In meso in situserial X-ray crystallography of soluble and membrane proteins at cryogenic temperatures

Abstract

A method for performing high-throughput in situ serial X-ray crystallography with soluble and membrane proteins in the lipid cubic phase at cryogenic temperatures (100 K) is described. It works with nanogram to single-digit microgram quantities of protein and lipid (and ligand when present), and is compatible with both high-resolution native data collection and experimental phasing without the need for crystal harvesting.

Tripeptide binding in a proton‐dependent oligopeptide transporter

“…During the transport cycle, these domains move relative to each other to allow alternate access from the cytoplasmic and extracellular sides . Several X‐ray structures of bacterial POTs have been reported, either in the apo‐form , peptide bound state , or in complex with the phosphonodipeptide alafosfalin (Table S1). However, only three structures have hitherto been reported in which a tripeptide is bound.…”

Microfluidics: From crystallization to serial time-resolved crystallography

“…In particular, COC has seen widespread adoption as the polymer film of choice for X-ray compatible devices, 46 including simple channel structures for counterdiffusion, 56,58,60,61,126 droplet-based devices, 108,109,118 and larger-scale X-ray compatible wellplates. [127][128][129][130][131][132][133][134][135][136][137][138][139][140] However, further decreasing the device thickness to achieve the signal-to-noise levels required for microcrystallography is a significant materials' challenge. Typical reports of X-ray compatible microfluidics describe results where the path length of the device materials is nearly twice that of the crystal of interest.…”

“…Typical reports of X-ray compatible microfluidics describe results where the path length of the device materials is nearly twice that of the crystal of interest. 18,19,40,128,129,141 Thus, the analysis of micronscale or smaller crystals would suggest the need to shrink device materials to a total thickness of 1-10 lm. Unfortunately, although ultra-thin polymeric films are available, such materials tend to suffer from poor mechanical stability and more critically show poor barrier performance against sample dehydration.…”

In situ crystallography as an emerging method for structure solution of membrane proteins: the case of CCR2A

“…Here, we review the current development status of in meso in situ serial X‐ray crystallization (IMISX), a specific example of a solid support crystal delivery and its recent application to the structure determination of the chemokine receptor 2A (CCR2A) at 2.7 Å resolution . CCL2/CCR2 signaling has been implicated in the pathogenesis of several neurological disorders and autoimmune diseases like multiple sclerosis, obesity, and atherosclerosis, suggesting that CCR2 is an attractive target for potential therapeutic intervention of these diseases .…”