Crystal Structure of Rhodopsin: A G Protein-Coupled Receptor
Tóm tắt
Từ khóa
Tài liệu tham khảo
Sakmar T. P., Prog. Nucleic Acid Res. Mol. Biol. 59, 1 (1998).
The tetragonal crystals of rhodopsin have been obtained by using purified protein from bovine rod outer segment (ROS) membranes (19 49) and derivatized by soaking for about 2 months in a few mM solution of mercury acetate. To avoid deterioration of crystals by visible light all of the experimental procedures involving rhodopsin were carried out under dim red light. The unit cell dimensions are a = b = ∼96.5 Å and c = 149.5 Å with larger deviations up to 1.5 Å in the a and b dimensions. The previous assignment of the space group as P4 1 22 or P4 3 22 (19) for native crystal is incorrect. Instead the crystals are merohedrally twinned with space group P4 1 with two rhodopsin molecules in the asymmetric unit. Table 1 contains information about the crystal the mercury heavy atom derivative the MAD phasing and the structure refinement. The least twinned data set was selected after the collection of several MAD data sets. Six-wavelength MAD data sets were collected using only one crystal with a twin ratio of 10% at SPring-8 BL45XU (50) on a R-AXIS IV after measurement of the XANES spectrum of another mercury derivative for the determination of the Hg absorption edge wavelength. The intensity data were integrated and scaled with DENZO and SCALEPACK (51). Twinning ratio of the data was estimated with CNS (52) but the data were processed as collected without any consideration for twinning. Crystallographic calculation was performed including the model-refinement using this 3.3 Å MAD data set. SOLVE (53) performed local scaling and found four mercury sites in the asymmetric unit and phase refinement was successively done by SHARP (54). The MAD phased electron density map could be traced and fit with 14 α helices from two monomers. The noncrystallographic symmetry (NCS) operator and molecular masks were obtained from the helices. Density modifications including NCS averaging were performed with DM/CCP4 (55). Most of the protein region including side chains could be recognized except loops C-II and C-III and the COOH-terminal portion in the map. The oligosaccharide chains from Asn 2 and Asn 15 were also partially found. Iterative simulated annealing and rebuilding of the model were carried out with CNS (52) and O (56). The R -value and free R were 23.9% and 28% for the 3.3 Å data at the final stage. The higher resolution model refinement has proceeded using a 2.8 Å data set collected at APS 19-ID using the 3.3 Å structure as the starting model and its MAD phased data with the experimental phase restraint option of CNS. The 2.8 Å data set was collected from a mercury-soaked crystal. The estimation of the twin fraction and detwining of the data were performed with CNS. The model structure was validated using PROCHECK (57).
Web figures 1 and 2 are available at www.sciencemag.org/feature/data/1053064.shl
Single-letter abbreviations for the amino acid residues are as follows: A Ala; C Cys; D Asp; E Glu; F Phe; G Gly; H His; I Ile; K Lys; L Leu; M Met; N Asn; P Pro; Q Gln; R Arg; S Ser; T Thr; V Val; W Trp; and Y Tyr. X indicates any residue.
Cowtan K., Joint CCP4 and ESF-EACBM Newsletter on Protein Crystallography 31, 34 (1994).
We are grateful to P. Van Hooser for sample preparation and support for this project; E. Merritt S. Turley M. Feese and S. Suresh for their help in the experiment at APS; and Y. Imamoto for the template artwork for Fig. 3. We thank the Stanford Synchrotron Radiation Laboratory for beam time for the initial stages of this study. Use of the Argonne National Laboratory Structural Biology Center beamlines at the Advanced Photon Source (APS) was supported by the U.S. Department of Energy Office of Biological and Environmental Research under contract W-31-109-ENG-38. Coordinates for bovine rhodopsin have been deposited in the Protein Data Bank (1F88). This research was supported by NIH grant EY09339 a grant from Research to Prevent Blindness Inc. (RPB Foundation) to the Department of Ophthalmology at the University of Washington and grants from Foundation Fighting Blindness Inc. the Ruth and Milton Steinbach Fund and the E. K. Bishop Foundation. This paper is dedicated to T. Yoshizawa.