Lipidic cubic phase serial femtosecond crystallography structure of a photosynthetic reaction centre

Abstract

Serial crystallography is a rapidly growing method that can yield structural insights from microcrystals that were previously considered to be too small to be useful in conventional X-ray crystallography. Here, conditions for growing microcrystals of the photosynthetic reaction centre of Blastochloris viridis within a lipidic cubic phase (LCP) crystallization matrix that employ a seeding protocol utilizing detergent-grown crystals with a different crystal packing are described. LCP microcrystals diffracted to 2.25 Å resolution when exposed to XFEL radiation, which is an improvement of 0.15 Å over previous microcrystal forms. Ubiquinone was incorporated into the LCP crystallization media and the resulting electron density within the mobile Q_B pocket is comparable to that of other cofactors within the structure. As such, LCP microcrystallization conditions will facilitate time-resolved diffraction studies of electron-transfer reactions to the mobile quinone, potentially allowing the observation of structural changes associated with the two electron-transfer reactions leading to complete reduction of the ubiquinone ligand.

Keywords: Blastochloris viridis; lipidic cubic phase; membrane-protein crystallization; microcrystals; photosynthetic reaction centre; serial crystallography.

Figure 1

LCP microcrystallization of RC _vir . (a) Early leads after screening. (b) Thin needle-like crystals which grew in syringes and diffracted to 2.4 Å resolution at SACLA but with a low hit rate. (c) Initial crystals grown in 800 µl glass wells. These crystals diffracted to 2.2 Å resolution at SACLA but had a long crystal axis and could not be indexed. (d) Seeded LCP crystals suitable for SFX and TR-SFX studies using XFEL radiation. These crystals diffracted to 2.25 Å resolution.

Figure 2

Electron density recovered for the SFX LCP structure of RC _vir . Diffraction data were recorded and processed to 2.25 Å resolution. Electron density recovered for the mobile ubiquinone (Q_B) was of similar quality to that recovered for other cofactors. 2F _obs − F _calc electron-density maps (blue) are contoured at 1σ, where σ represents the root-mean-square electron density of the map. The A-­branch leading to Q_A is on the right, whereas the M-branch leading to Q_B is on the left.

Figure 3

Hierarchal tree analysis of crystallographic structures of RC _vir deposited in the Protein Data Bank. Crystal structures are sorted according to the mean difference of their internal distances, as described by Wickstrand et al. (2015 ▸). The LCP SFX structures described here (PDB entry 7q7p, data collected at SACLA; PDB entry 7q7q, data collected at SwissFEL) form their own cluster, but have a mean internal distance-matrix difference on C^α atoms of 0.35 Å relative to all other RC _vir structures. Although the space group (P2₁2₁2) is the same as that found for earlier LCP crystal structures of RC _vir , the unit cell has a significantly shorter c axis than that recovered in the low-temperature LCP crystal structures.

Figure 4

Structure of the mobile ubiquinone Q_B binding site. (a) Superposition of ubiquinone observed here (black) with earlier proximal (red) and distal (blue) Q_B binding sites in RC_Sph (PDB entries 1aig and 1aij). (b) Superposition of ubiquinone observed here (black) with an earlier proximal (red) Q_B binding site in RC _vir (PDB entry 2i5n). (c) Electron density for the LCP SFX structure. (d) Electron density showing the presence of monoolein in an earlier LSP crystal form (PDB entry 2wjn). F _obs − F _calc omit electron-density maps (green) are contoured at 3σ. 2F _obs − F _calc electron-density maps (blue) are contoured at 1σ.

Figure 5

Structure of the surface-bound ubiquinone molecule. (a) The location of the surface-bound ubiquinone molecule is indicated in orange. (b) 2F _obs − F _calc electron-density map showing electron density for this ubiquinone. Two possible conformations are shown since the electron-density map does not yield a unique orientation for the surface-bound ubiquinone molecule. This map is contoured at 1σ.

Figure 6

A comparison of the waters (red) and lipids (blue) distributed within the two presented structures, with (a) showing the 2.4 Å resolution structure and (b) showing the 2.25 Å resolution structure. There are more lipids visible in (b), especially on the left side of the image corresponding to one of the contacts between the proteins within the crystal structure.