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The making of a molecular movie

January 3, 2017

Time-resolved serial femtosecond crystallography (TR-SFX)

Bacteriorhodopsin (bR) is a membrane protein that harvests the energy content from light to transport protons out of the cell against a transmembrane potential. Nango et al. used time-resolved serial femtosecond crystallography at an x-ray free electron laser to provide 13 structural snapshots of the conformational changes that occur in the nanoseconds to milliseconds following photoactivation. These changes begin at the active site, propagate toward the extracellular side of the protein, and mediate internal protonation exchanges that achieve proton transport.
The authors used time-resolved difference absorption spectra from bR microcrystals suspended in a lipidic cubic phase matrix (see earlier highlight) to create a molecular movie of the events. Obtaining the recordings required examining around 2 million small protein crystal samples with the free electron laser SACLA in Japan. The FEL hits the samples with very short, powerful X-rays, capturing precise moments. "It’s like a strobe light," says Jörg Standfuss, a biophysicist and head of the serial crystallography research group. "Unlike a flashlight, it reveals flickers of motion in the dark." The ultrashort light pulses make it possible to record data before the samples are destroyed by the powerful X-rays. Thanks to the new SwissFEL facility at PSI in Würenlingen, which opened on 5 December 2016, researchers at Swiss higher education institutions no longer have to go halfway around the world to conduct their experiments

Figure. Structure and function of bacteriorhodopsin (bR).(A) Schematic illustrating retinal covalently bound to Lys216 through a protonated Schiff base (SB) in an all-trans and a 13-cis configuration. (B) Proton-exchange steps (arrows) achieving proton pumping by bR. The primary proton-transfer step is from the SB to Asp85 and corresponds to the spectroscopic L-to-M transition. (C) 2mFobs – DFcalc electron density for the bR active site in its resting conformation. Electron density (gray) is contoured at 1.3σ (σ is the root mean square electron density of the map). W400, W401, and W402 denote water molecules.

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Reference: Nango, E., A. Royant, M. Kubo, T. Nakane, C. Wickstrand, T. Kimura, T. Tanaka, K. Tono, C. Song, R. Tanaka, T. Arima, A. Yamashita, J. Kobayashi, T. Hosaka, E. Mizohata, P. Nogly, M. Sugahara, D. Nam, T. Nomura, T. Shimamura, D. Im, T. Fujiwara, Y. Yamanaka, B. Jeon, T. Nishizawa, K. Oda, M. Fukuda, R. Andersson, P. Båth, R. Dods, J. Davidsson, S. Matsuoka, S. Kawatake, M. Murata, O. Nureki, S. Owada, T. Kameshima, T. Hatsui, Y. Joti, G. Schertler, M. Yabashi, A.-N. Bondar, J. Standfuss, R. Neutze and S. Iwata (2016). A three-dimensional movie of structural changes in bacteriorhodopsin. Science 354: 1552 (10.1126/science.aah3497) Nango-2016 (1.52 MB).

Also see the the SNF Press Release

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