Novel approaches for the lipid sponge phase crystallization of the Rhodobacter sphaeroides photosynthetic reaction center.

Georgii Selikhanov,Tatiana Fufina,Lyudmila Vasilieva,Azat Gabdulkhakov,Christian Betzel

doi:10.1107/s2052252520012142

Abstract

With the recent developments in the field of free-electron-laser-based serial femtosecond crystallography, the necessity to obtain a large number of high-quality crystals has emerged. In this work crystallization techniques were selected, tested and optimized for the lipid mesophase crystallization of the Rhodobacter sphaeroides membrane pigment-protein complex, known as the photosynthetic reaction center (RC). Novel approaches for lipid sponge phase crystallization in comparatively large volumes using Hamilton gas-tight glass syringes and plastic pipetting tips are described. An analysis of RC crystal structures obtained by lipid mesophase crystallization revealed non-native ligands that displaced the native electron-transfer cofactors (carotenoid sphero-idene and a ubi-quinone molecule) from their binding pockets. These ligands were identified and were found to be lipids that are major mesophase components. The selection of distinct co-crystallization conditions with the missing cofactors facilitated the restoration of sphero-idene in its binding site.

Highlights

Water-soluble proteins can be crystallized and studied by X-ray structural analysis with relative success, membrane proteins remain problematic objects for analysis of their spatial structure
We developed new techniques for the crystallization of reaction centers in the lipid sponge phase (LSP) in Hamilton gas-tight glass syringes and in plastic pipetting tips to increase the number of crystals obtained (Fig. 2)
In this work novel crystallization techniques for lipid mesophase crystallization of the Rba. sphaeroides reaction center (RC) were tested and optimized, and we obtained a considerable number of high-quality crystals

Summary

Introduction

Water-soluble proteins can be crystallized and studied by X-ray structural analysis with relative success, membrane proteins remain problematic objects for analysis of their spatial structure. One of techniques developed for membrane protein crystallization is crystallization using lipid mesophases. Later on its basis lipid sponge phase (LSP) crystallization method was introduced (Wadsten et al, 2006). In both cases, matrix lipids form an environment that mimics the cell membrane, which contributes to the crystallization process. Lipid mesophase crystallization has some advantages over crystallization by the detergent-based vapor-diffusion technique. Crystals obtained by this method often have less solvent in their overall composition, higher symmetry, increased durability and, most importantly, lipid mesophase crystallization is often the only effective crystallization method for some proteins. The proportion of new structures obtained by applying this technique has increased

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