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

Maike Bublitz; Karol Nass; Nikolaj D Drachmann; Anders J Markvardsen; Matthias J Gutmann; Thomas R M Barends; Daniel Mattle; Robert L Shoeman; R Bruce Doak; Sébastien Boutet; Marc Messerschmidt; Marvin M Seibert; Garth J Williams; Lutz Foucar; Linda Reinhard; Oleg Sitsel; Jonas L Gregersen; Johannes D Clausen; Thomas Boesen; Kamil Gotfryd; Kai-Tuo Wang; Claus Olesen; Jesper V Møller; Poul Nissen; Ilme Schlichting

doi:10.1107/S2052252515008969

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

Maike Bublitz, Karol Nass, Nikolaj D Drachmann, Anders J Markvardsen, Matthias J Gutmann, Thomas R M Barends, Daniel Mattle, Robert L Shoeman, R Bruce Doak, Sébastien Boutet, Marc Messerschmidt, Marvin M Seibert, Garth J Williams, Lutz Foucar, Linda Reinhard, Oleg Sitsel, Jonas L Gregersen, Johannes D Clausen, Thomas Boesen, Kamil GotfrydKai-Tuo Wang, Claus Olesen, Jesper V Møller, Poul Nissen, Ilme Schlichting

Publikation: Bidrag til tidsskrift/Konferencebidrag i tidsskrift /Bidrag til avis › Tidsskriftartikel › Forskning › peer review

19 Citationer (Scopus)

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.

Originalsprog	Engelsk
Tidsskrift	IUCrJ
Vol/bind	2
Nummer	Pt 4
Sider (fra-til)	409-420
Antal sider	12
ISSN	2052-2525
DOI	https://doi.org/10.1107/S2052252515008969
Status	Udgivet - 1 jul. 2015

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10.1107/S2052252515008969

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Bublitz, M., Nass, K., Drachmann, N. D., Markvardsen, A. J., Gutmann, M. J., Barends, T. R. M., Mattle, D., Shoeman, R. L., Doak, R. B., Boutet, S., Messerschmidt, M., Seibert, M. M., Williams, G. J., Foucar, L., Reinhard, L., Sitsel, O., Gregersen, J. L., Clausen, J. D., Boesen, T., ... Schlichting, I. (2015). Structural studies of P-type ATPase-ligand complexes using an X-ray free-electron laser. IUCrJ, 2(Pt 4), 409-420. https://doi.org/10.1107/S2052252515008969

@article{1918c96efcba4d3e8a7413d447ba2bf5,

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.",

author = "Maike Bublitz and Karol Nass and Drachmann, {Nikolaj D} and Markvardsen, {Anders J} and Gutmann, {Matthias J} and Barends, {Thomas R M} and Daniel Mattle and Shoeman, {Robert L} and Doak, {R Bruce} and S{\'e}bastien Boutet and Marc Messerschmidt and Seibert, {Marvin M} and Williams, {Garth J} and Lutz Foucar and Linda Reinhard and Oleg Sitsel and Gregersen, {Jonas L} and Clausen, {Johannes D} and Thomas Boesen and Kamil Gotfryd and Kai-Tuo Wang and Claus Olesen and M{\o}ller, {Jesper V} and Poul Nissen and Ilme Schlichting",

year = "2015",

month = jul,

day = "1",

doi = "10.1107/S2052252515008969",

language = "English",

volume = "2",

pages = "409--420",

journal = "IUCrJ",

issn = "2052-2525",

publisher = "INT UNION CRYSTALLOGRAPHY",

number = "Pt 4",

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Bublitz, M, Nass, K, Drachmann, ND, Markvardsen, AJ, Gutmann, MJ, Barends, TRM, Mattle, D, Shoeman, RL, Doak, RB, Boutet, S, Messerschmidt, M, Seibert, MM, Williams, GJ, Foucar, L, Reinhard, L, Sitsel, O, Gregersen, JL, Clausen, JD, Boesen, T, Gotfryd, K, Wang, K-T, Olesen, C, Møller, JV, Nissen, P & Schlichting, I 2015, 'Structural studies of P-type ATPase-ligand complexes using an X-ray free-electron laser', IUCrJ, bind 2, nr. Pt 4, s. 409-420. https://doi.org/10.1107/S2052252515008969

TY - JOUR

T1 - Structural studies of P-type ATPase-ligand complexes using an X-ray free-electron laser

AU - Bublitz, Maike

AU - Nass, Karol

AU - Drachmann, Nikolaj D

AU - Markvardsen, Anders J

AU - Gutmann, Matthias J

AU - Barends, Thomas R M

AU - Mattle, Daniel

AU - Shoeman, Robert L

AU - Doak, R Bruce

AU - Boutet, Sébastien

AU - Messerschmidt, Marc

AU - Seibert, Marvin M

AU - Williams, Garth J

AU - Foucar, Lutz

AU - Reinhard, Linda

AU - Sitsel, Oleg

AU - Gregersen, Jonas L

AU - Clausen, Johannes D

AU - Boesen, Thomas

AU - Gotfryd, Kamil

AU - Wang, Kai-Tuo

AU - Olesen, Claus

AU - Møller, Jesper V

AU - Nissen, Poul

AU - Schlichting, Ilme

PY - 2015/7/1

Y1 - 2015/7/1

N2 - 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.

AB - 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.

U2 - 10.1107/S2052252515008969

DO - 10.1107/S2052252515008969

M3 - Journal article

C2 - 26175901

SN - 2052-2525

VL - 2

SP - 409

EP - 420

JO - IUCrJ

JF - IUCrJ

IS - Pt 4

ER -

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

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