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.

Public Health 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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Program Projects (P01)
Project #
5P01GM062580-09
Application #
7817043
Study Section
Special Emphasis Panel (ZRG1-BST-A (41))
Program Officer
Flicker, Paula F
Project Start
2001-04-01
Project End
2012-04-30
Budget Start
2010-05-01
Budget End
2011-04-30
Support Year
9
Fiscal Year
2010
Total Cost
$2,041,273
Indirect Cost
Name
Harvard University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
047006379
City
Boston
State
MA
Country
United States
Zip Code
02115
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
Loveland, Anna B; Bah, Eugene; Madireddy, Rohini et al. (2016) Ribosomeā€¢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
Laxmikanthan, Gurunathan; Xu, Chen; Brilot, Axel F et al. (2016) Structure of a Holliday junction complex reveals mechanisms governing a highly regulated DNA transaction. Elife 5:
Ha, Jun Yong; Chou, Hui-Ting; Ungar, Daniel et al. (2016) Molecular architecture of the complete COG tethering complex. Nat Struct Mol Biol 23:758-60

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