In meso in situ serial X-ray crystallography of soluble and membrane proteins

Abstract

A method for performing high-throughput in situ serial X-ray crystallography with soluble and membrane proteins in the lipid cubic phase is described. It works with microgram quantities of protein and lipid (and ligand when present) and is compatible with the most demanding sulfur SAD phasing.

Advances in membrane protein crystallography: in situ and in meso data collection

“…In this issue of Acta Cryst. D, two independent groups, Axford et al (2015) and Huang et al (2015), have published methods that make a major contribution to addressing these problems, which will facilitate high-resolution data-collection of fragile crystals.In the methodology demonstrated by Axford et al (2015), a standard in situ 96-well sitting-drop crystallization plate was used to crystallize TehA from Haemophilus influenzae in a final volume of 200 nl. The plate was left for several days until crystals grew to their maximum size (up to 75 mm in the largest dimension).…”

“…In this issue of Acta Cryst. D, two independent groups, Axford et al (2015) and Huang et al (2015), have published methods that make a major contribution to addressing these problems, which will facilitate high-resolution data-collection of fragile crystals.…”

“…This study therefore provides a proof of principle that membrane protein structures can be determined at a synchrotron using in situ room temperature data collection strategies. Huang et al (2015) took the in situ approach one step further and showed the applicability of room temperature data collection for in meso/in situ crystallization (IMISX) and its use for high-throughput crystallography of membrane proteins crystallized in meso using the LCP technology. This study used two different membrane proteins (the -barrel AlgE and the -helical protein PepT St ) and lysozyme for their demonstration of proof-of-principle.…”

“…Two solutions for collecting in situ diffraction data from crystallization plates. (a) The set-up at SLS for IMISX (Huang et al, 2015). (b) The setup at Diamond for data collection from 96-well plates (Axford et al, 2015).…”

A Graphene-Based Microfluidic Platform for Electrocrystallization and In Situ X-ray Diffraction

“…Such platforms have been increasingly harnessed to facilitate the diffraction analysis of challenging targets for both static and dynamic structure determination. Various platforms have been developed to improve the growth and subsequent mounting of tiny and fragile crystals for X-ray diffraction analysis [24][25][26][27][28], including dense array-style devices [29][30][31][32][33][34][35][36][37][38][39][40][41], platforms for the lipidic cubic phase crystallization of membrane proteins [42,43], and thin-film sandwich devices [44]. In the meantime, the challenges of such platforms lie in the need to maintain a protected sample environment, as well as minimize the interference of device materials with the subsequent X-ray analysis.…”