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

Abstract

Membrane protein structural biology has made tremendous advances over the last decade as indicated by the exponential growth in the number of structures that have been published (http://blanco.biomol.uci.edu/mpstruc/). These advances are a result of many factors (Bill et al., 2011), including improvements in membrane protein overexpression, stabilization of proteins using antibodies or thermostabilizing mutations, and the enhancement of crystallization technologies such as crystallization in lipidic cubic phase (LCP, in meso crystallization). However, there are still many challenges associated with membrane protein crystallization, data collection and structure determination. Major problems often arise because membrane proteins frequently form tiny crystals, which either cannot be improved in size or which can be improved in size, but, as a consequence, lose diffraction quality. In addition, crystal handling, such as mounting the crystals and soaking in cryoprotectants, is often the reason for the loss of diffraction quality through mechanical shear-induced microlesions. This is particularly true for membrane protein crystals, which are often very fragile because of their high solvent content and being very thin in one 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.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). Instead of harvesting and mounting the crystals, the team mounted the entire plate on the beamline (in this case I24 at the Diamond Light Source; Fig. 1) and standard procedures were then used for data collection from the membrane protein crystals, i.e. each crystal was centered in the beam and wedges of data were collected at room temperature. This avoided simultaneously two major potential problems, namely crystal handling and cryocooling. Wedges of data were collected from multiple different crystals (30-50 images per wedge, 0.2 rotation each), thus reducing the effects of radiation damage, and then they were merged to obtain a data set at 2.3 Å resolution (90% complete). A direct comparison was performed between the structure of TehA determined from a cryocooled crystal at 1.5 Å resolution (98% complete, one crystal) and from the room temperature plate collection strategy (56 crystals); only minor changes were observed in flexible regions such as loop regions. 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...

Electron Paramagnetic Resonance as a Tool for Studying Membrane Proteins

“…In recent years, several biophysical techniques have been utilized to investigate the structural and dynamic properties of membrane proteins. The most popular biophysical techniques are X-ray crystallography, nuclear magnetic resonance (NMR), electron microscopy (Cryo-EM), Förster resonance energy transfer (FRET), and electron paramagnetic resonance (EPR) spectroscopy [13,[18][19][20][21][22][23]. X-ray crystallography is used to determine the highly resolved 3D structure of membrane proteins [24].…”

“…Figure 4 shows an illustration of CW-EPR spectra for the MTSL nitroxide spin label attached to KCNE1 reconstituted in different dynamic environments [69]. Another recent example of using nitroxide spin labeling CW-EPR spectroscopy is the study of pinholin S 21 68 [66]. Pinholin S 21 68 is an essential part of the phage Φ21 lytic protein system that releases the virus progeny at the end of the infection cycle.…”

“…Pinholin S 21 68 is an essential part of the phage Φ21 lytic protein system that releases the virus progeny at the end of the infection cycle. TMD1 of active pinholin S 21 68 externalizes very quickly to the periplasm resulting in the active dimer. Within seconds of pinholin triggering the system, it forms heptametric holes by rapid oligomerization and reorientation of TMD2.…”

“…Within seconds of pinholin triggering the system, it forms heptametric holes by rapid oligomerization and reorientation of TMD2. Ahammad et al analyzed CW-EPR spectra collected for spin-labeled active pinholin S 21 68 to investigate the dynamic properties of the active form of pinholin S 21 68 in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipid bilayers [66]. The CW-EPR spectral line shape analysis of the R1 side chain for 39 residue positions of S 21 68 suggested that the transmembrane domains (TMDs) have more restricted mobility when compared to the N-and C-termini.…”

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.…”