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

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.
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  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:
OSTI Identifier:
Grant/Contract Number:
Accepted Manuscript
Journal Name:
Additional Journal Information:
Journal Volume: 2; Journal Issue: 4; Journal ID: ISSN 2052-2525
International Union of Crystallography
Research Org:
SLAC National Accelerator Laboratory (SLAC), Menlo Park, CA (United States)
Sponsoring Org:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)
Contributing Orgs:
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.
Country of Publication:
United States
59 BASIC BIOLOGICAL SCIENCES XFEL; P-type ATPases; ligand screening; serial femtosecond crystallography