Computational protocol: Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser

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Protocol publication

[…] A partial 8.0 Å synchrotron dataset was collected at 100 K using an X-ray beam at the wavelength of 1.0 Å at the 21 ID-D beam line of LS-CAT and at 23-ID-D of GM-CAT at the Advanced Photon Source at Argonne National Laboratory. A full 7.7 Å dataset was collected from a single crystal (~20 microns in size) using 10 μm beam size and 0.1 s exposures per 0.1° oscillation with a Pilatus 6M pixel detector at the X10SA beam line at the Swiss Light Source. The observed reflections were reduced, merged, and scaled with XDS with statistics shown in Supplementary Table 1a. The L-test plot of the 7.7 Å dataset produced a curve that fits a perfectly twinned crystal. [...] LCP-SFX experiments were carried out at the Coherent X-ray Imaging (CXI) instrument at the Linac Coherent Light Source (LCLS) in the SLAC National Accelerator Laboratory (Menlo Park, California, USA). X-ray pulses of 50 fs duration at a wavelength of 1.3 Å (9.5 keV) were attenuated to ~3% (3·1010 photons/pulse) and focused to ~1.5 μm diameter at the interaction point using Kirkpatrick-Baez mirrors. Rhodopsin-arrestin complex crystals in LCP were injected across the XFEL beam using an LCP injector with a 50 μm diameter nozzle at a flow rate of ~0.2 μL/min. Diffraction patterns were collected at 120 Hz using the Cornell-SLAC Pixel Array Detector (CSPAD). Over 5 million data frames were collected corresponding to ~12 hours of data acquisition time. Of these frames, ~0.45% images contained potential crystal hits as identified using Cheetah (more than 40 Bragg peaks of 1–20 pixels in size and a signal to noise ratio better than 6 after local background subtraction). Of the potential crystal hits, 18,874 diffraction patterns could be auto-indexed by CrystFEL using a combination of MOSFLM, XDS and DirAx. An integration radius of only two pixels was used to avoid overlapping with neighboring peaks due to the high spot density resulting from the large unit cell dimensions. Partial reflections from different crystals in random orientations were merged using a Monte Carlo integration across the crystal rocking curve of each reflection. The resolution was anisotropic with ~3.3 Å resolution along the c*-axis and ~3.8 Å resolution along the a*/b* axes. The data used for the structure refinement were truncated at 3.8Å/3.8Å/3.3Å using the get_hkl program of CrystFEL based on the criteria of data correlation coefficient (CC*), which is 0.87 at the highest resolution shell (Supplementary Table 1a). The use of CC* of 0.5 as resolution cutoff has been recently recommended for X-ray diffraction. The resolution cutoff for several published XFEL structures follows this criterion, including the 5HT2B GPCR XFEL structure, which has a CC* of 0.74. The statistics of the final data used in structure refinement are shown in Supplementary Table 1b. [...] The XFEL data were initially merged according to the apparent Laue group of 4/mmm, and molecular replacement searches were performed in all possible space groups of 4/m and 4/mmm. The best structure solution was found in P43. Based on analysis of the Zanuda program, we determined that the most likely space group was P212121 and that the crystals were physically twinned. The data were reprocessed with the Laue group of mmm and molecular replacement searches were performed in P212121. This space group assignment resulted in the best statistics and map quality out of several possibilities of space groups (Supplementary Table 2).The crystals appeared to be pseudo-merohedrally twinned based on L-test analysis. Despite the challenge of twinned data, the rhodopsin-arrestin complex structure was solved by the molecular replacement method implemented in Phaser using the models of constitutively active rhodopsin, pre-activated arrestin, and T4L (PDB codes: 4A4M, 4J2Q1, and 3SN68, respectively). Four molecules of rhodopsin and four molecules of arrestin were found sequentially by molecular replacement search, resulting in four very similar rhodopsin-arrestin assemblies. Four T4Ls were also found in the aqueous layer with its C-terminal residue in a position to form a covalent bond with the first residue of rhodopsin, supporting the correct positioning of T4Ls by molecular replacement.The structure was initially refined against the XFEL data without twin law to an Rfree-factor of ~36% and the model maps from the data were of sufficient quality to interpret the overall structure of the rhodopsin-arrestin complex (). The model then underwent iterated cycles of manual building into 2Fo-Fc maps with Coot and refinement with REFMAC and the PHENIX, where rigid body, individual position, group B-factor, and TLS refinements were used along with NCS restraints and twin law (k, h, -l). The arrestin residues 70–78 and 165–175, which were not included in the molecular replacement model, were manually placed into the density map. These two regions of arrestin became visible because they are either engaged in direct interaction with rhodopsin or involved in crystal packing. Regions with poor density were removed from the final model, including T4L from the B complex and the N-domain of T4L (residues 13–57) from the C complex. We did not observe clear electron density for the all-trans retinal, which was thus not included in the structure. The structure has been carefully refined to the final state that has excellent geometry and refinement statistics (Supplementary Table 1b). Ramachandran plot analysis indicates that 100% of the residues are in favorable or allowed regions (no outliers). The final structure was validated with MolProbity, which revealed an all-atom-clash score of 1.47 and MolProbity score of 1.13.The real-space correlation coefficients against a 2mFo-DFc map for each chain of the structure on a per residue basis using the CCP4 EDSTATS program or the MolProbity program in Phenix indicated an overall good fit between the structure and the electron density map. The density fit correlation in Coot was low with the structure from Phenix twinned refinement because the map from the MTZ file with map coefficients produced by phenix.refine was not in absolute scale. This problem was overcome by using the 2Fo-Fc CNS format map from phenix.refine, which generated a normal correlation in Coot, similar to those from EDSTATS and MolProbity. All structural figures were prepared using PyMOL. [...] Samples were prepared as previously described. Briefly, the sample was applied to a freshly glow-discharged carbon coated copper grid and allowed to adhere for 10 seconds before being reduced to a thin film by blotting. Immediately after blotting 3 μl of a 1% solution of uranyl formate was applied to the grid and blotted off directly. This was repeated three times. Data were acquired using a Tecnai F20 Twin transmission electron microscope operating at 200 kV, with a dose of ~40 e−/Å2 and nominal underfocus ranging from 2 to 3 μm. Images were automatically collected at a nominal magnification of 62,000 × and pixel size of 0.273 nm. All images were recorded with a Tietz F416 4k × 4k pixel CCD camera utilizing Leginon data collection software. Experimental data were processed by the Appion software package, which interfaces with the Leginon database infrastructure. ~6000 particles were automatically extracted from 54 EM-micrographs and the particle stack was then aligned and sorted using the XMIPP reference-free maximum likelihood alignment. Several exemplary 2D averages are shown in , which were derived from class averages for the complex with or without T4L, computed from ~14000 particles selected from 155 EM-micrographs. […]

Pipeline specifications

Software tools XDS, iMosflm, Coot, PHENIX, MolProbity, CCP4, PyMOL
Applications Small-angle scattering, Protein structure analysis
Organisms Mus musculus, Dipturus trachyderma, Homo sapiens
Chemicals Rhodopsin, Arrestin