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Catching proteins in the act

August 22, 2016

Lipidic cubic phase injector is a viable crystal delivery system for time-resolved serial crystallography

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.

Figure. In side view (a) and perpendicular to the membrane (b). The retinal is shown as yellow sticks and the Lys216 side chain covalently binding retinal as blue sticks. Continuous 2FoFc electron density map around retinal and lysine is shown in magenta. Helices A–G are defined based on the header of pdb entry 1QHJ.

Reference: Nogly, P., V. Panneels, G. Nelson, C. Gati, T. Kimura, C. Milne, D. Milathianaki, M. Kubo, W. Wu, C. Conrad, J. Coe, R. Bean, Y. Zhao, P. Båth, R. Dods, R. Harimoorthy, K. R. Beyerlein, J. Rheinberger, D. James, D. DePonte, C. Li, L. Sala, G. J. Williams, M. S. Hunter, J. E. Koglin, P. Berntsen, E. Nango, S. Iwata, H. N. Chapman, P. Fromme, M. Frank, R. Abela, S. Boutet, A. Barty, T. A. White, U. Weierstall, J. Spence, R. Neutze, G. Schertler and J. Standfuss (2016). Lipidic cubic phase injector is a viable crystal delivery system for time-resolved serial crystallography. Nature Commun. 7: 12314. 10.1038/ncomms12314 Nogly-2016 (1.87 MB).

Also see the the ScienceDaily article

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