Abstracts & Bios

(Near-final)


Christopher BARNES – Where are we now? One year of COVID-19 structural biology

ABSTRACT: TBA

BIO: Dr. Christopher Barnes is a newly appointed Assistant Professor of Biology and ChEM-H Institute Scholar at Stanford University. Before arriving at Stanford, Dr. Barnes earned degrees in Chemistry and Psychology from the University of North Carolina at Chapel Hill and completed his PhD thesis at the University of Pittsburgh. It was during his doctoral work that Dr. Barnes begin to develop the structural tools necessary to understand the complexity of macromolecular interactions. Recently, in the lab of Dr. Pamela Bjorkman at the California Institute of Technology, he expanded his skillset to include cryo-electron microscopy techniques to help define the structural correlates of antibody-mediated neutralization of HIV-1 and SARS-CoV-2. As a recipient of the prestigious HHMI Hanna Gray Fellowship and Chan-Zuckerberg Biohub Investigator award, Dr. Barnes hopes to use structural methods to rationally design protein-based immunogens capable of eliciting broad and potent neutralizing antibodies against emerging zoonotic viruses.

Ariane BRIEGEL Exploring how bacteria interact with their environment using cryo-electron tomography

ABSTRACT: TBA

BIO: Ariane Briegel is a professor at the Leiden University (The Netherlands) and Co-director of The Netherlands Center for Electron Nanoscopy (NeCEN). She has over 20 years of experience using cryo-electron microscopy to study bacterial and archaeal ultrastructure. The Briegel laboratory focuses on investigating how microbes sense and respond to their environment. In order to gain insight into the structure and function of the molecular complexes involved in these behaviors, the lab uses electron cryotomography and correlative microscopy methods.

Mihnea BOSTINA – Structural basis for Seneca Valley virus cancer cells tropism

ABSTRACT: TBA

BIO: TBA

Shujun CAI – Cryo-electron tomography of the Parkinson’s disease-linked lipid transfer protein VPS13C in situ

ABSTRACT: VPS13 is a lipid-transfer protein expressed in all eukaryotes (Ugur et al. 2020, PMID: 32563856) and encoded by 4 distinct genes in mammals. Mutations in each of these genes result in neurological diseases. Loss of function mutations in the human VPS13C gene, which encodes a protein localized at contacts between the ER and late endosomes/lysosomes, are responsible for rare cases of early onset familial Parkinson’s disease (PD). A low-resolution negative-stain EM structure of purified yeast Vps13p reveals an elongated rod with a loop structure on one end (PMID: 28122955) and a cryo-EM structure of a portion of the rod shows the presence of a groove traveling along its length (PMID: 32182622). However, how VPS13 proteins are arranged at membrane contact sites in intact cells remains unclear. A precise elucidation of this arrangement is critical toward the understanding of the mechanisms of lipid transport by this family of proteins and to validate the hypothesis that they act by providing a hydrophobic bridge for the transfer of lipids between adjacent bilayers. To determine the precise localization and in-situ structure of full length VPS13C in mammalian cells, we used cryo-electron tomography (cryo-ET) combined with correlative light and electron microscopy (CLEM) and cryo-focused ion beam milling (cryo-FIB). Cryo-ET analysis of human cells overexpressing VPS13C and its ER binding partner VAP reveals abundant rod-shaped densities at contact of the ER with late endosome/lysosomes. Sub-tomogram averaging and classification reveals a 29-nm rod bridging the ER and endolysosome membranes, with a small gap between the N-terminus of the rod and the ER membrane. Taken together, our cryo-ET study supports the model according to which VPS13C bridges two membranes to channel lipids.

BIO: Shujun Cai is a postdoctoral fellow at Yale University. She was trained as a structural biologist in her PhD, with expertise in cellular cryo-electron tomography and chromatin biology. Her current work uses cryo-electron tomography as a tool to study lipid-transfer mechanisms in mammalian cells including neurons.

Yao CONG – Structural basis for TRiC-assisted substrate folding revealed by cryo-EM

ABSTRACT: Chaperonins, the protein folding nanomachine, play an essential role in maintaining cellular homeostasis and are important in all three domains of life, dysfunction of which is closely related to cancer and neurodegenerative diseases. Chaperonins provide a cage-like environment for proteins to fold in isolation, unimpaired by aggregation, and in some cases actively modulate the folding pathway of the encapsulated protein. The eukaryotic group II chaperonin TRiC/CCT assists the folding of ~10% of newly synthesized cytosolic proteins, including the key cytoskeletal proteins actin and tubulin, cell cycle regulator CDC20, and VHL tumor suppressor. Here, we present an ensemble of cryo-EM structures of mammalian TRiC throughout its ATPase cycle, with some of them revealing endogenously engaged substrate in different folding stages. Our study reveals a thorough picture on the conformational landscape of TRiC-mediated substrate folding accompanying its ATPase cycle, revealing the molecular mechanism of TRiC assisted substrate folding.

BIO: Professor Yao Cong graduated from Jilin University (China) and did her postdoctoral training at The Scripps Research Institute (USA) and Baylor College of Medicine (USA). In 2011, she was appointed as a Principle Investigator at Shanghai Institute of Biochemistry and Cell Biology, CAS. She was awarded the National Excellent Young Scientists Career Award of NSFC, the Hundred Talents Program of CAS, and the Academic Leader of Shanghai. As the Vice Chief Engineer, she set up the National Cryo-EM Facility at National Center for Protein Science (Shanghai). Her research is focused on elucidating the ATP-driven activation and substrate folding or degradation mechanisms of two protein quality control macromolecular machines including TRiC/CCT and proteasome, the structural basis for vaccine and drug development against COVID-19 and the causative agents of hand-foot and mouth disease, and the assemble and function of histone H3K4 methyltransferases COMPASS. Accompanying this is an effort to develop efficient and accurate 2D-image-alignment algorithm for cryo-EM 3D reconstruction and new technologies for accurate subunit identification through cryo-EM analysis.

Shangyu DANG – Application of MSBP improves particle distribution and orientation in single-particle cryo-EM

ABSTRACT: Recent technological breakthroughs in single-particle cryo-EM enabled structural determination of biological macromolecules at atomic resolution. However, due to the limited knowledge of air-water interface (AWI), preparation of cryogenic specimen is still a trial-and-error process and, in many cases, the bottleneck to get structural information. Among these, preferred orientation of specimen and attraction of particles in AWI are common problem in practice. Here we reported a new method to apply MSBP in the cryo-sample preparation process for high-resolution structural determination using single-particle cryo-EM. Further cryo-ET analysis demonstrated application of this new method can improve particle distribution in vitreous ice by protecting particles from AWI. Our methods can also change particles orientation in vitreous ice thus alleviate preferred orientation problem as exemplified by single-particle analysis of Hemagglutinin trimer. Together, our MSBP method is a simple method and could be applied to improve particle distribution for high-resolution structure determination using single-particle cryo-EM.

BIO: Shangyu Dang is an assistant professor in the Division of Life Science at the Hong Kong University of Science and Technology. The research in Dang lab focuses on molecular mechanisms of the biological macromolecules, with particular interests in membrane proteins, protein-DNA complex, through multiple approaches including x-ray crystallography, cryo-EM, cryo-ET, biochemistry, and electrophysiology. In addition, we also interested in new method development in single particle cryo-EM and cryo-ET field to solve the recurring problems and expand their application.

Danny HSU – Game of spike: the battle between mutations and antibodies

ABSTRACT: TBA

BIO: Danny obtained his PhD in Chemistry in 2004 from the Utrecht University, the Netherlands, specializing in the applications of solution state NMR spectroscopy and molecular dynamics to study bimolecular interactions. He coined the term pyrophosphate cage to describe how a highly cyclized lanthionine-containing antibiotic, nisin, targets bacterial cell wall precursor, Lipid II, to achieve efficient membrane pore formation. He was awarded a Royal Netherlands Ramsay Memorial Fellowship and subsequently a Long-term fellowship from the Human Frontier Science Program to study co-translational folding of nascent polypeptide chains on the ribosome by NMR at Cambridge University. His work led to a Young Investigator Award from the International Society of Magnetic Resonance in 2007. He also applied NMR spectroscopy to investigate the folding dynamics of DNA and RND G-quadruplexes, and how ions and small molecules modulate the folding topologies and stabilities. He returned to Taiwan in 2010 with a Career Development Award from the Human Frontier Science Program to study folding mechanisms and functions of topologically knotted proteins. Danny is since 2017 a tenured Associate Research Fellow at the Institute of Biological Chemistry, Academia Sinica. His recent research interests focus on the integration of cryo-EM, mass spectrometry and complementary biophysical tools to study highly glycosylated proteins, including the spike protein of SARS-CoV-2.

Duane LOH – Decoding the messages stored by electron puddles in transmission electron microscopy

ABSTRACT: Direct electron detection (DED) helped drive the "resolution revolution" in cryo-electron microscopy. Many extolled DEDs' increased detection efficiency for boosting contrast for particle detection. Currently, it is common to count individual electrons arriving on detectors with high frame rates. Yet the shapes and sizes of individual "electron puddles" generated by each incident electron are discarded. In this talk, I will describe an efficient scheme [1] to record the shapes and sizes of these electron puddles. I will also discuss insights about DEDs enabled by this scheme, opportunities for new forms of statistical learning, and our experience with their early demonstrations.

[1] Datta A, Ng KF, Balakrishnan D, Ding M, Chee SW, Ban Y, et al. A data reduction and compression description for high throughput time-resolved electron microscopy. Nat Commun. 2021;12: 664.

BIO: Duane Loh is a faculty member in the departments of Physics and Biological Sciences at the National University of Singapore (NUS). He is interested in combining machine learning with scientific and instrument priors to create computational lenses that help make sense of the chaotic and nearly invisible dynamics that occur at the nanometer-scale. Duane earned his PhD in Physics from Cornell University for his feasibility studies of single-particle X-ray diffractive imaging. He extended these methods to unsupervised statistical learning to discover transient intermediate states and spontaneous order formation in highly heterogeneous and dynamic systems using both for X-ray and electron-based imaging sciences. Since starting his research group NUS in 2016, Duane continues to develop core ideas and technologies towards far-reaching computational lenses that are too challenging for hardware-based microscopy alone.

Shee-Mei LOK – The interplay between dengue morphological diversity and antibody recognition

ABSTRACT: Dengue virus (DENV) is major human pathogens infecting approximately 400 million people annually worldwide. However, currently there is no highly effective vaccine and therapeutics are non-existent. Here we show that DENV in addition to the presence of four different serotypes, within each serotypes, there is also variation of particle morphology between strains. The changes in particle morphology thus antigenicity, is due to the rearrangement of the surface viral envelope (E) proteins allowing DENV to evade host immunity. This will thus complicated the development of effective vaccines and therapeutics. We will discuss the extensive studies that we have done to examine the different morphologies displayed by various DENV strains and how we identify molecular determinants on the E proteins that lead to different structural changes. By studying the different morphological variants displayed by strains within each serotypes, we may be able to incorporate “representative morphologies” for each serotypes to make a more effective vaccine with greater coverage against all DENV serotypes antigenic structures. We will also discuss how highly potent human antibodies can neutralize all or some of the morphological variants of four DENV serotypes.

BIO: Dr Shee-Mei Lok is a Professor in the Emerging Infectious program in Duke-NUS, Singapore. She was also a National Research Foundation (NRF) fellow (2009-2014) and is now a NRF Investigator. She is a structural virologist specializing in x-ray crystallography and cryo-electron microscopy. Her research interest focus on the structural changes of flavivirus particles during its infection cycle and the effect of anti-viral therapeutics on them. She obtained her Msc and PhD in NUS and did her post-doctoral training in Purdue University under the supervision of the late Hanley Prof Michael Rossmann. Her laboratory made significant discoveries in the understanding of the structures of the Zika and dengue viruses, the morphological variants of dengue viruses, also how potent human antibodies neutralize flaviviruses and the structural changes of flavivirus during maturation.

Naoko MIZUNO – In situ cryo-electron tomography reveals local orchestration of cellular machineries for axon branch development

ABSTRACT: Neurons are highly polarized cells whose shape is controlled by cytoskeleton networks. The formation of neuronal protrusions such as dendrites and axons is mediated by the dynamic organization of the actin and microtubule cytoskeleton network, and it is the basis of neuronal development. Particularly at axon branches, signaling processes trigger actin re-formation leading to the recruitment of microtubules to reinforce the branching site. However, little is known about this remodeling mechanism. Combining interdisciplinary methods of cryo-EM, biophysics, and cell biology, we focus on elucidating the mechanisms of neuronal cell shape formation and accompanying cytoskeleton remodeling. I will present our recent observations of the socialization of molecules and organelles with the cytoskeleton at axon branching sites using cryo-electron tomography of primary neurons.

BIO: Naoko Mizuno is an NIH investigator since 2020. She graduated from the University of Tokyo in 1999, and received her Ph.D. in biophysics from the University of Tokyo/University of Texas Southwestern Medical Center in 2005. Prior to joining the NHLBI, she spent 8 years as an independent group leader at the Max Planck Institute of Biochemistry, Martinsried in Germany. Dr. Mizuno received several European awards and honors, including EMBO Young Investigators award, Boheringer Ingelheim Plus3 programme, and ERC consolidator grant.

Kelly NGUYEN – Replenishing the ends: Visualization of human telomerase holoenzyme by cryo-EM

ABSTRACT: Linear eukaryotic chromosome ends are capped with telomeres, which play key roles in maintaining genome integrity. Telomeres are, however, progressively shortened because of incomplete replication by the replisome. Critically short telomere length leads to proliferative senescence and cell death. To balance this loss, telomerase ribonucleoprotein (RNP) synthesizes telomeric repeats through its telomerase reverse transcriptase (TERT) and an integral RNA subunit (hTR) carrying the template for repeat synthesis. In addition to TERT and hTR, human telomerase holoenzyme consists of a number of other protein factors required for RNP assembly and localization. We recently determined the structure of DNA substrate-bound human telomerase holoenzyme at sub-4Å resolution. Here I will discuss the mechanistic insight on telomerase active site, assembly and regulation gained by our recent structural work.

BIO: TBA

Gaia PIGINO – The molecular structure of kidney primary cilia by cryo-electron tomography

ABSTRACT: TBA

BIO: TBA

Isabelle ROUILLER – Analyzing the dynamic properties of the AAA ATPase p97 using single particle cryo-EM

ABSTRACT: The dynamic properties of molecular machines are inextricable to the way proteins function. These dynamic properties define the range of conformations the protein sample adopts as well as how fast the protein switches between conformations. Sample preparation for cryo-EM should theoretically capture all the various conformations a protein complex adopts in solution and as such should provide the critical information required to understand how proteins function as dynamic molecular machines. To achieve atomic resolution of 3D reconstruction from cryo-EM images, classical approaches rely on the selection of most consistent views and conformations and consequently often result in the loss of information regarding the conformational variability of the proteins. Through these approaches, dynamic regions of proteins are not visible in the calculated density maps as the densities from these regions are “averaged out” during image processing. My group has been interested for several years in a protein complex formed by the AAA ATPase p97 for which the problem of “disappearing densities” during averaging is particularly severe: p97 assembles at a 582 KDa complex and yet typically approximately a third of the protein mass is not accounted for in the 3 dimensional averages. Here, I will present advances we have made to better understand the dynamic properties of p97 in solution. Worth noting is that single amino acid substitutions in p97 cause severe neurological diseases and this by modifying the dynamic properties of the p97 complex, indicating the biological and medical relevance of studies characterising protein dynamics.

BIO: Isabelle Rouiller obtained her PhD in 1998 for her studies at The Pirbright laboratories (England). After training as a postdoctoral fellow at the Scripps Research Institute and at the Burnham Institute (San Diego, USA), she directed her research group at McGill University (Montreal Canada) where she was the Associate Director (cryo-EM) of the Facility for Electron Microscopy Research. In 2018, she was recruited to The Department of Biochemistry and Pharmacology/ Bio21 Molecular Science and Biotechnology Institute at The University of Melbourne (Australia). She is currently Centre Deputy and Node Leader of the ARC Industrial Transformation Training Centre for Cryo-electron Microscopy of Membrane Proteins (CCeMMP). Isabelle has over 20 years of experience in using single particle cryo-EM and molecular electron tomography to study molecular machines, including ATPases and membrane proteins.

Carsten SACHSE – Cryo-EM structures of ESCRT membrane remodeling complexes

ABSTRACT: TBA

BIO: Carsten Sachse is heading the Structural Biology division of the Ernst-Ruska-Centre at the Forschungszentrum Jülich (Germany). He was a Group Leader at the EMBL in Heidelberg from 2010 to 2018. His group determines the three-dimensional cryo-EM structures of multi-protein complexes in isolation and their biological context with a particular focus on elucidating the mechanism of membrane biological processes such as autophagy and endocytosis. In order to further advance the cryo-EM method, he is interested in applying novel hardware and developing software for high-resolution single-particle as well as cellular cryo-EM.

Moran SHALEV-BENAMI – The ‘Hunger Games’ - Structural studies of the MC4 receptor reveal mechanism for satiety

ABSTRACT: Obesity is a global epidemic causing increased morbidity and impaired quality of life. The melanocortin receptor 4 (MC4R) is at the crux of appetite, energy homeostasis, and body-weight control in the central nervous system and is a prime target for anti-obesity drugs. MC4R is a member of the GPCR superfamily and signals primarily through the heterotrimeric G protein Gs. Here, we used cryo-EM to determine the structure of the human MC4R-Gs signaling complex. The work reveals the mechanism of MC4R activation, highlighting a molecular switch that signals satiation. These results fill a major gap in understanding MC4R activation and guide the design of future weight management drugs.

BIO: Dr. Shalev-Benami received her bachelor's degree in Molecular Biochemistry from the Technion Institute of Technology in 2006. She holds a M.Sc. degree in Biochemistry from the Hebrew University in Jerusalem, where she conducted her graduate research under the mentorship of Prof. Miriam Altstein. After receiving her M.Sc. in 2008, she returned to the Technion to peruse her Ph.D. in Structural Biology under the supervision of Prof. Timor Baasov and Prof. Noam Adir. Her Ph.D. studies focused on X-ray crystallographic studies of RNA and structure-based drug design. In 2013, she joined the lab of Prof. Ada Yonath at the Weizmann Institute of Science as a postdoctoral fellow, working on structural investigations of eukaryotic ribosomes. In 2015, she joined the lab of Prof. Georgios Skiniotis at the University of Michigan where she further perused her structural studies of eukaryotic ribosomes through single particle electron cryo-microscopy (cryo-EM). In 2017, Dr. Shalev-Benami continued her studies under the supervision of Prof. Skiniotis at Stanford University where she focused on structural characterization of membrane proteins using cryo-EM. In December 2018, Dr. Shalev-Benami joined the Weizmann Institute of Science, where her research focuses on the visualization of macromolecular complexes involved in cell-cell communication by cryo-EM.

Minhaj SIRAJUDDIN – Helical reconstruction of actin cytoskeleton bound with cognate binding proteins

ABSTRACT: TBA

BIO: Our lab is interested in understanding the mechanism of molecular machinery that mediate biological motility, including but not limited to muscle contraction, cell motility and intracellular cargo transport. We employ biochemistry, in vitro reconstitution, light and electron microscopy to study these active matters across dimensions from molecular, mesoscopic, cellular to tissue and organ scale.

Olga SOKOLOVA – Structures of viral chaperonins in complex with substrate proteins

ABSTRACT: Chaperonins are ubiquitously present protein complexes, which assist in the proper folding of newly synthesized proteins and prevent aggregation of denatured proteins in an ATP-dependent manner. They are classified into group I (bacterial, mitochondrial, chloroplast chaperonins) and group II (archaeal and eukaryotic cytosolic variants). Recently, viral chaperonins were discovered, which differ from both group I and II chaperonins. Gene 246 of bacteriophage OBP P. fluorescens encodes a single-ring chaperonin with a unique subunit arrangement: three pairs of subunits and one unpaired subunit. Each pair combines subunits in two possible conformations differing in nucleotide-binding affinity. The binding of nucleotides results in the increase of subunits’ conformational variability. When chaperonin bind the substrate, it undergoes a conformational change. We studied the interactions between chaperonins and substrates. E. coli chaperonin, GroEL, when it binds the prion protein, and PrPC could lead to pathogenic transformation of the latter to the aggregation-prone PrPSc form. We studied the molecular basis of the interactions in the GroEL–PrP complex using cryo-EM and molecular dynamics approaches. The obtained cryo-EM structure shows the PrP to be bound to several subunits of GroEL at the level of their apical domains. According to MD simulations, the disordered N-domain of PrP forms an increased number of intermolecular contacts with GroEL.

BIO: Olga S. Sokolova, PhD, D.Sc., Professor of Russian Academy of Sciences. Her main interest lies in the field of structural biology and cryo-electron microscopy. She has a background in biochemistry and structural biology/biophysics, with specific postdoctoral training in single particle EM in the laboratory of Dr Nikolaus Grigorieff (HHMI). As a postdoctoral fellow, she has conducted research on the 3D structure of the Shaker Kv channel. After starting her own laboratory in 2006 in Moscow Lomonosov University, she began to develop an integrated approach to study the structures of large protein complexes using cryo-EM and molecular modeling. She collaborates with numerous scientists from USA and Europe and participates in large multinational consortia that develop methods for investigating the structure of transmembrane proteins: “European Drug Initiative of Channels and Transporters” and “Integrated Biology Infrastructure Life-Science Facility at the European XFEL”. She was awarded the honorary title of Professor of Russian Academy of Sciences in 2016.

Ji-Joon SONG – Visualizing molecular architectures of endogenous protein complexes: finding a needle in a haystack

ABSTRACT: In cells, proteins form macromolecular complexes to execute their own unique roles in biological processes. Conventional structural biology methods adopt a bottom-up approach starting from defined sets of proteins to investigate their structures and interactions of protein complexes. However, this approach does not reflect the diverse and complex landscape of endogenous molecular architectures. Here, we introduce a top-down approach called EMPAS (Electron Microscopy screening for endogenous Protein ArchitectureS) to investigate the diverse and complex landscape of endogenous macromolecular architectures in an unbiased manner. By applying EMPAS, we discovered a spiral architecture to identify it as AdhE. Aldehyde-alcohol dehydrogenase (AdhE) is a key enzyme in bacterial fermentation, converting acetyl-CoA to ethanol, via two consecutive catalytic reactions. We determine a 3.45 Å resolution cryo-EM structure of full-length AdhE revealing a high-order spirosome architecture. The structure shows that the aldehyde dehydrogenase (ALDH) and alcohol dehydrogenase (ADH) active sites reside at the outer surface and the inner surface of the spirosome respectively, thus topologically separating these two activities. Furthermore, mutations disrupting the helical structure abrogate enzymatic activity, implying that formation of the spirosome structure is critical for AdhE activity.

BIO: Ji-Joon Song is a Professor in the Department of Biological Sciences at KAIST. He received his. B.S. at Seoul National University, and his Ph.D. in structural biology with Leemor Joshua-Tor at the Cold Spring Harbor Laboratory. During his Ph.D., he determined the first structure of Argonaute involved in RNAi. He then joined in Bob Kingston group as a research fellow at Massachusetts General Hospital, Harvard Medical School. The research in Song Lab at KAIST focuses on understanding the molecular mechanisms of epigenetic gene regulation using cryo-EM and integrative structural approaches.

Elitza TOCHEVA – Applying super-resolution confocal cryo-CLEM, cryo-FIB milling and cryo-ET methods to study the cellular architecture of Deinococcus radiodurans

ABSTRACT: D. radiodurans is a radiation-resistant bacterium with unique structural and functional properties. The bacterium is known to have a unique cell envelope with an outer membrane that lacks the typical lipopolysaccharide (LPS) lipids, and a surface S-layer composed of multiple protein complexes. The extreme resistance of the D. radiodurans cells to UV light and desiccation has been attributed to the cell envelope and in particular the S-layer. Due to its size, however, direct imaging of D. radiodurans with cryo-ET has not been feasible to date. By applying a combination of advanced imaging approaches we were able to reveal the native in vivo structure of the cell envelope and division sites of D. radiodurans.

BIO: TBA

Kuang-lei TSAI – Structure of the Mediator Kinase module

ABSTRACT: The eukaryotic Mediator complex, including a Core and a Cdk8 kinase module (CKM), plays an essential role in the regulation of RNA polymerase II transcription. A detailed understanding of Mediator’s structure and interactions is critical for determining how the complex regulates gene transcription. My talk will focus on the structure of the CKM and describe how Cdk8 is non-canonically activated by Med12.

BIO: My research interests focus on the use of single-particle cryo-electron microscopy (cryo-EM) and computational image analysis to determine the structures of macromolecular complexes that carry out key biological functions, such as transcription. In particular, I am interested in the functional relevance of large-scale conformational changes in macromolecular complexes. My research group uses advanced cryo-EM, X-ray crystallography and biochemical approaches to understand how dysfunction of specific macromolecular complexes drives cancer cell development.

Ming-Daw TSAI – Cryo-EM in enzymology

ABSTRACT: Although not yet widely recognized, cryo-EM is also breaking new grounds in the fields of mechanistic enzymology and protein dynamics. The goal of this lecture is to illustrate these applications of cryo-EM with three enzymes: (1) Ketol-acid reductoisomerase (KARI) from the thermophilic archaeon Sulfolobus solfataricus (Sso), which is a dodecamer, and Sulfolobus acidocaldarius (Sac), particularly in the use of cryo-EM to dissect intermediate structures of temperature-dependent conformational changes. (2) Human DNA recombinase DMC1. We used cryo-EM, MD simulations, and functional assays to suggest that the recombination fidelity is governed primarily by two structural factors: flexibility of the boundary gate to the base-pair triplet, and strength of support for the DNA backbone by a structural wall. (3) In an unpublished work, we discovered a new ATP allosteric regulation mechanism used by a wide variety of microorganisms for sensitive regulation of carbon fixation during energy shortage, and used cryo-EM to demonstrate the mechanism of these processes.

BIO: Professor Ming-Daw Tsai received B.S. degree from National Taiwan University (1972) and Ph.D. from Purdue University (1978), and served in the faculty of the Department of Chemistry and Biochemistry, The Ohio State University in 1981-2006. Subsequently he moved to the Institute of Biological Chemistry of Academia Sinica, Taiwan. His research interests include mechanistic enzymology of phosphoryl transfer enzymes including DNA polymerases, kinases and phospholipases, and structure-function relationship of proteins in DNA damage response and cancer signaling, including ankyrin repeat proteins and FHA domain proteins. A recent focus is on the roles of TIFA in cancer and immunity. He probes mechanistic problems by applying emerging methodologies in structural biology, including NMR, X-ray crystallography, MS, and recently cryo-EM, leading to close to 300 publications. He was elected to Fellow, American Association for the Advancement of Science (AAAS, 1992), Academician, Academia Sinica (2012), and Fellow, The World Academy of Science (TWAS, 2014).

Lexi WALLS – Visualizing the SARS-CoV-2 spike in complex with neutralizing antibodies

ABSTRACT: TBA

BIO: Lexi received her bachelors of science degree in Biochemistry from the University of Massachusetts Amherst and worked with Dr. Mary Munson and Dr. Lila Gierasch during her time there. She moved to Seattle to pursue her PhD in Biochemistry from the University of Washington in Seattle where she is currently working as a scientist in David Veesler's laboratory. Her work has been foundational for understanding coronavirus structure, function and developing therapeutics both before and during the current pandemic. She has co-authored more than 28 scientific publications, contributed to four US patents, and her work has led to two clinical trials currently being evaluated in humans. When she isn't in the laboratory, she spends her free time hiking and climbing the beautiful mountains of the Pacific Northwest.

Elizabeth WRIGHT – Structural studies of virus assembly using cryo-electron tomography

ABSTRACT: Respiratory syncytial virus (RSV) is a common respiratory virus that impacts both pediatric and geriatric populations. There are currently no approved treatments or vaccines. RSV virions are enveloped filamentous virions that are highly pleomorphic and range in length from 1-4 µm with a diameter of ~130 nm. The RSV matrix protein (M) has been crystallized as both a monomer and a dimer and experiments have shown that dimerization is required to assemble filamentous virions. However, the organization of matrix within the virion has been elusive. To address this challenge, we used whole-cell cryo-electron tomography and sub-tomogram averaging to show that M is arranged in a packed helical-like lattice of M-dimers ordered at an angle to the viral long axis. Sub-tomogram averages including F and M indicate that the position of F on the viral surface is correlated with the underlying M lattice. We also observed that RSV F is present as pairs, with the F trimers oriented in an anti-parallel conformation that may support potential interaction between trimers. Our results provide insight into RSV assembly and virion organization and may aid in the identification and development of RSV vaccines and anti-viral targets. Cryo-ET technology developments will also be highlighted throughout the talk.

BIO: Elizabeth Wright is a professor at the University of Wisconsin, Madison and Director of the Cryo-EM Research Center and NIH-funded Midwest Center for Cryo-ET. She has over 20 years of experience using cryo-EM to study the structure of macromolecules, viruses, eukaryotic cells, and bacteria. The Wright lab develops and uses correlative and cryo-electron microscopy technologies to explore the nano-scale architecture of bacteria, viruses, and human cells. Our goal is to use this information to aid in the development of novel antimicrobials, therapeutics, and vaccines.

Min XU – Automatic analysis of cryo-electron tomography using computer vision and machine learning

ABSTRACT: The cell is the basic structural and functional unit of all living organisms. Understanding how cells function is fundamental to life science. Macromolecules are nano-machines inside cells that govern the cellular processes. To fully understand such processes, it is necessary to know the native structures and spatial organizations of macromolecules inside single cells, and their interactions with other subcellular components. Such information has been extremely difficult to obtain due to a lack of suitable data acquisition techniques. The recent revolution of Cryo-electron tomography (cryo-ET) 3D imaging technology has made collecting such information possible. Cryo-ET captures a 3D image of a single cell's subcellular structures at sub-molecular resolution and in a near-native state. It provides unprecedented opportunities for systematically studying the native spatial organization of subcellular structures, especially macromolecules. However, cryo-ET has a high degree of structural complexity and imaging limits, such as high structural diversity and crowding, low signal-to-noise ratio, and missing values. These have made the automated systematic analysis of such images extremely difficult. Since 2008, we have been developing image analysis methods to address this challenge. In particular, we focus on systematic recognition and recovery of the structures of large numbers (millions) of macromolecules captured by cryo-ET, without relying on external structural knowledge. To do so, we have developed different cryo-ET image registration, classification, segmentation restoration techniques. Our effort is a key step for systematic analysis of macromolecules' structures and spatial organizations inside single cells captured by cryo-ET.

BIO: Dr. Min Xu is an Assistant Professor at the Computational Biology Department in the School of Computer Science at Carnegie Mellon University. He serves as training faculty at the Joint CMU-Pitt Ph.D. Program in Computational Biology. He also serves as a training faculty Master of Science in Computer Vision Program at Robotics Institute. He is an investigator at the National Center for Multiscale Modeling of Biological Systems. Dr. Xu’s career has centered on developing computational methods to study cellular systems using imaging and omics data. He started his research career in the field of Computational Biology and Bioinformatics since 2000. He developed machine learning methods for gene selection for classifying cancer samples, for cancer gene network module discovery, and sample phenotype prediction by integrating hundreds of gene expression datasets. Since 2008, he started working in computational analysis of Cellular Cryo-Electron Tomography (Cryo-ET) data. He designed structural pattern mining methods and first demonstrated the feasibility of De Novo extraction of structures and spatial organizations of macromolecular complexes in single cells using Cryo-ET data. His current research focus on developping computer vision and machine learning techniques for Cryo-ET-derived modeling of the cellular organization at molecular resolution. Dr. Xu has published over 60 research papers. Example publications include top image analysis conference papers such as CVPR and MICCAI, as well as prestigious journals and conference papers in computational biology, such as PNAS and ISMB. He is a recipient of NIH R01, NSF IIBR, and IIS awards. He is currently serving in the editorial board of Statistical Methods in Medical Research. Dr. Xu received an B.E. in Computer Science from the Beihang University, M.Sc from School of Computing at the National University of Singapore, M.A. in Applied Mathematics from the University of Southern California (USC), and Ph.D. in Computational Biology and Bioinformatics from USC. He was a postdoctoral researcher at USC.

Koji YONEKURA – Two-way application of cryo-EM to high-resolution and high-precision SPA and EX – Visualization of hydrogen and beyond

ABSTRACT: We have introduced a cryo-EM system based on the 1st machine of JEOL CRYO ARM 300 equipped with a cold field emission (CFE) gun and an in-column energy filter, and been carrying out R&D for cryo-electron crystallography (EX) and single particle analysis (SPA) using this system. The former has been applied to small and thin crystals of various molecules including proteins, polypeptides, drugs and organic materials. We have shown that combination of 300 kV electrons and energy filtration is highly beneficial to achieve high-quality structure analysis. For SPA, the CFE beam enables higher-resolution structure analysis with a less amplitude decay from smaller numbers of images than the conventional Schottky emission does. Our targets include spherical viruses, membrane protein complexes and protein-nucleic acids complexes.

Automated and remote data collection improves throughputs in both single particle cryo-EM and EX. Machine-learning approaches further accelerate these applications. In this symposium, I will report our recent results and advances by complementary use of the two technologies.

BIO: TBA