ELECTRON-CRYSTALLOGRAPHIC REFINEMENT OF THE STRUCTURE OF BACTERIORHODOPSIN

Using electron diffraction data corrected for diffuse scattering toget her with additional phase information from 30 new images of tilted spe cimens, an improved experimental density map has been calculated for b acteriorhodopsin. The atomic model has then been rebuilt into this new map with particular attention to the surface loops. All the residues from 7 to 227 as well as ten lipid molecules are now included, althoug h a few amino acid residues in three of the six surface loops, about h alf of the lipid hydrophobic chains and all of the lipid head groups a re disordered. The model has then been refined against the experimenta l diffraction amplitudes to an X-factor of 28% at 3.5 Angstrom resolut ion with strict geometry (0.005 Angstrom bond length deviation) using the improvement of the ''free'' phase residual between calculated and experimental phases from images as an objective criterion of accuracy. For the refinement some new programs were developed to restrain the n umber of parameters, to be compatible with the limited resolution of o ur data. In the final refined model of the protein (2BRD), compared wi th earlier co-ordinates (1BRD), helix D has been moved towards the cyt oplasm by almost 4 Angstrom, and the overall accuracy of the co-ordina tes of residues in the other six helices has been improved. As a resul t the positions of nearly all the important residues in bacteriorhodop sin are now well determined. In particular, the buried, protonated Asp 115 is 7 Angstrom from, and so not in contact with, the retinal and Me t118 forms a cap on the pocket occupied by the beta-ionone ring. No cl ear density exists for the side-chain of Arg82, which forms a central part of the extracellular half-channel. The only arginine side-chain b uilt into good density is that of Arg134 at the extracellular end of h elix E, the others being disordered near one of the two surfaces. The interpretation of the end of helix F on the extracellular surface is n ow clearer; an extra loose helical turn has been built bringing the si de-chain of Glu194 close to Arg134 to form a probable salt bridge. The model provides an improved framework for understanding the mechanism of the light-driven proton pumping. A number of cavities that could co ntain water molecules were found by searching the refined model, most of them above or below the Schiff base in the half-channels leading to the two surfaces. The ordered and disordered regions of the structure are described by the temperature factor distribution. (C) 1996 Academ ic Press Limited