PubMed · 7020584
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W Hoppe. 1981. Three-dimensional electron microscopy.. https://doi.org/10.1146/annurev.bb.10.060181.003023
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A high resolution projection at 2.6 A of deoxycholate-treated purple membrane using only images has been obtained with a 200 keV FEG microscope operated at liquid helium temperature. Examination of this high quality map has allowed the following conclusions to be made: Comparison between the internal structure of the trimers of the native and the deoxycholate-treated crystal forms shows that almost every detail of the structure at high resolution is identical. The cell dimension change from 62.4 A to 57.9 A is accompanied by a loss of about half the normal lipids and a 2 degrees anticlockwise rotation of the trimer as a rigid body. Three of the lipids per bacteriorhodopsin molecule remain in identical positions relative to the trimer. In addition, from the projection map together with a packing analysis using the atomic model for bacteriorhodopsin, space for three further lipids has been identified making a total of six lipids per bacteriorhodopsin molecule in this crystal form. Finally, the surprisingly small rotation of the trimer between the two crystal forms with completely different Van der Waals contacts suggest that the crystals are held together by strong, long-range electrostatic interactions.
Bacteriorhodopsin is a membrane protein that functions as a light-driven proton pump. Each cycle of proton transport is initiated by the light-induced isomerization of retinal from the all-trans to 13-cis configuration and is completed by the protein-driven reisomerization of retinal to the all-trans configuration. Previous studies have shown that replacement of Leu-93, a residue in close proximity to the 13-methyl group of retinal, by alanine, resulted in a 250-fold increase in the time required to complete each photocycle. Here, we show that the kinetic defect in the photocycle of the Leu-93-->Ala mutant occurs at a stage after the completion of proton transport and can be overcome in the presence of strong background illumination. Time-resolved retinal-extraction experiments demonstrate the continued presence of a 13-cis intermediate in the photocycle of the Leu-93-->Ala mutant well after the completion of proton release and uptake. These results indicate that retinal reisomerization is kinetically the rate-limiting step in the photocycle of this mutant and that the slow thermal reisomerization can be bypassed by the absorption of a second photon. The effects observed for the Leu-93-->Ala mutant are not observed upon replacement of any other residue in van der Waals contact with retinal or upon replacement of Leu-93 by valine. We conclude that the contact between Leu-93 and the 13-methyl group of retinal plays a key role in controlling the rate of protein conformational changes associated with retinal reisomerization and return of the protein to the initial state.
The crystal structure of halorhodopsin was determined in (centrosymmetric) projection to 6-A resolution by direct methods that use only the amplitudes of the electron diffraction pattern. A multisolution technique was used to generate initial 15-A-resolution basis sets, and after selection of the best phase set (by the closest match of magnitude of Eobs and magnitude of Ecalc), annealing of individual reflections was used to improve its accuracy. The Sayre equation was then used to expand the phase terms to 10 A, followed again by phase annealing. A final expansion with the Sayre equation enlarged this corrected phase set to 6 A. When the condition of density flatness was used to locate the best phase solution after each extension, a final structure could be observed that was quite similar to the one found earlier by analysis of electron micrographs.