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Research Themes Membrane proteins

Membrane Proteome: Microcrystals Yield Big Data

SBKB [doi:10.1038/sbkb.2014.195]
Technical Highlight - April 2014
Short description: An X-ray free-electron laser helps determine the first room-temperature structure of a G protein-coupled receptor.

Experimental setup. Microcrystals (circle, right) dispersed in LCP (second circle) are injected inside a vacuum chamber and intersected with pulsed XFEL beam. Figure courtesy of Vadim Cherezov.

Crystallographic studies of membrane proteins are notoriously challenging, due to the difficulty of producing sufficient amounts of soluble protein and generating large crystals that can withstand the high-intensity X-ray beams required for data collection. Structural information on G protein-coupled receptors (GPCRs) is particularly valuable, as they comprise nearly 4% of the human proteome and are estimated to be the targets of around 40% of all modern drugs. Although recent advances in high-intensity microfocus X-ray beams have facilitated the determination of GPCR structures from microcrystals, these specialized beamlines are still incapable of collecting data from crystals smaller than around 1,000 μm3.

Recently, Cherezov and colleagues (PSI GPCR) employed serial femtosecond crystallography (SFX) to determine the structure of the human serotonin 5-HT2B receptor bound to the agonist ergotamine, from microcrystals averaging about 125 μm3 in size. Key to this study was the use of an X-ray free-electron laser (XFEL), which generates extremely high-intensity (∼2 mJ) and ultrashort (∼50 femtosecond) X-ray pulses, and of a specially designed injector system that allowed for continuous extrusion of a matrix of microcrystals grown in lipidic cubic phase through the X-ray beam. By slowly streaming a small volume (∼100 μL) of crystal-filled matrix through the beam, the authors were able to collect single diffraction images from 152,651 microcrystals, and could index and integrate a complete data set from 32,819 of these patterns, to determine the structure at 2.8-Å resolution (PDB 4NC3).

Because these data were collected at room temperature, the 5-HT2B structure determined in this study had slightly higher average B-factors—indicative of increased thermal motions—than those in the previous 5-HT2B structure obtained from large cryo-cooled crystals (∼10,000 μm3) and synchrotron radiation. Notably, the largest B-factor deviations were seen in the extracellular and intracellular loop regions of the receptor, where protein flexibility is expected to affect the binding of ligands and intracellular binding proteins, respectively. These results indicate that SFX may allow researchers not only to determine structures of membrane proteins from traditionally unsuitable crystals, but also to model the conformational dynamics of these proteins in a more physiological state.

Timothy Silverstein

References

  1. W. Liu et al. Serial Femtosecond Crystallography of G Protein-Coupled Receptors.
    Science. 342, 1521-4 (2013). doi:10.1126/science.1244142

  2. U. Weierstall et al. Lipidic cubic phase injector facilitates membrane protein serial femtosecond crystallography.
    Nat Commun. 5, 3309 (2014). doi:10.1038/ncomms4309

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