JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS 2019, VOL. 37, NO. S1, 1–92 https://doi.org/10.1080/07391102.2019.1604468 Book of Abstracts. Albany 2019: The 20th Conversation Abstracts 1. The structure and dynamics of 2. Cryo-EM and drug discovery biomolecules in their native states Sriram Subramaniam Joachim Frank Djavad Mowafaghian Centre for Brain Health, 2215 Wesbrook Mall, Department of Biochemistry and Molecular Biophysics, Columbia University of British Columbia, Vancouver, BC, V6T 1Z3, Canada University, New York, NY 10032, USA [email protected] [email protected] Cryo-EM has transitioned rapidly in the last few years from being The aim of structural biology is to explain life processes in amethodthatwasonlycapableofhandlingasmallsubsetof terms of macromolecular interactions in the cell. These inter- cryo-EM worthy specimens, to one that is useful for analysis of a actions typically involve more than two partners, and can very large spectrum of protein complexes (Subramaniam, 2019). run up to dozens. A full description will need to characterize This transition of cryo-EM from being a technology that was billed all structures on the atomic level, and the way these struc- as a tool to analyze large and/or highly symmetric specimens, to tures change in the process. Because of the crowded envir- one that can successfully tackle a range of proteins and protein onment of the cell, such characterization is presently (but complexes of broad general interest has been transformative. The see below) only possible when the group of interacting mol- structures of an impressive number of proteins, small and large, ecules (often organized into processive ‘molecular machines’) sometimes with extensive conformational spread, have been suc- is isolated and studied in vitro. While X-ray crystallography cessfully analyzed by cryo-EM. Many of these protein complexes has provided structures of a large number of molecular may never be coaxed to produce well-ordered crystals for study structures, the need for crystals diffracting to high resolution by X-ray crystallography. It is important to recognize that in has severely limited the number of supramolecular assem- almost every instance, these selected successes in the application blies and the range of conformers that can be studied with of cryo-EM rest on decades of advances in biochemistry, biophys- this technique. Single-particle cryo-electron microscopy is ics and protein science that laid the necessary groundwork. Nor about to fill this gap, allowing functional processes to be can we overlook the fact that the landscape of macromolecular studied in great detail without imposing restraints on the entities that are still intractable to analysis by cryo-EM remains structures. There are many examples now for this expansion immense. Yet, the future looks bright, and there is every reason of Structural Biology toward a full characterization of a func- to hope that an increasingly complex array of biological assem- tional process. Future developments of single-particle cryo- blies will be tackled by cryo-EM. EM include the study of short-lived intermediates in a none- Work in my laboratory has focused on the application of quilibrium system by time-resolved techniques, and the char- cryo-EM to small dynamic protein assemblies, with particular acterization of continuous structural changes using data emphasis on its use for drug discovery and therapeutic applica- mining from large ensembles of molecule images. It is very tions, and for the development of image processing methods to interesting and promising that another technique of cryo-EM, improve resolution (Banerjee et al., 2016; Chittori et al., 2018; cryo-electron tomography of FIB-milled cell sections, has Guo et al., 2017; Kang et al., 2018; Meyerson et al., 2016). In my started to contribute information about processes even presentation, I will discuss recent examples where we have used within the cellular context. Distinct ribosome states, for cryo-EM in this context to study metabolic enzyme complexes, instance, have already been identified and localized by sub- ion channels, nucleic-acid protein complexes and intact viruses. tomogram averaging. With this advance, we get closer to the fulfillment of the most ambitious aim of Structural Biology, References the visualization and interpretation of molecular interactions Banerjee, S., Bartesaghi, A., Merk, A., Rao, P., Bulfer, S. L., Yan, Y., Green, in situ. N., … Subramaniam, S. (2016). 2.3 Å resolution cryo-EM structure of 2 BOOK OF ABSTRACTS. ALBANY 2019: THE 20TH CONVERSATION human p97 and mechanism of allosteric inhibition. Science, 351(6275), 871–875. 4. Distance sensitive D-loop dynamics Chittori, S., Hong, J., Saunders, H., Feng, H., Ghirlando, R., Kelly, A. E., … and near-atomic resolution of F-actin Subramaniam, S. (2018). Structural mechanisms of centromeric chromosome recognition by the kinetochore protein CENP-N. Science, phalloidin structure by cryoEM 359(6373), 339–343. Guo, T., Bartesaghi, A., Yang, H., Falconieri, V., Rao, P., Merk, A., … Sanchaita Dasa , Peng Gec,d , Zeynep A. Oztug Subramaniam, S. (2017). Cryo-EM structures reveal mechanism and a à Ãa c,d inhibition of DNA targeting by a CRISPR-Cas surveillance complex. Durer , Elena E. Grintsevich ,Z.HongZhou and à a,b Cell, 171(2), 414–426. Emil Reisler Kang, Y., Kuybeda, O., de Waal, P. W., Mukherjee, S., Van Eps, N., Dutka, aDepartment of Chemistry and Biochemistry, UCLA, Los Angeles, P., … Xu, H. E. (2018). Cryo-EM structure of human rhodopsin bound CA, USA; bMolecular Biology Institute, UCLA, Los Angeles, CA, to an inhibitory G protein. Nature, 558(7711), 553–558. USA; cDepartment of Microbiology, Immunology and Molecular Meyerson, J. R., Chittori, S., Merk, A., Rao, P., Han, T. H., Serpe, M., … Genetics, University of California, Los Angeles (UCLA), Los Angeles, Subramaniam, S. (2016). Structural basis of kainate subtype glutamate CA, USA; dCalifornia NanoSystems Institute (CNSI), UCLA, Los receptor desensitization. Nature, 537(7621), 567–571. Angeles, CA, USA [email protected] Subramaniam, S. (2019). The cryo-EM revolution: Fueling the next phase. Contributed equally. IUCrJ, 6(Pt 1), 1–2. à Actin is indispensable for eukaryotic cells. Therefore, molecular details of F-actin structure and dynamics are essential for our understanding of its key cellular functions. It is well established 3. Deadly spiders and scary zombies— that nucleotide-bound state of F-actin (ATP/ADP-Pi or ADP) Not a Halloween story a near-atomic defines its dynamic properties, stability and interactions with regulatory factors. Previous studies also indicate that an resolution glance into the CNS innately flexible DNase I binding loop (D-loop, residues 40–50) plays a major role in such conformational dynamics. Recent Moran Shalev-Benami advances in cryoEM provide high-resolution information about Department of Structural Biology, Weizmann Institute of Science, nucleotide bound states of actin, however, the role of D loop in Rehovot, 7610001, Israel [email protected] monomer to polymer transition still remains elusive. Intriguingly, phalloidin, a ‘gold standard’ for actin staining in Synapses are specialized junctions between neurons that trans- vivo and in vitro, is also known to stabilize actin filaments and mit and compute information in the central nervous system affect the D-loop. Specifically, it can also convert polymeriza- (CNS). The establishment, properties, and dynamics of synapses tion-defective D loop mutants into stable polymers, thereby are governed by diverse trans-synaptic signaling molecules that bypassing D loop dependency. By utilizing a multidisciplinary communicate their signal via multifarious interactions with their approach of mutational disulfide crosslinking, light scattering synaptic partners. Mutations in the genes encoding these mole- measurements and cryoEM, we probe the structural mecha- cules have been associated with diverse neuropsychiatric and nisms that govern D-loop transitions in actin dynamics and neurodegenerative disorders thus highlighting their crucial how phalloidin stabilizes F-actin. Our biochemical data provides importance for normal brain function. Over the past few deca- a molecular ruler-based model of how intraprotomer distance des, tremendous efforts have been made to structurally char- between two D-loop residues facilitates a transition from G to acterize the trans-synaptic signaling molecules as well as their F-actin and vice versa. Additionally, we report the first 3.7 Å interacting partners. Nevertheless, their low expression levels resolution structure of Phalloidin bound F-actin in the ADP-Pi and high structural complexity has posed a great challenge to state. Our structure supports (i) the role of methylation of His traditional structural methods, such as NMR and X-ray crystal- 73 on actin in Pi binding, (ii) shows that phalloidin inhibits Pi lography. Advances in single particle electron cryo-microscopy (cryo-EM) now allow the capture of such complexed macro- molecular assemblies in great details, providing snapshots of these fascinating molecules in action. Here we present the near-atomic resolution structures of two such synaptic compo- nents, the cannabinoid receptor 1 (CB1R), and teneurin, two transmembrane receptors that are primarily expressed in neu- rons and are considered to mediate various functions in syn- apse formation and maintenance. The structures provide a high-resolution glance into the receptors’ architectures and present structural