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. 2015 Aug 4;2(Pt 5):545-51.
doi: 10.1107/S2052252515013160. eCollection 2015 Sep 1.

Serial femtosecond crystallography of soluble proteins in lipidic cubic phase

Raimund Fromme  1 , Andrii Ishchenko  2 , Markus Metz  3 , Shatabdi Roy Chowdhury  1 , Shibom Basu  1 , Sébastien Boutet  4 , Petra Fromme  1 , Thomas A White  5 , Anton Barty  5 , John C H Spence  6 , Uwe Weierstall  6 , Wei Liu  1 , Vadim Cherezov  2
Affiliations

Serial femtosecond crystallography of soluble proteins in lipidic cubic phase

Raimund Fromme et al. IUCrJ. .

Abstract

Serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs) enables high-resolution protein structure determination using micrometre-sized crystals at room temperature with minimal effects from radiation damage. SFX requires a steady supply of microcrystals intersecting the XFEL beam at random orientations. An LCP-SFX method has recently been introduced in which microcrystals of membrane proteins are grown and delivered for SFX data collection inside a gel-like membrane-mimetic matrix, known as lipidic cubic phase (LCP), using a special LCP microextrusion injector. Here, it is demonstrated that LCP can also be used as a suitable carrier medium for microcrystals of soluble proteins, enabling a dramatic reduction in the amount of crystallized protein required for data collection compared with crystals delivered by liquid injectors. High-quality LCP-SFX data sets were collected for two soluble proteins, lysozyme and phycocyanin, using less than 0.1 mg of each protein.

Keywords: X-ray free-electron laser; lipidic cubic phase; serial femtosecond crystallography; soluble protein.

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Figures

Figure 1
Figure 1
Representative crystal images and diffraction patterns. (a, b) Pictures of crystals embedded in LCP for lysozyme (a) and PC (b). (c, d) Full diffraction patterns for lysozyme (c) and PC (d). Parts of the diffraction images, outlined by squares in (c) and (d), are enlarged in (e) and (f), respectively, to facilitate visualization of individual diffraction peaks. Diffraction peaks identified by Cheetah are circled.
Figure 2
Figure 2
Lysozyme structure. (a) Overall structure of lysozyme showing 2mF o − DF c electron density around several residues in the active site contoured at 1σ. (b) Difference electron density between the data collected in this work and those presented in Boutet et al. (2012 ▸), contoured at 3σ. Positive difference density is green and negative is red.
Figure 3
Figure 3
Phycocyanin structure. Simulated-annealing composite OMIT 2mF o − DF c electron-density map contoured at the 1.5σ level for the chromophore phycobilin inside its binding pocket. The map was built using 1.75 Å resolution XFEL data from PC crystals delivered in LCP.
See this image and copyright information in PMC

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