH DRUG WITH ITS SURROUNDINGS, IN THE COURSE WE RECOGNIZED SOMETHING IMPORTANT, WHY THIS MOLECULE WAS SO DIFFICULT TO CRYSTALLIZE IN THE PRESENCE OF ATP. WHAT YOU SEE HERE IS BECAUSE IN THE CRYO-EM WORK WE WERE ABLE TO RESOLVE SIMULTANEOUSLY PRESENCE OF MULTIPLE CONSERVATIONS, SHOW THE BINDING OF ATP, TWO MOLECULES OF ATF FIRST LED TO CHANGE IN ONE OF THE LIGAND BINDING DOMAINS, FOLLOWED BY SECOND SET OF CHANGES, THESE CONSUMMATION CHANGES THAT TAKE PLACE IN THE ENZYMES THAT REALLY IS THE POWER OF CRYO-EM. THE APPLICATION OF MEMBRANE PROTEINS IS VERY EXCITING, AND THIS WAS ENABLED ESSENTIALLY SOLELY REALLY WITH THE COLLABORATION WE HAD WITH MARK MAYER AT THE NICHD, WHO IS PIONEERING THE WORK ON GLUTAMATE RECEPTORS FOR A LONG TIME. IN WORK THAT JOEL MIRESON AND SALVATORE LOOK THE LEAD IN MY LAB, ABLE TO APPLY METHODS TO DETERMINE STRUCTURES OF GLUTAMATE RECEPTORS, AND LED TO A NUMBER OF INSIGHTS IN THE ARCHITECTURE AND GATING CYCLE OF THESE RECEPTORS. I WANT TO HIGHLIGHT TWO THINGS THAT ARE RELEVANT TO OUR OWN ADVANCE IN USING THESE METHODS, ONE OF THEM BEING THAT THIS IS THE FIRST INSTANCE IN OUR LAB WHERE WE WERE ABLE TO USE NATIVE PROTEINS AND DEMONSTRATE DENSITY FOR SUGAR MOLECULES, THE LOWER RIGHT ARE DENSITIES FOR SUGAR MOLECULES AND CARBOHYDRATES, I THINK THIS ABILITY TO VISUALIZE AT LEAST THE ORDERED PIECES WILL PROBABLY BE VERY IMPORTANT IN STUDIES OF MEMBRANE PROTEINS. INSIGHT UNEXPECTED, UNDERSTANDING OF THE EXTENT OF THESE CHANGES THAT TAKE PLACE WITH DESENSITIZATION OF RECEPTOR, CERTAINLY GOING INTO THIS PROJECT WE HAD NO EXPECTATION THAT WE WOULD UNCOVER THESE TYPES OF VERY LARGE CHANGES, AGAIN WERE ABLE TO DO THIS WITH CRYO-EM BECAUSE WE CAN CLASSIFY MOLECULES, COMPUTATIONALLY, AND DETERMINE THE PRESENCE OF MULTIPLE CONFIRMATIONS. ONE FINAL EXAMPLE OF WHERE MEMBRANE PROTEIN CONFIRMATION CHANGES WERE ANALYZED BY CRYO-EM IN THE WORK THAT WE WERE DOING, AND THIS BEGAN AS A COLLABORATION EDUARDO, TRIGGERED BY AN INTERESTING CONUNDRUM IN THIS FIELD BECAUSE EDUARDO HAD BEEN STUDYING THE MAGNESIUM CHANNEL FOR A LONG TIME, AND HAD POINTED OUT TO US THAT THE STRUCTURES OF THIS ION CHANNEL, WERE THERE MANY BY CRYSTALLOGRAPHY, ESSENTIALLY IDENTICAL WHETHER OR NOT MAGNESIUM WAS PRESENT, FLEW IN THE FACE OF BIOLOGICAL WORK THAT SUGGESTED LARGE CHANGES INDUCED BY THE PRESENCE OR ABSENCE OF MAGNESIUM, AND THIS WAS A PROBLEM THAT WE WORKED ON IN THE LAB, AND THIS WAS LED BY DOREEN MATTHEWS, POSTDOC IN THE LAB, WHERE WE SYSTEMATICALLY WENT AFTER LOOKING AT THE STRUCTURES OF THESE COR-A, IN LIPIDS AND DETERGENTS, PUBLISHED IN "CELL" IN 2016. WE WERE ABLE TO SHOW AT FIRST IN THE MAGNESIUM BOUND STATE THE STRUCTURE OF COR-A, SMALLER PROTEIN, CONSIDERED WELL OUT OF REACH A YEAR AGO FOR CRYO-EM BUT 200 KILODALTON MEMBRANE PROTEIN COULD BE STUDIED BY CRYO-EM, THE STRUCTURE IN MAGNESIUM BOUND STATE WAS SIMILAR TO X-RAY CRYSTALLOGRAPHY BEFORE. WHEN YOU TAKE THE MAGNESIUM AWAY, THE PROTEIN APPEARS TO FALL APART. DOREEN SHOWED THIS WAS ONE OF MANY OPEN STATES POPULATED ESSENTIALLY ENSEMBLE OF STATES ESSENTIALLY POPULATED LIKELY BECAUSE OF MAGNESIUM BINDING SITES ARE AT INTERFACE BETWEEN THE PROTIMERS, ENTITIES THAT OPEN AND CLOSE SYMMETRICALLY CLEARLY NO LONGER THE CASE, MAY WELL BE TRUE THAT OTHER ION CHANNELS ALSO DISPLAYED THIS TYPE OF ASYMMETRIC OPENING MECHANISMS. THE THIRD EXAMPLE OF PROTEINS WE WANTED TO ACTUALLY DEMONSTRATE THAT WE COULD TACKLE IN OUR LAB WERE NUCLEIC'S ACID PROTEIN COMPLEXES, COLLABORATIVE FROM MEMORIAL SLOAN-KETTERING WHERE WE BEGAN TO WORK ON CRISPR/CAS SURVEILLANCE COMPLEX, A FIELD THIS AUDIENCE NEEDS NO INTRODUCTION TO, IT'S DIFFICULT TO KEEP UP CAN SUBTYPES BEING DISCOVERED EVERY WEEK IT TURNS OUT. BUT THE WORK IN THE LAB FOCUSED AT THE TIME, WE BEGAN THIS ON A PARTICULAR CLASS OF THESE COMPLEXES, TYPE 1F, AND IN THE LAB, A POSTBAC STUDENT, LED THIS WORK, WAS ABLE TO WORK WITH THE LAB, OTHER PEOPLE IN THE LAB, TO DETERMINE THE STRUCTURE OF THIS COMPLEX IN THE PRESENCE AND ABSENCE OF BOUND DNA. AND I HAVE TO SAY THIS IS AMONG THE MOST CHALLENGING PROBLEMS THAT WE WORKED ON, BECAUSE THIS IS AN ASYMMETRIC COMPLEX, NINE POLYPEPTIDES, DOUBLE STRAND DNA, SHE AND ALBERTO AND ALLEN WORKED TOGETHER TO GET TO THESE RESOLUTIONS, THREE ANGSTROMS, VISUALIZE THE ENTIRE COMPLEX. YET AGAIN HERE DYNAMICS IS REALLY WHAT WAS REALLY INTERESTING WHEN WE COMPARE THE BINDING OF THE -- COMPARE THE STRUCTURE BEFORE AND AFTER BOUND DNA THERE'S DRAMATIC CHANGE IN PITCH THAT YOU CAN APPRECIATE THE STRUCTURE ON THE RIGHT-HAND SIDE ESSENTIALLY THE BINDING OF THE DNA LEADS TO REDUCTION IN PITCH, ESSENTIALLY CLOSES AND OPENS WITH THE BINDING OF DNA, AND THIS ABILITY OF THESE COMPLEXES, THESE ABILITIES TO ESSENTIALLY BE MODULATED WITH DNA BINDING IS VERY LIKELY SIGNIFICANT FUNCTIONALLY. IN ADDITION TO BEING ABLE TO LOOK AT THE RESULTS OF DNA BINDING WE WERE ALSO ABLE TO LOOK AT A NUMBER OF INHIBITORS, THESE ARE DEVELOPED BY ALAN DAVIDSON IN TORONTO. AND AMONG THE MANY STRUCTURES THAT WE DID WITH THESE INHIBITORS, WHAT'S MECHANISTICALLY SATISFYING IS WE CANNOT ONLY DETERMINE THE STRUCTURE BUT NOW COMPARE WHERE THIS SITS IN RELATION TO THE DNA MOLECULE AND IN THE CASE OF ACRF 10 AND OTHERS THE BINDING SITE OF THESE INHIBITORS IS ALMOST EXACTLY WHERE THE DNA BINDS. ONCE AGAIN, THESE ARE PHASE DRUG INHIBITORS SO INTERPLAY BETWEEN BIOLOGY AND PHAGE IS FASCINATING. FINAL EXAMPLE OF NUCLEIC ACID COMPLEX, PUBLISHED IN "SCIENCE," LED BY A POSTDOC IN THE LAB, AN ALL CCR COLLABORATION WITH ALEX KELLY'S LAB, ANOTHER LAB AND MY LAB FOCUSING TO TAKE ON AN INTERESTING PROBLEM TO LOOK AT THE STRUCTURE OF PROTEIN COMPLEX OF THE CENTROMERE, RECOGNITION OF THE CENTROMERE DRIVEN BY NUCLEOSOME, ONE OF THE CHALLENGES IN THE FIELD IS THE UNIQUE ASPECT, SUSPECTED TO DO WITH SPECIFIC RESIDUES AT INTERFACE BUT THE STRUCTURE OF CENP ITSELF WAS UNKNOWN, YOU SEE THE PARTIAL IN ORANGE, 30 KILODALTONS OR SO, WE'RE WERE ABLE TO USE CRYO-EM METHODS TO GET AT THE STRUCTURE OF CENP-N AND POSITION IN THE CONTEXT OF THE CENP-N NUCLEOSOME. TECHNICAL AS ASPECT, ABILITY TO BUILD POLYPEPTIDE, FOLD INTO THE DENSITY MAP DESPITE LOW RESOLUTION, CHANGED THE WAY -- REFLECTIVE OF THE WAY CRYO-EM IS NOW BEING USED, WITHOUT BIOCHEMISTRY AND BIOLOGY WE WOULD HAVE NO IDEA HOW MECHANISTIC ASPECTS OF THIS COMPLEX. ANOTHER INTERESTING ASPECT OF THIS WORK AS WE WERE DOING THIS, TO PUBLISH THIS, A FEW DAYS LATER WORK APPEARED FROM CALVIN AND ANDREA'S LAB, WE WERE NERVOUS WHETHER WE HAD DONE THIS RIGHT OR WRONG BUT FORTUNATELY AND WE COULD SLEEP WELL AFTER THIS WE COULD SHOW THE STRUCTURES INDEED MATCHED VERY WELL. SO THESE ARE SEVERAL EXAMPLES WHERE I THINK IT'S HOPEFULLY CLEAR CRYO-EM METHODS CAN TAKE ON RELATIVELY CHALLENGING EXAMPLES, THINGS THAT MIGHT BE DIFFICULT TO CRYSTALLIZE, MIGHT HAVE MULTIPLE CONFIRMATIONS, BUT ONE OF THE THINGS WE'VE ALWAYS WORRIED ABOUT IS TO SEE IF WE COULD ACTUALLY DO BETTER WITH RESOLUTION. THIS IS WORK ALBERTO IN THE LAB HAS BEEN REALLY EXPLORING AND PIONEERING FOR MANY YEARS, WHETHER WE CAN ACTUALLY MAKE BETTER USE OF THE IMAGES THAT COME OUT OF ELECTRON DETECTORS, AND CORRECT FOR THINGS THAT GO BEYOND CORRECTING AT THE WHOLE IMAGE LEVEL, SLIGHTLY TECHNICAL, BUT IN THESE IMAGES IN PRINCIPLE BECAUSE EACH ONE OF THESE MOLECULES EXPERIENCES ELECTRON BEAM IN A SLIGHTLY DIFFERENT ENVIRONMENT, IF YOU COULD FIGURE OUT HOW TO CORRECT FOR THE LOCAL VARIATIONS, THE HOPE WAS WE MIGHT BE ABLE TO DO SLIGHTLY BETTER, ALBERTO WAS SUCCESSFUL IN THIS EXERCISE AND THIS PAPER HAS JUST BEEN ACCEPTED A COUPLE DAYS AGO. AND WE'VE NOW BEEN ABLE TO ADVANCE RESOLUTION OF FURTHER BY MAKING THESE CORRECTIONS, TO GIVE YOU A FEELING FOR WHERE WE'VE COME COME SINCE EARLY WORK IN 2015 YOU SEE THE TOP ROW, BASICALLY WHAT WE WERE EXCITED ABOUT, NOW IN THE BOTTOM YOU SEE THIS IS WHETHER WE -- WHETHER WE ARE, DELINEATION OF INDIVIDUAL CONTOURS OF ATOMS, A WAY TO ILLUSTRATE IS IN THIS ANIMATION WHAT I'M SHOWING YOU EXAMPLES OF EACH TYPE OF AMINO ACID, WHERE THE DENSITY IS VERY, VERY CLEAR, AND FOLLOWS THE SHAPES, THE INDIVIDUAL CONTOURS OF THE NON-HYDROGEN ATOMS, YOU CAN SEE THIS ALSO FOR RESIDUE IN PARTICULAR WHERE HOLES IN THE MIDDLE OF THE RINGS ARE CLEAR, YOU CAN SEE SLIGHTLY LARGER DENSITY OF SULPHUR ATOMS, EVEN FOR NEGATIVELY CHARGED RESIDUES WHERE PREVIOUSLY HIGHER DOSES WE LOST DENSITY IN THE CARBOSYLATE, NOW RECOVER AT MOIETIES, SUCH AS LYSINE WE CAN SEE DENSITY TO THE END SUGGESTING THEY ARE REALLY WELL ORDERED. WE'RE AFTER TO DO THIS AND LOCATE DRUG MOLECULES AT HIGH HIGH RESOLUTION, NOW LETTING US MAP BINDING SITE IN MORE DETAIL. WHAT YOU SEE AT THE LEFT IS VISUALIZATION OF THE BINDING SITE OF PET-G, REPRESENTING A SINGLE CARBON ATOM, DENSITY FOR THE DRUG IS WEAKER BECAUSE IT'S BOUND NON-COVALENTLY. THE VERY LAST EXAMPLE IS A PAPER JUST ACCEPTED PROVISIONALLY YESTERDAY, IT'S WORK ON HUMAN RHODOPSIN, INHIBITORY G-PROTEIN WITH SPECIAL SIGNIFICANCE FOR ME BECAUSE I CAME TO THE NIH AND ACTUALLY WAS GIVEN A JOB TO WORK ON RHODOPSIN, SO FINALLY IN SOME SMALL MEASURE WE CAN ACTUALLY SHOW WE CAN MAKE A CONTRIBUTION TO STRUCTURE OF RHODOPSIN AND THE SECOND REASON THIS IS VERY SIGNIFICANT IS THIS COLLABORATION WITH ERIC'S LAB GREW OUT OF A VISIT THERE MANY MONTHS AGO, THE MIDDLE OF LAST YEAR, AND THE PERSON WHO FOUNDED THIS INSTITUTE WAS GEORGE VANDERBILT WHO WAS THE ONE WHO WAS A DIRECTOR OF THE NCI, THE CCR, THE INTRAMURAL PROGRAM WHEN I CAME. AND I THINK THIS IS WHAT I TOLD HIM WE WOULD DO WHEN I CAME TO NIH SO I FEEL PARTICULARLY SATISFIED. FINALLY, IT'S INTERESTING TO COMPARE STRUCTURAL CHANGES IN THE 7 HELIX PROTEINS, WHEN I FIRST CAME TO NIH THE WORK I HAD DONE ON BACTERIAL RHODOPSIN LED TO DISCOVERY OF CHANGES WE THOUGHT WERE LARGE AT THAT TIME, A FEW ANGSTROMS, BUT CHANGES NOW THAT TAKE PLACE WITH THE G-PROTEIN COUPLED RECEPTORS ARE SUBSTANTIALLY LARGE, BUT THE INTERESTING MECHANISTIC INSIGHT IS THE HELICES ARE SIMILAR AS THE HELIX 6 PLAYS AN IMPORTANT ROLE, AS DO SOME OTHER HELICES, BUT IT'S BEEN A LONG -- CERTAINLY BEEN AN IMPORTANT ADVANCE TO NO LONGER NEED TO DEPEND ON 2D CRYSTALS TO GET AT THESE KINDS OF STRUCTURES. THE LAST COUPLE MINUTES I'D LIKE TO SHARE MY THOUGHTS ON HOW THIS TECHNOLOGY MIGHT BE DISSEMINATED MORE GENERALLY, AND FOR SOME YEARS NOW IN DISCUSSIONS I'VE HAD WITH THE LEADERSHIP OF NIH, FRANCIS AS WELL AS MANY INSTITUTE DIRECTORS IT WAS CLEAR CERTAINLY WHAT I BENEFITED MIGHT BE USEFUL FOR THE NIH TO EXPAND THESE EFFORTS. THIS IS NOT JUST NIH EFFORT. IN A PIECE THAT I WROTE WITH DAVID STEWART AND NIKOLAI BRESIA IN SPAIN AND DAVID IN THE U.K., THERE ARE EFFORTS ACROSS THE WORLD TO MAKE THESE TECHNOLOGIES MORE EASILY AVAILABLE TO DEMOCRATIZE ESSENTIALLY AND THIS LED TO THE FOUNDING OF CENTERS EUROPE AND THE U.K. AND NIH IS TAKING A MAJOR LEAP FORWARD AND HOPEFULLY ANNOUNCING OPENING OF SEVEN CENTERS. A SMALL WAY WE ACTUALLY ALREADY HAVE STARTED TO DO THIS AT THE NCI, THANKS TO THE SUPPORT OF HAROLD VARMUS AND DOUG LOWY WHO SUPPORTED A PROJECT TO LAUNCH A NATIONAL FACILITY AT NCI TO DISSEMINATE TO THE EXTRAMURAL WORLD. RIGHT NOW WE HAVE A SINGLE MICROSCOPE, TWO THIS SUMMER, PERHAPS THREE THE NEXT YEAR AND PEOPLE DO THIS. THIS IS DONE UNDER THE AUSPICES OF THE LIDOS, ETHAN IS THE DIRECTOR OF THE FREDERICK NATIONAL LAB PROGRAM, DWIGHT OVERSEES SOME, ULRICH AND MATT ARE THE TEAM THAT RUN THIS. WHAT'S REALLY SATISFYING IS THAT IN THE SHORT TIME WE'VE BEEN OPERATIONAL IT'S BEEN OPEN TO THE ENTIRE EXTRAMURAL ESTABLISHMENT IN THE U.S., E'VE HAD OVER 20 INSTITUTIONS COME TO USE IT, OVER 80 PROJECTS, AND THE USE OF THE FACILITY GROWS UNABATED. I THINK THAT IN THE YEARS TO COME NIH HOPEFULLY WILL INVEST MORE IN DISSEMINATING TECHNOLOGY AND MAKE SURE THAT IT ACTUALLY CAN GET TO BIOLOGISTS WHO CAN BENEFIT FROM THIS THE MOST. TO SUMMARIZE, THE CRYO-EM REVOLUTION IS HERE, A GOAL IN STRUCTURAL BIOLOGY, BUT ITS REAL GOAL IN PROVIDING INSIGHTS INTO MECHANISM AND ESPECIALLY STRUCTURAL ASPECT, THAT MIGHT NOT BE POSSIBLE BY THE OTHER VERY POWERFUL METHODS CRYSTALLOGRAPHY, SO LOOKING INTO THE FUTURE IT WILL BECOME AN IMPORTANT TOOL IN ADDITION TO ALL THE ONES THAT WE HAVE, AND FINALLY I DO WANT TO SAY THAT IN THE BROADER SENSE OF THE WORD SOME METHODS WE USED TO INTERROGATE VIRUSES, CELLS AND TISSUE, DON'T NECESSARILY USE CRYOMICROSCOPY. IT'S STILL USEFUL TO VIEW THIS AS A BROAD DISCIPLINE WHERE WE ARE USING ELECTRONS TO INTERROGATE 3D STRUCTURES, PROTEINS, OR OF VIRUSES OR CELLS, AND THE ABILITY TO GO INTO 3D IS OBVIOUSLY VERY POWERFUL. I'D LIKE TO END WITH MY DEEP GRATITUDE TO THE MANY LAB MEMBERS WHO WORK WITH ME ON ALL OF THESE PROJECTS, AND REALLY LED MANY OF THESE PROJECTS, CURRENT ONES ARE HIGHLIGHTED IN BLUE, QUICKLY MENTION THEM, ALBERTO HAS LED THE WORK ON THE HIV WORK, SINGLE PARTICLE WORK, INSTRUMENTAL IN MANY TECHNICAL DEVELOPMENTS IN THE LAB. BRIAN CAFFREY Ph.D. STUDENT, AND JEAN PHILIPPE, POSTDOCS IN THE LAB WORKING ON VARIOUS OTHER PROJECTS. VERONICA HAS BEEN MOST OF THE ANIMATIONS FOR THE CRYO-EM WORK SHE MADE, I'M GRATEFUL TO HER FOR WORKING WITH ME AND MEMBERS OF THE LAB TO BE ABLE TO PRESENT APPEALING IMAGES OF THE WORK THAT WE DO, AND IN THE SAME VEIN GRATEFUL TO DONNY BLISS WHO MAY BE IN THE AUDIENCE WHO FOR MANY YEARS HELPED US PUT A FACE TO THE STRUCTURES, DENDRITIC, HIV, ALL OF THAT WAS REALLY DONNY'S WORK. JOSEPH, ANDRE, WORK IN THE LAB AND OTHER PROJECTS, I MENTIONED ALISA WORK WITH MUSCLE TISSUE, DOREEN'S WORK ON COR-A, ALAN MERCK, A BRILLIANT MICROSCOPIST, REALLY HELPED US ALL THE WAY TO REALLY HARNESS THE POWER OF THE RESOLUTION REVOLUTION BY WORKING AT THE MICROSCOPE AND ALSO GENERAL INSIGHT HOW TO ADVANCE TO HIGH RESOLUTION, YANIS IS A POSTDOC IN THE LAB, THAT I HAVE NOT MENTIONED, DISCUSSED HER WORK, ERIN TRAN WITH US FOR A DECADE PUSHED MUCH OF THE HIV TOMOGRAPHY, AND ANDREW A POSTBA WORKING WITH HER IN THE LAB. COLLABORATORS, TOO MANY TO GO THROUGH, SUFFICE IT TO SAY THAT ALL THE WORK THAT I WAS ABLE DO HERE WAS BECAUSE OF MY COLLABORATION WITH JACQUELINE MILNE, ALSO HAPPENS TO BE MY WIFE. WHEN I FIRST CAME GEORGE RANDALL SAID AS CONSENTING ADULTS WE HAD PERMISSION TO WORK TOGETHER. I'M REALLY GRATEFUL FOR THAT OPPORTUNITY, AND ALSO THE MANY COLLEAGUES AT THE NIH I'VE MENTIONED, OVER THE YEARS, FINALLY THIS IS THE PRESENT COMPOSITION OF MY LAB WITH SOME OF HER COLLEAGUES AT HER ANNUAL RETREAT, MY WEBSITE, AND I'M HAPPY TO TAKE ANY QUESTIONS. THANK YOU. [APPLAUSE] >> YES, DO YOU HAVE ANY PLANNED APPLICATIONS FOR THIS FROM A DIAGNOSTIC REALM? YOU WERE TALKING ABOUT MELANOMA, ET CETERA. ARE THERE APPLICATIONS PENDING THAT YOU'RE STUDYING IN TERMS OF DIAGNOSIS, IN TERMS OF SOME TUMORS, TUMOR MARGINS, IT GOES INTO MORE DEPTH THAN CONVENTIONAL EM BUT MIGHT BE USEFUL IN SOME TYPE OF SECRETIONS BY TUMORS AND MARGINS, ET CETERA, VERY -- CASES, WOULD THIS BE ECONOMICAL TO BE USED IN CASES SUCH AS THAT? >> THE QUESTION IS WHETHER 3D METHODS USING FOCUSED ION BEAMS MIGHT BE APPLICABLE IN A CLINICAL SETTING. ACTUALLY WHEN WE BEGAN TO DEVELOP THIS THAT WAS THE PRIMARY GOAL. WE QUICKLY REALIZED THAT EVEN THEN THE METHODS WERE VERY SLOW AND TEDIOUS, STILL ARE. CERTAINLY NOT ECONOMICAL. SO I THINK THERE NEEDS TO BE INCREASE IN SPEED, IN SEVERAL ORDERS OF MAGNITUDE AT DATA COLLECTION AND DATA ANALYSIS STAGE. IT ADDS A NEW DIMENSION, NEW LAYER OF INFORMATION, THAT DOES NOT COME OUT OF THE CONVENTIONAL PATHOLOGICAL ASSAYS. THAT'S MY HOPE. >> OKAY. THANK YOU. >> GREAT TALK. CAN YOU COMMENT ON FUTURE PROSPECTS OF AREAS WHERE IONATION MAY HELP AND REDUCE COST AUTOMATION, WHERE CAN YOU AUTOMATE? >> THE QUESTION IS IS THERE ANYTHING LEFT TO BE AUTOMATED, IS THAT WHAT YOU'RE ASKING? I THINK JOB SECURITY IS GOOD. I THINK LEARNING AS WE GO, MANY THINGS WE NOW DO ARE MORE AUTOMATED THAN BEFORE, BUT OBVIOUSLY I THINK THE -- IF YOU LOOK AT OTHER TECHNOLOGIES, THE ONE THING THAT SEEMS CERTAIN IS THAT THINGS GET QUICKER, WHETHER THEY GET BETTER WE DON'T KNOW BUT CERTAINLY THEY ARE QUICKER. >> THERE'S BEEN SOME DISCUSSION BETWEEN THE CRYSTALLOGRAPHER AND CRYO-EM TEAM ON DEFINITION RESOLUTION. DO YOU CARE TO COMMENT? >> YOU'RE DOING ME NO FAVORS ASKING THIS QUESTION. THE CRYSTALLOGRAPHERS ARE USED TO DEFINING RESOLUTION IN MATHEMATICAL USE, DIFFRACTION PATTERNS. IN THE CRYO-EM WORLD RESOLUTION IS A LOOSE MEASURE, AND THERE'S A PIECE THAT I WROTE, PUBLISHED A YEAR OR SO AGO, WHETHER WE ACTUALLY POINT OUT WHAT MANY OF US HAVE KNOWN ACTUALLY ALMOST EVERYBODY IN THE FIELD KNOWS, THE WAY WE MEASURE RESOLUTION IN THE EM FIELD IS ESSENTIALLY A MEASURE OF CONSISTENCY, AND NOT TRUE MEASURE OF RESOLUTION. SO THERE'S MANY WAYS YOU CAN CORRELATE SIGNAL, NOISE. SO THE CONVENTIONAL MAPS, PERHAPS REFERRING TO SHELL CORRELATION, IT'S A PRIMITIVE MEASURE OF RESOLUTION. THE LAST PIECE OF PRESENTED, IF YOU CAN SEE THE ATOMS THAT'S ATOMIC RESOLUTION. THE NUMBERS DON'T MATTER SO MUCH. I THINK THIS IS A DEBATE THAT GOES ON AND ON. BUT I THINK IF YOU KEEP IN MIND THAT ALMOST ALL OF THESE MEASURES ARE JUST BEING PROVIDING INDICATIONS OF QUALITY OF MAP, NOT TO BE USED AS ABSOLUTE NUMBERS, THAT'S PROBABLY A GOOD WAY TO BE. >> NO FURTHER QUESTIONS, LET'S THANK THE SPEAKERS. THERE'S A RECEPTION IN THE LIBRARY.