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Serial Femtosecond Crystallography of G Protein–Coupled Receptors

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Science  20 Dec 2013:
Vol. 342, Issue 6165, pp. 1521-1524
DOI: 10.1126/science.1244142

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

    5-HT2B receptor microcrystals (first zoom level) dispersed in LCP (second zoom level) are injected as a continuous column of 20 to 50 μm in diameter—stabilized by a co-axial gas flow (blue dash curved lines)—inside a vacuum chamber and intersected with 1.5-μm-diameter pulsed XFEL beam focused with Kirkpatrick-Baez (K-B) mirrors. Single-pulse diffraction patterns were collected at 120 Hz by using a CSPAD detector. The entire XFEL beam path and CSPAD are under vacuum.

  2. Fig. 2 Comparison between 5-HT2B-XFEL (light red) and 5-HT2B-SYN (teal) 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.phar.umich.edu) (28). (A) Electron density for the Glu212 side chain is missing in 5-HT2B-SYN and fully resolved in 5-HT2B-XFEL. (B) A salt bridge between Glu319 and Lys247 links intracellular parts of helices V and VI in the 5-HT2B-XFEL structure. In the 5-HT2B-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-HT2B-XFEL with Thr114, making a stabilizing hydrogen bond with the backbone carbonyl, whereas in 5-HT2B-SYN, a water-stabilized kink is introduced at this position. (D) Tyr87 forms a hydrogen bond with Asn90 in 5-HT2B-XFEL; this hydrogen bond is broken, and Tyr87 adopts a different rotamer conformation in the 5-HT2B-SYN structure. 2mFobs-DFcalc maps (contoured at 1σ level) are shown only around described residues.

  3. Fig. 3 Comparison of B-factors between 5-HT2B-XFEL and 5-HT2B-SYN structures.

    (A) B-factors difference (BXFEL-BSYN) for Cα atoms plotted versus residue number. (B) View of the 5-HT2B-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 to 170 Å2). Loops for which the B-factor difference is above 50 Å2 are labeled in red, and those with a difference below 50 Å2 are in blue in (A) and (C). Helices are labeled in (B).

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