This is a first competitive renewal of a Program Project Grant with the goal of developing experimental and computational tools of cryo-electron microscopy in the context of a widening range of biological applications. Atomic resolution pictures of complex assemblies are the ultimate objectives. The renewal has three central themes. The first is a focus on membrane proteins and membrane-associated assemblies. We will expand work from the first grant period on 2-D membrane protein crystals, on clathrin-coated vesicles, and on single particle analysis as applied to membrane proteins. The second theme is development of maximum likelihood methods in single-particle reconstructions, with the goal of extending our ability to obtain molecular structural information from conformationally heterogeneous specimens. The results of the previous grant period demonstrate that x-ray crystallography and cryo-EM form a smooth continuum of methods that indeed can achieve atomic resolution for a great variety of homogeneous samples. The goal is now to improve the robustness of single-particle reconstruction algoirthms and to deal with conformationally heterogeneous single particles. Cryo-electron tomography of macromolecular complexes will also have a role in bridging between lower resolution analyses of the assemblies and high-resolution analyses of their substructures. The third theme is precisely the application of these methods to transient, multi-state assemblies, such as spliceosomes and kinetochore substructures. ? ? Relevance: Each of the assemblies we study has an important connection with a human disease, as outlined in each of the project sections. More generally, """"""""molecular machines"""""""", rather than single enzymes, will be targets of future interventions, and learning how to relate structure and function of such assemblies will underlie such translational developments. ? ? ?
| Close, William; Neumann, Matthias; Schmidt, Andreas et al. (2018) Physical basis of amyloid fibril polymorphism. Nat Commun 9:699 |
| Loveland, Anna B; Demo, Gabriel; Grigorieff, Nikolaus et al. (2017) Ensemble cryo-EM elucidates the mechanism of translation fidelity. Nature 546:113-117 |
| Liu, Yuhang; Pan, Junhua; Jenni, Simon et al. (2017) CryoEM Structure of an Influenza Virus Receptor-Binding Site Antibody-Antigen Interface. J Mol Biol 429:1829-1839 |
| Abeyrathne, Priyanka D; Koh, Cha San; Grant, Timothy et al. (2016) Ensemble cryo-EM uncovers inchworm-like translocation of a viral IRES through the ribosome. Elife 5: |
| Schmidt, Andreas; Annamalai, Karthikeyan; Schmidt, Matthias et al. (2016) Cryo-EM reveals the steric zipper structure of a light chain-derived amyloid fibril. Proc Natl Acad Sci U S A 113:6200-5 |
| Chou, Hui-Ting; Dukovski, Danijela; Chambers, Melissa G et al. (2016) CATCHR, HOPS and CORVET tethering complexes share a similar architecture. Nat Struct Mol Biol 23:761-3 |
| Dimitrova, Yoana N; Jenni, Simon; Valverde, Roberto et al. (2016) Structure of the MIND Complex Defines a Regulatory Focus for Yeast Kinetochore Assembly. Cell 167:1014-1027.e12 |
| van der Feltz, Clarisse; Pomeranz Krummel, Daniel (2016) Purification of Native Complexes for Structural Study Using a Tandem Affinity Tag Method. J Vis Exp : |
| Loveland, Anna B; Bah, Eugene; Madireddy, Rohini et al. (2016) Ribosome•RelA structures reveal the mechanism of stringent response activation. Elife 5: |
| Baytshtok, Vladimir; Fei, Xue; Grant, Robert A et al. (2016) A Structurally Dynamic Region of the HslU Intermediate Domain Controls Protein Degradation and ATP Hydrolysis. Structure 24:1766-1777 |
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