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Title: Structural studies of P-type ATPase–ligand complexes using an X-ray free-electron laser

Abstract

Membrane proteins are key players in biological systems, mediating signalling events and the specific transport ofe.g.ions and metabolites. Consequently, membrane proteins are targeted by a large number of currently approved drugs. Understanding their functions and molecular mechanisms is greatly dependent on structural information, not least on complexes with functionally or medically important ligands. Structure determination, however, is hampered by the difficulty of obtaining well diffracting, macroscopic crystals. Here, the feasibility of X-ray free-electron-laser-based serial femtosecond crystallography (SFX) for the structure determination of membrane protein–ligand complexes using microcrystals of various native-source and recombinant P-type ATPase complexes is demonstrated. The data reveal the binding sites of a variety of ligands, including lipids and inhibitors such as the hallmark P-type ATPase inhibitor orthovanadate. By analyzing the resolution dependence of ligand densities and overall model qualities, SFX data quality metrics as well as suitable refinement procedures are discussed. Even at relatively low resolution and multiplicity, the identification of ligands can be demonstrated. This makes SFX a useful tool for ligand screening and thus for unravelling the molecular mechanisms of biologically active proteins.

Authors:
 [1];  [2];  [1];  [3];  [3];  [2];  [1];  [2];  [4];  [5];  [5];  [5];  [5];  [2];  [1];  [1];  [1];  [1];  [1];  [6] more »;  [1];  [1];  [1];  [1];  [2] « less
+ Show Author Affiliations
  1. Aarhus Univ., Aarhus (Denmark)
  2. Max Planck Institute for Medical Research, Heidelberg (Germany)
  3. Rutherford Appleton Lab., Didcot (England)
  4. Max Planck Institute for Medical Research, Heidelberg (Germany); Arizona State Univ., Tempe, AZ (United States)
  5. SLAC National Accelerator Lab., Menlo Park, CA (United States)
  6. Univ. of Copenhagen, Copenhagen (Denmark)
Publication Date:
Research Org.:
SLAC National Accelerator Laboratory (SLAC), Menlo Park, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES)
Contributing Org.:
Parts of this research were carried out at the LCLS, a National User Facility operated by Stanford University on behalf of the US Department of Energy (DOE), Office of Basic Energy Sciences (OBES). The CXI instrument was funded by the LCLS Ultrafast Science Instruments (LUSI) project funded by DOE, OBES.
OSTI Identifier:
1208835
Grant/Contract Number:  
AC03-76SF00515
Resource Type:
Accepted Manuscript
Journal Name:
IUCrJ
Additional Journal Information:
Journal Volume: 2; Journal Issue: 4; Journal ID: ISSN 2052-2525
Publisher:
International Union of Crystallography
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; XFEL; P-type ATPases; ligand screening; serial femtosecond crystallography

Citation Formats

Bublitz, Maike, Nass, Karol, Drachmann, Nikolaj D., Markvardsen, Anders J., Gutmann, Matthias J., Barends, Thomas R. M., Mattle, Daniel, Shoeman, Robert L., Doak, R. Bruce, Boutet, Sébastien, Messerschmidt, Marc, Seibert, Marvin M., Williams, Garth J., Foucar, Lutz, Reinhard, Linda, Sitsel, Oleg, Gregersen, Jonas L., Clausen, Johannes D., Boesen, Thomas, Gotfryd, Kamil, Wang, Kai -Tuo, Olesen, Claus, Møller, Jesper V., Nissen, Poul, and Schlichting, Ilme. Structural studies of P-type ATPase–ligand complexes using an X-ray free-electron laser. United States: N. p., 2015. Web. doi:10.1107/S2052252515008969.
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Bublitz, Maike, Nass, Karol, Drachmann, Nikolaj D., Markvardsen, Anders J., Gutmann, Matthias J., Barends, Thomas R. M., Mattle, Daniel, Shoeman, Robert L., Doak, R. Bruce, Boutet, Sébastien, Messerschmidt, Marc, Seibert, Marvin M., Williams, Garth J., Foucar, Lutz, Reinhard, Linda, Sitsel, Oleg, Gregersen, Jonas L., Clausen, Johannes D., Boesen, Thomas, Gotfryd, Kamil, Wang, Kai -Tuo, Olesen, Claus, Møller, Jesper V., Nissen, Poul, & Schlichting, Ilme. Structural studies of P-type ATPase–ligand complexes using an X-ray free-electron laser. United States. https://doi.org/10.1107/S2052252515008969
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Bublitz, Maike, Nass, Karol, Drachmann, Nikolaj D., Markvardsen, Anders J., Gutmann, Matthias J., Barends, Thomas R. M., Mattle, Daniel, Shoeman, Robert L., Doak, R. Bruce, Boutet, Sébastien, Messerschmidt, Marc, Seibert, Marvin M., Williams, Garth J., Foucar, Lutz, Reinhard, Linda, Sitsel, Oleg, Gregersen, Jonas L., Clausen, Johannes D., Boesen, Thomas, Gotfryd, Kamil, Wang, Kai -Tuo, Olesen, Claus, Møller, Jesper V., Nissen, Poul, and Schlichting, Ilme. Thu . "Structural studies of P-type ATPase–ligand complexes using an X-ray free-electron laser". United States. https://doi.org/10.1107/S2052252515008969. https://www.osti.gov/servlets/purl/1208835.
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@article{osti_1208835,
title = {Structural studies of P-type ATPase–ligand complexes using an X-ray free-electron laser},
author = {Bublitz, Maike and Nass, Karol and Drachmann, Nikolaj D. and Markvardsen, Anders J. and Gutmann, Matthias J. and Barends, Thomas R. M. and Mattle, Daniel and Shoeman, Robert L. and Doak, R. Bruce and Boutet, Sébastien and Messerschmidt, Marc and Seibert, Marvin M. and Williams, Garth J. and Foucar, Lutz and Reinhard, Linda and Sitsel, Oleg and Gregersen, Jonas L. and Clausen, Johannes D. and Boesen, Thomas and Gotfryd, Kamil and Wang, Kai -Tuo and Olesen, Claus and Møller, Jesper V. and Nissen, Poul and Schlichting, Ilme},
abstractNote = {Membrane proteins are key players in biological systems, mediating signalling events and the specific transport ofe.g.ions and metabolites. Consequently, membrane proteins are targeted by a large number of currently approved drugs. Understanding their functions and molecular mechanisms is greatly dependent on structural information, not least on complexes with functionally or medically important ligands. Structure determination, however, is hampered by the difficulty of obtaining well diffracting, macroscopic crystals. Here, the feasibility of X-ray free-electron-laser-based serial femtosecond crystallography (SFX) for the structure determination of membrane protein–ligand complexes using microcrystals of various native-source and recombinant P-type ATPase complexes is demonstrated. The data reveal the binding sites of a variety of ligands, including lipids and inhibitors such as the hallmark P-type ATPase inhibitor orthovanadate. By analyzing the resolution dependence of ligand densities and overall model qualities, SFX data quality metrics as well as suitable refinement procedures are discussed. Even at relatively low resolution and multiplicity, the identification of ligands can be demonstrated. This makes SFX a useful tool for ligand screening and thus for unravelling the molecular mechanisms of biologically active proteins.},
doi = {10.1107/S2052252515008969},
journal = {IUCrJ},
number = 4,
volume = 2,
place = {United States},
year = {Thu Jun 11 00:00:00 EDT 2015},
month = {Thu Jun 11 00:00:00 EDT 2015}
}
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https://doi.org/10.1107/S2052252515008969
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