Richard Henderson

Model for the structure of bacteriorhodopsin based on high-resolution electron cryo-microscopy

The light-driven proton pump bacteriorhodopsin occurs naturally as two-dimensional crystals. A three-dimensional density map of the structure, at near-atomic resolution, has been obtained by studying the crystals using electron cryo-microscopy to obtain electron diffraction patterns and high-resolution micrographs. New methods were developed for analysing micrographs from tilted specimens, incorporating methods previously developed for untilted specimens that enable large areas to be analysed and corrected for distortions. Data from 72 images, from both tilted and untilted specimens, were analysed to produce the phases of 2700 independent Fourier components of the structure. The amplitudes of these components were accurately measured from 150 diffraction patterns. Together, these data represent about half of the full three-dimensional transform to 3.5 A. The map of the structure has a resolution of 3.5 A in a direction parallel to the membrane plane but lower than this in the perpendicular direction. It shows many features in the density that are resolved from the main density of the seven alpha-helices. We interpret these features as the bulky aromatic side-chains of phenylalanine, tyrosine and tryptophan residues. There is also a very dense feature, which is the beta-ionone ring of the retinal chromophore. Using these bulky side-chains as guide points and taking account of bulges in the helices that indicate smaller side-chains such as leucine, a complete atomic model for bacteriorhodopsin between amino acid residues 8 and 225 has been built. There are 21 amino acid residues, contributed by all seven helices, surrounding the retinal and 26 residues, contributed by five helices, forming the proton pathway or channel. Ten of the amino acid residues in the middle of the proton channel are also part of the retinal binding site. The model also provides a useful basis for consideration of the mechanism of proton pumping and allows a consistent interpretation of a great deal of other experimental data. In particular, the structure suggests that pK changes in the Schiff base must act as the means by which light energy is converted into proton pumping pressure in the channel. Asp96 is on the pathway from the cytoplasm to the Schiff base and Asp85 is on the pathway from the Schiff base to the extracellular surface.

Three-dimensional model of purple membrane obtained by electron microscopy

A 7-Å resolution map of the purple membrane has been obtained by electron microscopy of tilted, unstained specimens. The protein in the membrane contains seven, closely packed, α-helical segments which extend roughly perpendicular to the plane of the membrane for most of its width. Lipid bilayer regions fill the spaces between the protein molecules.

Structure of a Beta1-Adrenergic G-Protein-Coupled Receptor.

G-protein-coupled receptors have a major role in transmembrane signalling in most eukaryotes and many are important drug targets. Here we report the 2.7 Å resolution crystal structure of a β1-adrenergic receptor in complex with the high-affinity antagonist cyanopindolol. The modified turkey (Meleagris gallopavo) receptor was selected to be in its antagonist conformation and its thermostability improved by earlier limited mutagenesis. The ligand-binding pocket comprises 15 side chains from amino acid residues in 4 transmembrane α-helices and extracellular loop 2. This loop defines the entrance of the ligand-binding pocket and is stabilized by two disulphide bonds and a sodium ion. Binding of cyanopindolol to the β1-adrenergic receptor and binding of carazolol to the β2-adrenergic receptor involve similar interactions. A short well-defined helix in cytoplasmic loop 2, not observed in either rhodopsin or the β2-adrenergic receptor, directly interacts by means of a tyrosine with the highly conserved DRY motif at the end of helix 3 that is essential for receptor activation.

Molecular structure determination by electron microscopy of unstained crystalline specimens.

Abstract The projected structures of two unstained periodic biological specimens, the purple membrane and catalase, have been determined by electron microscopy to resolutions of 7 A and 9 A, respectively. Glucose was used to facilitate their in vacuo preservation and extremely low electron doses were applied to avoid their destruction. The information on which the projections are based was extracted from defocussed bright-field micrographs and electron diffraction patterns. Fourier analysis of the micrograph data provided the phases of the Fourier components of the structures; measurement of the electron diffraction patterns provided the amplitudes. Large regions of the micrographs (3000 to 10,000 unit cells) were required for each analysis because of the inherently low image contrast ( Our methods appear to be limited in resolution only by the performance of the microscope at the unusually low magnifications which were necessary. Resolutions close to 3 A should ultimately be possible.

The potential and limitations of neutrons, electrons and X-rays for atomic resolution microscopy of unstained biological molecules

Radiation damage is the main problem which prevents the determination of the structure of a single biological macromolecule at atomic resolution using any kind of microscopy. This is true whether neutrons, electrons or X-rays are used as the illumination. For neutrons, the cross-section for nuclear capture and the associated energy deposition and radiation damage could be reduced by using samples that are fully deuterated and 15N-labelled and by using fast neutrons, but single molecule biological microscopy is still not feasible. For naturally occurring biological material, electrons at present provide the most information for a given amount of radiation damage. Using phase contrast electron microscopy on biological molecules and macromolecular assemblies of approximately 10(5) molecular weight and above, there is in theory enough information present in the image to allow determination of the position and orientation of individual particles: the application of averaging methods can then be used to provide an atomic resolution structure. The images of approximately 10,000 particles are required. Below 10(5) molecular weight, some kind of crystal or other geometrically ordered aggregate is necessary to provide a sufficiently high combined molecular weight to allow for the alignment. In practice, the present quality of the best images still falls short of that attainable in theory and this means that a greater number of particles must be averaged and that the molecular weight limitation is somewhat larger than the predicted limit. For X-rays, the amount of damage per useful elastic scattering event is several hundred times greater than for electrons at all wavelengths and energies and therefore the requirements on specimen size and number of particles are correspondingly larger. Because of the lack of sufficiently bright neutron sources in the foreseeable future, electron microscopy in practice provides the greatest potential for immediate progress.

Structure of purple membrane from halobacterium halobium: recording, measurement and evaluation of electron micrographs at 3.5 Å resolution

Abstract Electron micrographs of the purple membrane have been recorded using liquid nitrogen and liquid helium cooling on three cryoelectron microscopes. The best micrographs show optical diffraction spots, arising from the two-dimensional crystal, out to resolutions of around 6 A. Large areas of several of these micrographs have been analysed using a procedure which determines the strength of the very weak high resolution Fourier components of the image of the crystal. The procedure consists of reciprocal space filtering followed by real space correlation analysis to characterise image distortions, removal of the distortions by interpolation, and finally extraction of the amplitudes and phases of the Fourier components from the distortion-corrected image of the crystal. These raw image amplitudes and phases are then used, together with previously measured amplitude and phase information, to refine the beam tilt and crystal tilt, phase origin and amount of defocus and astigmatism of the image. The phases can then be corrected for the effects of the contrast transfer function, beam tilt and phase origin. The amplitudes of all the spots which are expected to be strong from their known electron diffraction intensity are observed to be significantly above the background noise level, and the independent phases from different images, and from symmetry-related directions in the same image, show excellent agreement out to a resolution of 3.5 A. Although only images from untilted or slightly tilted (

Electron diffraction analysis of structural changes in the photocycle of bacteriorhodopsin.

Structural changes are central to the mechanism of light‐driven proton transport by bacteriorhodopsin, a seven‐helix membrane protein. The main intermediate formed upon light absorption is M, which occurs between the proton release and uptake steps of the photocycle. To investigate the structure of the M intermediate, we have carried out electron diffraction studies with two‐dimensional crystals of wild‐type bacteriorhodopsin and the Asp96‐‐>Gly mutant. The M intermediate was trapped by rapidly freezing the crystals in liquid ethane following illumination with a xenon flash lamp at 5 and 25 degrees C. Here, we present 3.5 A resolution Fourier projection maps of the differences between the M intermediate and the ground state of bacteriorhodopsin. The most prominent structural changes are observed in the vicinity of helices F and G and are localized to the cytoplasmic half of the membrane.

Structure of crystalline α-chymotrypsin: II. A preliminary report including a hypothesis for the activation mechanism

An electron density map of tosyl-α-chymotrypsin at 2 A resolution is presented, which shows the conformation of the polypeptide chain. An electron density map of the differences between the tosylated and the native enzyme has also been calculated. These maps lead to the following conclusions. Histidine 57 and serine 195 are known to be part of the active site. In the native enzyme, these are in an environment open to the solvent and in a conformation consistent with the existence of a hydrogen bond between them. In the tosylated enzyme, small movements of histidine 57, serine 195 and methionine 192 bring histidine 57 into a position where it can interact with the sulphonyl group. No other conformational changes are observed. The enzyme contains an ion pair between the α-amino group of isoleucine 16 and the β-carboxyl group of aspartate 194, located in an otherwise non-polar cavity. In conjunction with kinetic and spectroscopic studies (especially Oppenheimer, Labouesse & Hess, 1966) and X-ray analysis of chymotrypsinogen and δ-chymotrypsin at low resolution (Kraut, Sieker, High & Freer, 1962; Kraut, Wright, Kellerman & Freer, 1967), our results lead to a hypothesis for the stereochemistry of the activation process. It is proposed that (i) activation of the zymogen involves no gross reorganization of the main chain nor a significant helix-coil transition; (ii) activation involves a structural change of the enzyme, caused by the formation to an ion-pair between isoleucine 16 and aspartate 194. This structural change would be reversed when the positively charged α-amino group of isoleucine 16 is deprotonated at high pH. The reversal seems to be sterically blocked when the enzyme is inhibited by a bulky group on serine 195. In the light of our model, the location of the disulphide bridges in the chemical sequence of trypsin suggests that trypsin and chymotrypsin have nearly identical tertiary structures, and that their disulphide bridges serve to stabilize rather than to determine the structure. The interactions between the molecules related by the non-crystallographic dyad axes in the crystal are described. One of these involves the active site, but does not inhibit enzymic activity. Of the heavy atoms used for phase determination by the method of isomorphous replacement, platinum(II)- and mercury-containing substituents are bound to sites containing cystine and methionine residues.

Molecular mechanism of vectorial proton translocation by bacteriorhodopsin.

Bacteriorhodopsin, a membrane protein with a relative molecular mass of 27,000, is a light driven pump which transports protons across the cell membrane of the halophilic organism Halobacterium salinarum. The chromophore retinal is covalently attached to the protein via a protonated Schiff base. Upon illumination, retinal is isomerized. The Schiff base then releases a proton to the extracellular medium, and is subsequently reprotonated from the cytoplasm. An atomic model for bacteriorhodopsin was first determined by Henderson et al, and has been confirmed and extended by work in a number of laboratories in the last few years. Here we present an atomic model for structural changes involved in the vectorial, light-driven transport of protons by bacteriorhodopsin. A ‘switch’ mechanism ensures the vectorial nature of pumping. First, retinal unbends, triggered by loss of the Schiff base proton, and second, a protein conformational change occurs. This conformational change, which we have determined by electron crystallography at atomic (3.2 Å in-plane and 3.6 Å vertical) resolution, is largely localized to helices F and G, and provides an ‘opening’ of the protein to protons on the cytoplasmic side of the membrane.

The structure of the purple membrane from Halobacterium halobium: Analysis of the X-ray diffraction pattern

Abstract An X-ray diffraction analysis of oriented specimens of the purple membrane from Halobacterium halobium shows that the protein and lipid components are packed in a P 3 hexagonal lattice, with one protein molecule per asymmetric unit. The structure is made up of a single layer of the protein molecules, oriented vectorially in the same direction across the membrane. The presence of strong diffraction peaks equatorially centred at 10 A, and axially at 5 A and 1.5 A, show that the protein molecules, which make up most of the mass of the membrane, are composed to a considerable extent of α-helices, 25 to 35 A long, arranged roughly perpendicular to the plane of the membrane to form superhelical groupings of the “coiled-coil” type. The surface of the membrane is flat, with no bumps or dimples large enough to affect the X-ray pattern when the electron density of the suspending medium is altered. The phospholipids may be less exactly positioned in the lattice than the protein, since the presence of uranyl acetate, which is expected to co-ordinate with the acidic phosphate groups, produces intensity changes only at low resolution.