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 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.
authors.affiliation: 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.
authors.forename: Maike
authors.initials: M
authors.lastname: Bublitz
authors.affiliation: Max Planck Institute for Medical Research , Jahnstrasse 29, 69120 Heidelberg, Germany.
authors.forename: Karol
authors.initials: K
authors.lastname: Nass
authors.affiliation: 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.
authors.forename: Nikolaj D
authors.initials: ND
authors.lastname: Drachmann
authors.affiliation: Rutherford Appleton Laboratory, ISIS Facility , Chilton, Didcot OX11 0QX, England.
authors.forename: Anders J
authors.initials: AJ
authors.lastname: Markvardsen
authors.affiliation: Rutherford Appleton Laboratory, ISIS Facility , Chilton, Didcot OX11 0QX, England.
authors.forename: Matthias J
authors.initials: MJ
authors.lastname: Gutmann
authors.affiliation: Max Planck Institute for Medical Research , Jahnstrasse 29, 69120 Heidelberg, Germany.
authors.forename: Thomas R M
authors.initials: TR
authors.lastname: Barends
authors.affiliation: 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.
authors.forename: Daniel
authors.initials: D
authors.lastname: Mattle
authors.affiliation: Max Planck Institute for Medical Research , Jahnstrasse 29, 69120 Heidelberg, Germany.
authors.forename: Robert L
authors.initials: RL
authors.lastname: Shoeman
authors.affiliation: Max Planck Institute for Medical Research , Jahnstrasse 29, 69120 Heidelberg, Germany ; Department of Physics, Arizona State University , Tempe, AZ 85287, USA.
authors.forename: R Bruce
authors.initials: RB
authors.lastname: Doak
authors.affiliation: Linac Coherent Light Source, LCLS, SLAC National Accelerator Laboratory , 2575 Sand Hill Road, Menlo Park, CA 94025, USA.
authors.forename: Sébastien
authors.initials: S
authors.lastname: Boutet
authors.affiliation: Linac Coherent Light Source, LCLS, SLAC National Accelerator Laboratory , 2575 Sand Hill Road, Menlo Park, CA 94025, USA.
authors.forename: Marc
authors.initials: M
authors.lastname: Messerschmidt
authors.affiliation: Linac Coherent Light Source, LCLS, SLAC National Accelerator Laboratory , 2575 Sand Hill Road, Menlo Park, CA 94025, USA.
authors.forename: Marvin M
authors.initials: MM
authors.lastname: Seibert
authors.affiliation: Linac Coherent Light Source, LCLS, SLAC National Accelerator Laboratory , 2575 Sand Hill Road, Menlo Park, CA 94025, USA.
authors.forename: Garth J
authors.initials: GJ
authors.lastname: Williams
authors.affiliation: Max Planck Institute for Medical Research , Jahnstrasse 29, 69120 Heidelberg, Germany.
authors.forename: Lutz
authors.initials: L
authors.lastname: Foucar
authors.affiliation: 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.
authors.forename: Linda
authors.initials: L
authors.lastname: Reinhard
authors.affiliation: 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.
authors.forename: Oleg
authors.initials: O
authors.lastname: Sitsel
authors.affiliation: 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.
authors.forename: Jonas L
authors.initials: JL
authors.lastname: Gregersen
authors.affiliation: 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 Biomedicine, Aarhus University , Ole Worms Allé 3, 8000 Aarhus C, Denmark.
authors.forename: Johannes D
authors.initials: JD
authors.lastname: Clausen
authors.affiliation: 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.
authors.forename: Thomas
authors.initials: T
authors.lastname: Boesen
authors.affiliation: Department of Neuroscience and Pharmacology, University of Copenhagen , Blegdamsvej 3B, 2200 Copenhagen, Denmark.
authors.forename: Kamil
authors.initials: K
authors.lastname: Gotfryd
authors.affiliation: 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.
authors.forename: Kai-Tuo
authors.initials: KT
authors.lastname: Wang
authors.affiliation: 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 Biomedicine, Aarhus University , Ole Worms Allé 3, 8000 Aarhus C, Denmark.
authors.forename: Claus
authors.initials: C
authors.lastname: Olesen
authors.affiliation: 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 Biomedicine, Aarhus University , Ole Worms Allé 3, 8000 Aarhus C, Denmark.
authors.forename: Jesper V
authors.initials: JV
authors.lastname: Møller
authors.affiliation: 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 ; DANDRITE, Nordic-EMBL Partnership for Molecular Medicine, Aarhus University, Gustav Wieds Vej 10C, DK-8000 Aarhus C, Denmark.
authors.forename: Poul
authors.initials: P
authors.lastname: Nissen
authors.affiliation: Max Planck Institute for Medical Research , Jahnstrasse 29, 69120 Heidelberg, Germany.
authors.forename: Ilme
authors.initials: I
authors.lastname: Schlichting
keywords: P-type ATPases; XFEL; ligand screening; serial femtosecond crystallography