Serial femtosecond crystallography of G protein-coupled receptors

Abstract

X-ray crystallography of G protein-coupled receptors and other membrane proteins is hampered by difficulties associated with growing sufficiently large crystals that withstand radiation damage and yield high-resolution data at synchrotron sources. We used an x-ray free-electron laser (XFEL) with individual 50-femtosecond-duration x-ray pulses to minimize radiation damage and obtained a high-resolution room-temperature structure of a human serotonin receptor using sub-10-micrometer microcrystals grown in a membrane mimetic matrix known as lipidic cubic phase. Compared with the structure solved by using traditional microcrystallography from cryo-cooled crystals of about two orders of magnitude larger volume, the room-temperature XFEL structure displays a distinct distribution of thermal motions and conformations of residues that likely more accurately represent the receptor structure and dynamics in a cellular environment.

Fig. 1. Experimental setup for SFX data collection using an LCP injector

5-HT_2B receptor microcrystals (first zoom level) dispersed in LCP (second zoom level) are injected as a continuous column of 20–50 µm diameter inside a vacuum chamber and intersected with 1.5 µm diameter pulsed XFEL beam focused by Kirkpatrick-Baez (K-B) mirrors. Single pulse diffraction patterns are collected at 120 Hz using a CSPAD detector.

Fig. 2. Comparison between 5-HT_2B-XFEL (light red) and 5-HT_2B-SYN (cyan) structures

Central image represents a backbone overlay of the two structures. Dashed lines correspond to membrane boundaries defined by the Orientation of Proteins in Membrane database (http://opm.pharm.umich.edu) (28). (A) Electron density for the Glu212 side chain is missing in 5-HT_2B-SYN and fully resolved in 5-HT_2B-XFEL. (B) A salt bridge between Glu319 and Lys247 links intracellular parts of helices V and VI in the 5-HT_2B-XFEL structure. In the 5-HT_2B-SYN structure Lys247 makes a hydrogen bond with Tyr1105 from the BRIL fusion protein. (C) Extracellular tip of helix II forms a regular helix in 5-HT_2B-XFEL with Thr114 making a stabilizing hydrogen bond with the backbone carbonyl, while in 5-HT_2B-SYN a water stabilized kink is introduced at this position. (D) Tyr87 forms a hydrogen bond with Asn90 in 5-HT_2B-XFEL; this hydrogen bond is broken and Tyr87 adopts a different rotamer conformation in 5-HT_2B-SYN structure. 2mF_obs-F_calc maps (contoured at 1 σ level) are shown only around described residues.

Fig. 3. Differences in B-factors between 5-HT_2B-XFEL and 5-HT_2B-SYN structures

(A) B-factors difference (B_XFEL-B_SYN) for Cα atoms plotted vs. residue number. (B) View of the 5-HT_2B-XFEL structure from the extracellular side and (C) in the lateral to membrane orientation. Structure in B and C is shown in putty representation and colored in rainbow colors by the Cα B-factors (range 60 – 170 Ų). Loops for which B-factors increased above average are labeled in red and those that have about an average increase are labeled in blue in (A) and (C). Helices are labeled in (B).