The overall objective of the proposed research is to develop enhanced anomalous diffraction methods for analyzing structures of biological macromolecules. We build on our recent accomplishments at New York Structural Biology Center (NYSBC) beamlines at Brookhaven's National Synchrotron Light Source (NSLS) and worldwide experience showing that multiwavelength anomalous diffraction (MAD) and SAD, MAD's single-wavelength counterpart, now predominate for de novo determinations of three- dimensional structures for biological macromolecules. We propose to optimize SAD and MAD phasing procedures by meeting the demands of compelling applications to current problems of biological significance. Biologically exciting problems motivate the development of appropriate tools, and forefront methods accelerate the solution of structures for systems of biological and medical significance. The overall objective is embodied in four specific aims: (1) we propose to enhance SAD phasing procedures for challenging problems such as selenomethionyl proteins at low resolution and only-light-atom native structures. A focus is on improved methods for increasing signal-to-noise ratios for anomalous diffraction by combining data from many crystals. (2) We propose to enhance MAD procedures for accurate experimental phase evaluation. Data collection strategies will be devised to mitigate radiation damage and minimize systematic errors. (3) We propose to develop procedures for low-energy anomalous diffraction experiments. We will develop experimental procedures and design instrumentation for low-energy (3 - 7 keV) experiments aiming to enhance anomalous signal from light atoms such as sulfur and phosphorous. (4) We propose to develop automation and enhanced procedures to facilitate SAD and MAD analyses from many crystals. A first priority is to encode and disseminate our current multi-crystal SAD process in convenient software.

Public Health Relevance

This is a project in technology development for basic science, but applications of expected to result from these studies are likely to have profound relevance for human health. Biological structures to be determined in atomic detail include molecular chaperones with impact for neurodegenerative diseases and cancer, calcium-release channel receptors with impact for heart failure, and histidine kinase receptors with relevance for microbial infection.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM107462-03
Application #
9024582
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Edmonds, Charles G
Project Start
2014-06-03
Project End
2018-02-28
Budget Start
2016-03-01
Budget End
2017-02-28
Support Year
3
Fiscal Year
2016
Total Cost
$385,209
Indirect Cost
$107,912
Name
Columbia University (N.Y.)
Department
Biochemistry
Type
Schools of Medicine
DUNS #
621889815
City
New York
State
NY
Country
United States
Zip Code
10032
Assur Sanghai, Zahra; Liu, Qun; Clarke, Oliver B et al. (2018) Structure-based analysis of CysZ-mediated cellular uptake of sulfate. Elife 7:
Su, Min; Gao, Feng; Yuan, Qi et al. (2017) Structural basis for conductance through TRIC cation channels. Nat Commun 8:15103
Liu, Qun; Hendrickson, Wayne A (2017) Contemporary Use of Anomalous Diffraction in Biomolecular Structure Analysis. Methods Mol Biol 1607:377-399
Hendrickson, Wayne A (2016) Atomic-level analysis of membrane-protein structure. Nat Struct Mol Biol 23:464-7
Ranaivoson, Fanomezana M; Liu, Qun; Martini, Francesca et al. (2015) Structural and Mechanistic Insights into the Latrophilin3-FLRT3 Complex that Mediates Glutamatergic Synapse Development. Structure 23:1665-1677
Liu, Qun; Hendrickson, Wayne A (2015) Crystallographic phasing from weak anomalous signals. Curr Opin Struct Biol 34:99-107
Guo, Youzhong; Kalathur, Ravi C; Liu, Qun et al. (2015) Protein structure. Structure and activity of tryptophan-rich TSPO proteins. Science 347:551-5
Zhang, Zhen; Liu, Qun; Hendrickson, Wayne A (2014) Crystal structures of apparent saccharide sensors from histidine kinase receptors prevalent in a human gut symbiont. FEBS J 281:4263-79
Chang, Yanqi; Bruni, Renato; Kloss, Brian et al. (2014) Structural basis for a pH-sensitive calcium leak across membranes. Science 344:1131-5
Yang, Tingting; Liu, Qun; Kloss, Brian et al. (2014) Structure and selectivity in bestrophin ion channels. Science 346:355-9

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