Membrane protein structure determination - the next generation

Abstract

The field of Membrane Protein Structural Biology has grown significantly since its first landmark in 1985 with the first three-dimensional atomic resolution structure of a membrane protein. Nearly twenty-six years later, the crystal structure of the beta2 adrenergic receptor in complex with G protein has contributed to another landmark in the field leading to the 2012 Nobel Prize in Chemistry. At present, more than 350 unique membrane protein structures solved by X-ray crystallography (http://blanco.biomol.uci.edu/mpstruc/exp/list, Stephen White Lab at UC Irvine) are available in the Protein Data Bank. The advent of genomics and proteomics initiatives combined with high-throughput technologies, such as automation, miniaturization, integration and third-generation synchrotrons, has enhanced membrane protein structure determination rate. X-ray crystallography is still the only method capable of providing detailed information on how ligands, cofactors, and ions interact with proteins, and is therefore a powerful tool in biochemistry and drug discovery. Yet the growth of membrane protein crystals suitable for X-ray diffraction studies amazingly remains a fine art and a major bottleneck in the field. It is often necessary to apply as many innovative approaches as possible. In this review we draw attention to the latest methods and strategies for the production of suitable crystals for membrane protein structure determination. In addition we also highlight the impact that third-generation synchrotron radiation has made in the field, summarizing the latest strategies used at synchrotron beamlines for screening and data collection from such demanding crystals. This article is part of a Special Issue entitled: Structural and biophysical characterisation of membrane protein-ligand binding.

Keywords: Crystal dehydration; Crystal seeding; In situ data collection; Macromolecular crystallography; Membrane protein; XFEL.

Graphical abstract

Fig. 1

Bottlenecks in membrane protein structure determination. Picture courtesy of Prof. So Iwata.

Fig. 2

Analyzing current trends in alpha helical membrane protein crystallization. Inset, pie charts showing the change in the proportion of structures between 2008 and 2012 and used in the analysis of their crystallization conditions. Respiratory complexes (brown), Channels (black), Transporters (green), Photosynthetic and Light Harvesting Complexes (purple), GPCRs (red), ATPases (orange), Bacterial Rhodopsins (blue) and the Others category (olive), those not fitting the seven main groupings. Stacked bar chart showing the breakdown of successful detergents used for crystallization is shown, subdivided into the eight MP families. Analyses such as these can help formulate successful strategies for crystallizing new MP targets.

Fig. 3

Picture showing the environment setup used for the in situ data collection at I24 beamline, Diamond Light Source. Inset, on-axis microscope image of an in situ crystal hit is shown. The red circle and cross-hair represent the beam size and position.