Lipidic phase membrane protein serial femtosecond crystallography

Abstract

X-ray free electron laser (X-FEL)-based serial femtosecond crystallography is an emerging method with potential to rapidly advance the challenging field of membrane protein structural biology. Here we recorded interpretable diffraction data from micrometer-sized lipidic sponge phase crystals of the Blastochloris viridis photosynthetic reaction center delivered into an X-FEL beam using a sponge phase micro-jet.

Figure 1

LSP batch crystallization of RC_vir. (a) A 250-μl batch-crystallization setup in a glass vial with the sponge phase containing RC_vir (brown) floating on top. (b) Optical microscopy image of the sponge phase showing crystals. Larger crystals are ~20 μm long.

Figure 2

Serial femtosecond crystallography of RC_vir crystals grown in a LSP. (a) Liquid jet formed by the sponge phase containing RC_vir crystals. The X-FEL beam interacting with the liquid jet is visible as a white fluorescent spot (white arrow). (b) Bragg diffraction spots (dark spots) recorded from a single RC_vir crystal using a single X-FEL pulse of 70 fs. (c) An identical diffraction image as shown in b but with the predicted spot positions after data indexing shown as circles. The resolution was limited to 7.4 Å in the corners of the lower pnCCD detector panel.

Figure 3

Electron density for the LSP serial femtosecond crystallography RC_vir structure at 8.2 Å resolution. (a) Stereo view of the 2m F_obs - DF_calc electron density map where m is the figure of merit and D is estimated from coordinate errors (contoured at 1.0 σ) recovered from 265 processed RC_vir diffraction images. (b) Stereo view of the mF_obs - DF_calc omit electron density map (contoured at 2.0 σ), calculated with the four heme groups of the cytochrome subunit removed from the structural model. This figure was generated with Pymol (DeLano Scientific LLC).