Matching Items (4)
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Description
Serial femtosecond crystallography (SFX) using X-ray free-electron laser sources is an emerging method with considerable potential for time-resolved pump-probe experiments. Here we present a lipidic cubic phase SFX structure of the light-driven proton pump bacteriorhodopsin (bR) to 2.3 Å resolution and a method to investigate protein dynamics with modest sample requirement.

Serial femtosecond crystallography (SFX) using X-ray free-electron laser sources is an emerging method with considerable potential for time-resolved pump-probe experiments. Here we present a lipidic cubic phase SFX structure of the light-driven proton pump bacteriorhodopsin (bR) to 2.3 Å resolution and a method to investigate protein dynamics with modest sample requirement. Time-resolved SFX (TR-SFX) with a pump-probe delay of 1 ms yields difference Fourier maps compatible with the dark to M state transition of bR. Importantly, the method is very sample efficient and reduces sample consumption to about 1 mg per collected time point. Accumulation of M intermediate within the crystal lattice is confirmed by time-resolved visible absorption spectroscopy. This study provides an important step towards characterizing the complete photocycle dynamics of retinal proteins and demonstrates the feasibility of a sample efficient viscous medium jet for TR-SFX.
ContributorsNogly, Przemyslaw (Author) / Panneels, Valerie (Author) / Nelson, Garrett (Author) / Gati, Cornelius (Author) / Kimura, Tetsunari (Author) / Milne, Christopher (Author) / Milathianaki, Despina (Author) / Kubo, Minoru (Author) / Wu, Wenting (Author) / Conrad, Chelsie (Author) / Coe, Jesse (Author) / Bean, Richard (Author) / Zhao, Yun (Author) / Bath, Petra (Author) / Dods, Robert (Author) / Harimoorthy, Rajiv (Author) / Beyerlein, Kenneth R. (Author) / Rheinberger, Jan (Author) / James, Daniel (Author) / Deponte, Daniel (Author) / Li, Chufeng (Author) / Sala, Leonardo (Author) / Williams, Garth J. (Author) / Hunter, Mark S. (Author) / Koglin, Jason E. (Author) / Berntsen, Peter (Author) / Nango, Eriko (Author) / Iwata, So (Author) / Chapman, Henry N. (Author) / Fromme, Petra (Author) / Frank, Matthias (Author) / Abela, Rafael (Author) / Boutet, Sebastien (Author) / Barty, Anton (Author) / White, Thomas A. (Author) / Weierstall, Uwe (Author) / Spence, John (Author) / Neutze, Richard (Author) / Schertler, Gebhard (Author) / Standfuss, Jorg (Author) / College of Liberal Arts and Sciences (Contributor) / Department of Physics (Contributor) / Department of Chemistry and Biochemistry (Contributor) / Biodesign Institute (Contributor) / Applied Structural Discovery (Contributor) / School of Molecular Sciences (Contributor)
Created2016-08-22
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Description
Recently we have seen rapid progress in the serial crystallography (SC) method at X-ray free-electron lasers (XFELs). Injection of thousands of protein microcrystals into the ∼10[superscript 12] photons of few-femtosecond XFEL pulses has allowed the structure determination of crystals grown in vivo, or of submicron size, and from challenging targets

Recently we have seen rapid progress in the serial crystallography (SC) method at X-ray free-electron lasers (XFELs). Injection of thousands of protein microcrystals into the ∼10[superscript 12] photons of few-femtosecond XFEL pulses has allowed the structure determination of crystals grown in vivo, or of submicron size, and from challenging targets such as membrane proteins. For time-resolved studies, the small crystal size allows for rapid diffusive saturation in mix-and-inject analysis of biochemical reactions, and full optical saturation of the sample by a pump laser in studies of light-driven proteins. The ability to outrun most radiation damage avoids the need for sample cooling and its artifacts, allowing studies of molecular machines at work in their correct room-temperature thermal bath or a controlled chemical environment.
ContributorsStandfuss, Jorg (Author) / Spence, John (Author) / College of Liberal Arts and Sciences (Contributor) / Department of Physics (Contributor)
Created2017-03
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Description
Diacylglycerol kinase catalyses the ATP-dependent conversion of diacylglycerol to phosphatidic acid in the plasma membrane of Escherichia coli. The small size of this integral membrane trimer, which has 121 residues per subunit, means that available protein must be used economically to craft three catalytic and substrate-binding sites centred about the

Diacylglycerol kinase catalyses the ATP-dependent conversion of diacylglycerol to phosphatidic acid in the plasma membrane of Escherichia coli. The small size of this integral membrane trimer, which has 121 residues per subunit, means that available protein must be used economically to craft three catalytic and substrate-binding sites centred about the membrane/cytosol interface. How nature has accomplished this extraordinary feat is revealed here in a crystal structure of the kinase captured as a ternary complex with bound lipid substrate and an ATP analogue. Residues, identified as essential for activity by mutagenesis, decorate the active site and are rationalized by the ternary structure. The γ-phosphate of the ATP analogue is positioned for direct transfer to the primary hydroxyl of the lipid whose acyl chain is in the membrane. A catalytic mechanism for this unique enzyme is proposed. The active site architecture shows clear evidence of having arisen by convergent evolution.
ContributorsLi, Dianfan (Author) / Stansfeld, Phillip J. (Author) / Sansom, Mark S. P. (Author) / Keogh, Aaron (Author) / Vogeley, Lutz (Author) / Howe, Nicole (Author) / Lyons, Joseph A. (Author) / Aragao, David (Author) / Fromme, Petra (Author) / Fromme, Raimund (Author) / Basu, Shibom (Author) / Grotjohann, Ingo (Author) / Kupitz, Christopher (Author) / Rendek, Kimberley (Author) / Weierstall, Uwe (Author) / Zatsepin, Nadia (Author) / Cherezov, Vadim (Author) / Liu, Wei (Author) / Bandaru, Sateesh (Author) / English, Niall J. (Author) / Gati, Cornelius (Author) / Barty, Anton (Author) / Yefanov, Oleksandr (Author) / Chapman, Henry N. (Author) / Diederichs, Kay (Author) / Messerschmidt, Marc (Author) / Boutet, Sebastien (Author) / Williams, Garth J. (Author) / Seibert, M. Marvin (Author) / Caffrey, Martin (Author) / College of Liberal Arts and Sciences (Contributor) / School of Molecular Sciences (Contributor) / Biodesign Institute (Contributor) / Applied Structural Discovery (Contributor) / Department of Physics (Contributor)
Created2015-12-17