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

IUCrJ

Schlichting, I.¹  Gotfryd, K.¹  Drachmann, N.D.²  Mattle, D.²  Møller, J.V.²  Gregersen, J.L.²  Boesen, T.²  Wang, K.-T.²  Bublitz, M.²  Clausen, J.D.²  Nissen, P.²  Sitsel, O.²  Olesen, C.³  Boutet, S.⁴  Williams, G.J.⁵  Seibert, M.M.⁵  Messerschmidt, M.⁵  Foucar, L.⁵  Reinhard, L.⁶  Shoeman, R.L.⁶  Nass, K.⁶  Doak, R.B.⁶  Barends, T.R.M.⁶  Markvardsen, A.J.⁷  Gutmann, M.J.⁷ 

[1] Department of Biomedicine, Aarhus University, Ole Worms Allé 3, 8000 Aarhus C, Denmark;Department of Molecular Biology and Genetics, Centre for Membrane Pumps in Cells and Disease – PUMPkin, Danish National Research Foundation, Aarhus University, Gustav Wieds Vej 10c, 8000 Aarhus C, Denmark;Department of Neuroscience and Pharmacology, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark;Department of Physics, Arizona State University, Tempe, AZ 85287, USA;Linac Coherent Light Source, LCLS, SLAC National Accelerator Laboratory, 2575 Sand Hill Road, Menlo Park, CA 94025, USA;Max Planck Institute for Medical Research, Jahnstrasse 29, 69120 Heidelberg, Germany;Rutherford Appleton Laboratory, ISIS Facility, Chilton, Didcot OX11 0QX, England

关键词: XFEL;    P-TYPE ATPASES;    LIGAND SCREENING;    SERIAL FEMTOSECOND CRYSTALLOGRAPHY;   

DOI  :  10.1107/S2052252515008969

学科分类：数学（综合）

来源: International Union of Crystallography

【 摘 要 】

Membrane proteins are key players in biological systems, mediating signalling events and the specific transport of e.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.

【 授权许可】

CC BY