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

Abstract

The in meso in situ serial X‐ray crystallization method (Huang et al., (2015) Acta Crystallogr D Biol Crystallogr 71, 1238) combines lipid cubic phase crystallization, direct freezing of the crystallization droplet without handling of the crystals, and data collection in situ. Recently, this method was used to overcome the mechanical fragility of crystals which enabled the X‐ray structure determination of chemokine receptor 2A (Apel et al., (2019) Structure 27, 427) at 2.7 Å resolution. The CCR2 structure provides the structural basis for ligand selectivity of CCR2 against chemokine receptor 5 and provides insights into the residence time of MK‐0812 analogs based on molecular dynamics simulations. These findings offer new opportunities for drug discovery targeting chemokine receptors.

Plant transporters involved in combating boron toxicity: beyond 3D structures

“…To access the high-resolution and accurate structural information of transporters and to derive data of functional proteins, a combination of X-ray diffraction and micro-crystal electron diffraction [ 80 ] — using crystalline samples in native lipids and combined with in meso in situ serial X-ray crystallography [ 81 ] — is the preferred method. Furthermore, these analyses could be combined with cryo-electron microscopy [ 82 ] that images membrane proteins in lipid environments without crystallisation requirements.…”

“…These regions may be required for NIPs to fold and form diffracting crystals with interpretable electron density maps. Including these regions together with testing different fusions with or without binding partners, combined with SMALP and LCP platforms and in situ and microfocus beamline X-ray crystallography or cryo-electron microscopy [80][81][82], could lead to success. Other options include using cost-effective expression systems for high yields of folded NIPse.g.…”

3D-printed holders forin meso in situfixed-target serial X-ray crystallography

“…The cubic phase has also found application as a native-like medium in which to port crystals across beams for time-resolved serial crystallography at synchrotron (Nogly et al, 2015;Weinert et al, 2017) and free-electron laser X-ray sources (Weierstall et al, 2014;White et al, 2016;Nogly et al, 2018;Cheng, 2020). The recently developed in situ in meso serial X-ray crystallography (IMISX) method uses perforated double-stick tape to create wells for crystallization and supporting sachets (IMISX wells) in which to deliver the crystal-laden mesophase to the X-ray beam without the need for direct crystal harvesting (Huang et al, 2015(Huang et al, , 2016(Huang et al, , 2018El Ghachi et al, 2018;Apel et al, 2019;Cheng et al, 2019). The original IMISX method involved securing a slightly curved thin-film sachet to a pin on a goniometer (Fig.…”