Matching Items (12)
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Description
Serial femtosecond crystallography (SFX) has opened a new era in crystallo­graphy by permitting nearly damage-free, room-temperature structure determination of challenging proteins such as membrane proteins. In SFX, femtosecond X-ray free-electron laser pulses produce diffraction snapshots from nanocrystals and microcrystals delivered in a liquid jet, which leads to high protein consumption.

Serial femtosecond crystallography (SFX) has opened a new era in crystallo­graphy by permitting nearly damage-free, room-temperature structure determination of challenging proteins such as membrane proteins. In SFX, femtosecond X-ray free-electron laser pulses produce diffraction snapshots from nanocrystals and microcrystals delivered in a liquid jet, which leads to high protein consumption. A slow-moving stream of agarose has been developed as a new crystal delivery medium for SFX. It has low background scattering, is compatible with both soluble and membrane proteins, and can deliver the protein crystals at a wide range of temperatures down to 4°C. Using this crystal-laden agarose stream, the structure of a multi-subunit complex, phycocyanin, was solved to 2.5 Å resolution using 300 µg of microcrystals embedded into the agarose medium post-crystallization. The agarose delivery method reduces protein consumption by at least 100-fold and has the potential to be used for a diverse population of proteins, including membrane protein complexes.
ContributorsConrad, Chelsie (Author) / Basu, Shibom (Author) / James, Daniel (Author) / Wang, Dingjie (Author) / Schaffer, Alexander (Author) / Roy Chowdhury, Shatabdi (Author) / Zatsepin, Nadia (Author) / Aquila, Andrew (Author) / Coe, Jesse (Author) / Gati, Cornelius (Author) / Hunter, Mark S. (Author) / Koglin, Jason E. (Author) / Kupitz, Christopher (Author) / Nelson, Garrett (Author) / Subramanian, Ganesh (Author) / White, Thomas A. (Author) / Zhao, Yun (Author) / Zook, James (Author) / Boutet, Sebastien (Author) / Cherezov, Vadim (Author) / Spence, John (Author) / Fromme, Raimund (Author) / Weierstall, Uwe (Author) / Fromme, Petra (Author) / Department of Chemistry and Biochemistry (Contributor) / Biodesign Institute (Contributor) / Applied Structural Discovery (Contributor) / College of Liberal Arts and Sciences (Contributor) / Department of Physics (Contributor) / School of Molecular Sciences (Contributor)
Created2015-06-30
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Description

Structural studies on living cells by conventional methods are limited to low resolution because radiation damage kills cells long before the necessary dose for high resolution can be delivered. X-ray free-electron lasers circumvent this problem by outrunning key damage processes with an ultra-short and extremely bright coherent X-ray pulse. Diffraction-before-destruction

Structural studies on living cells by conventional methods are limited to low resolution because radiation damage kills cells long before the necessary dose for high resolution can be delivered. X-ray free-electron lasers circumvent this problem by outrunning key damage processes with an ultra-short and extremely bright coherent X-ray pulse. Diffraction-before-destruction experiments provide high-resolution data from cells that are alive when the femtosecond X-ray pulse traverses the sample. This paper presents two data sets from micron-sized cyanobacteria obtained at the Linac Coherent Light Source, containing a total of 199,000 diffraction patterns. Utilizing this type of diffraction data will require the development of new analysis methods and algorithms for studying structure and structural variability in large populations of cells and to create abstract models. Such studies will allow us to understand living cells and populations of cells in new ways. New X-ray lasers, like the European XFEL, will produce billions of pulses per day, and could open new areas in structural sciences.

Contributorsvan der Schot, Gijs (Author) / Svenda, Martin (Author) / Maia, Filipe R. N. C. (Author) / Hantke, Max F. (Author) / DePonte, Daniel P. (Author) / Seibert, M. Marvin (Author) / Aquila, Andrew (Author) / Schulz, Joachim (Author) / Kirian, Richard (Author) / Liang, Mengning (Author) / Stellato, Francesco (Author) / Bari, Sadia (Author) / Iwan, Bianca (Author) / Andreasson, Jakob (Author) / Timneanu, Nicusor (Author) / Bielecki, Johan (Author) / Westphal, Daniel (Author) / Nunes de Almeida, Francisca (Author) / Odic, Dusko (Author) / Hasse, Dirk (Author) / Carlsson, Gunilla H. (Author) / Larsson, Daniel S. D. (Author) / Barty, Anton (Author) / Martin, Andrew V. (Author) / Schorb, Sebastian (Author) / Bostedt, Christoph (Author) / Bozek, John D. (Author) / Carron, Sebastian (Author) / Ferguson, Ken (Author) / Rolles, Daniel (Author) / Rudenko, Artem (Author) / Epp, Sascha W. (Author) / Foucar, Lutz (Author) / Rudek, Benedikt (Author) / Erk, Benjamin (Author) / Hartmann, Robert (Author) / Kimmel, Nils (Author) / Holl, Peter (Author) / Englert, Lars (Author) / Loh, N. Duane (Author) / Chapman, Henry N. (Author) / Andersson, Inger (Author) / Hajdu, Janos (Author) / Ekeberg, Tomas (Author) / College of Liberal Arts and Sciences (Contributor)
Created2016-08-01