J.M. Baldwin

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.

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 (

Haemoglobin: the structural changes related to ligand binding and its allosteric mechanism.

Abstract The structural changes that occur on ligand binding to haemoglobin have been studied by comparison of the atomic co-ordinates of human deoxy, horse met and human carbonmonoxy haemoglobin, using computer graphics and least-squares fitting methods. The changes that occur on going from deoxy to either of the liganded forms are very similar. These include tertiary structure changes within the α1β1 dimer and a quaternary structure change in which the packing of α1β1 against α2β2 alters. On going from deoxy to liganded haemoglobin, no significant structural change occurs in the central regions of the α1β1 dimer, including the α1β1 interface and nearby helices B, C, G and H in both subunits. Movements occur in the outer parts of the dimer, where the haems, F helices and FG corners of both subunits move towards the centre of the molecule. The two haems and the two FG corners come ~2 A closer together. One important effect of the changes in both subunits is to translate the F helix across the face of the haem by ~1 A. This moves the haem-linked histidine F8 from a position that is asymmetric with respect to the porphyrin nitrogens in deoxy to a more symmetric position in liganded haemoglobin. The motion of the β haem removes the ligand-binding site from the vicinity of ValβE11, which hinders ligand binding in deoxy. The changes in tertiary structure are linked to the quaternary change through the motion of the FG corners. The C helices and FG corners of α1β1 are in contact with the FG corners and C helices of α2β2 in both quaternary structures. In the quaternary change the contacts between α1FG and β2C and between α2FG and β1C act as “flexible joints” allowing small relative motions. The same side-chains are involved in the contacts in both structures. The contacts between α1C and β2FG and between α2C and β1FG act as “switch” regions, having two different stable positions with different side-chains in contact. The change between the two positions involves a relative movement of ~6 A. The quaternary structure change to liganded haemoglobin destroys the contacts made by the C-terminal residues of each subunit in deoxy haemoglobin, and these residues rotate freely in the liganded form. These structural results, together with other work, particularly the calculations of Gelin & Karplus and of Warshel, support a description of the haemoglobin mechanism in which (1) the binding of ligand to the deoxy form is accompanied by steric strain, originating from the particular position of the F helix and of His F8 relative to the haem. (2) The strain leads to decreased stability of the deoxy quaternary structure relative to the liganded quaternary structure, so that the proportion of molecules in the high-affinity form increases as successive ligands bind. (3) The quaternary structure change to the high-affinity form induces tertiary structure changes that reposition the F helix and HisF8 relative to the haem and there is then no strain on ligand binding. In the absence of ligand the deoxy structure is favoured by the greater surface area buried between α1β1 and α2β2 in this quaternary structure. Further implications of the structural results are discussed.

Arrangement of rhodopsin transmembrane α-helices

Rhodopsins, the photoreceptors in rod cells, are G-protein-coupled receptors with seven hydrophobic segments containing characteristic conserved sequence patterns that define a large family,. Members of the family are expected to share a conserved transmembrane structure. Direct evidence for the arrangement of seven α-helices was obtained from a 9å projection map of bovine rhodopsin. Structural constraints inferred from a comparison of G-protein-coupled receptor sequences were used to assign the seven hydrophobic stretches in the sequence to features in the projection map. A low-resolution three-dimensional structure of bovine rhodopsin and two projection structures of frog rhodopsin confirmed the position of the three least tilted helices, 4, 6 and 7. A more elongated peak of density for helix 5 indicated that it is tilted or bent,, but helices 1, 2 and 3 were not resolved. Here we have used electron micrographs of frozen-hydrated two-dimensional frog rhodopsin crystals to determine the structure of frog rhodopsin. Seven rods of density in the map are used to estimate tilt angles for the seven helices. Density visible on the extracellular side of the membrane suggests a folded domain. Density extends from helix 6 on the intracellular side, and a short connection between helices 1 and 2, and possibly a part of the carboxy terminus, are visible.

Structure and function of receptors coupled to G proteins

Direct structural data on receptors coupled to G proteins were obtained last year in the form of a low resolution projection map of rhodopsin. A large number of receptor sequences have now been determined and detailed analysis of these has provided structural information about the receptors. New results from site-directed mutagenesis experiments can be examined in conjunction with the structural information from sequence analysis and the rhodopsin map. The identification of constitutively active mutated receptors has influenced our understanding of normal receptor equilibria.

The structure of human carbonmonoxy haemoglobin at 2.7 A resolution.

Abstract The structure of human carbonmonoxy haemoglobin has been determined to 2.7 A resolution using X-ray data for the native protein only. The atomic co-ordinates were refined from those of an initial model based on the co-ordinates of the closely related protein horse methaemoglobin whose structure is known at high resolution (Ladner et al., 1977). The space group of the new unit cell (P41212) is such that the location of the haemoglobin molecules is specified by two parameters only, and the values of these were found through a search for the best initial R-factor. The refined structure of human carbonmonoxy haemoglobin is, as expected, generally similar to that of horse methaemoglobin. The root-meansquared shift for all atoms between the initial model and the final co-ordinates was 1.35A. The new structure confirms that the CO ligand lies off the normal to the haem plane in both α and β subunits, as indicated previously by a difference Fourier map of CO versus horse methaemoglobin (Heidner et al., 1976). The Fe-C-O group, assumed to be linear, makes an angle of about 13 ° with the haem normal and it points towards the inside of the haem pocket. In the α subunit the iron atom lies in the mean plane of the haem within experimental error. In the β subunit the situation is less clear in that unconstrained refinement put the iron atom 0.22 A from the haem plane, a distance that is 3 2 times the expected error. The new structure also confirms that in carbonmonoxy haemoglobin the side chain of cysteine β93(F9) points away from the surface of the molecule into the pocket between helices F, G and H that, in deoxyhaemoglobin, is occupied by the side chain of tyrosine β145(HC2). A detailed comparison of the structures of the deoxy and liganded forms from the same species is now possible and the conclusions drawn from this comparison are given in a separate publication (Baldwin & Chothia, 1979).

Three-dimensional structure of the bacterial multidrug transporter EmrE shows it is an asymmetric homodimer.

The small multidrug resistance family of transporters is widespread in bacteria and is responsible for bacterial resistance to toxic aromatic cations by proton‐linked efflux. We have determined the three‐dimensional (3D) structure of the Escherichia coli multidrug transporter EmrE by electron cryomicroscopy of 2D crystals, including data to 7.0 Å resolution. The structure of EmrE consists of a bundle of eight transmembrane α‐helices with one substrate molecule bound near the centre. The substrate binding chamber is formed from six helices and is accessible both from the aqueous phase and laterally from the lipid bilayer. The most remarkable feature of the structure of EmrE is that it is an asymmetric homodimer. The possible arrangement of the two polypeptides in the EmrE dimer is discussed based on the 3D density map.

Images of purple membrane at 2.8 Å resolution obtained by cryo-electron microscopy

Improvements in technique have produced electron micrographs of purple membrane that provide, after computer analysis, reproducibly measurable diffraction peaks extending to 2.8 A (1 A = 0.1 nm). The improvements include better specimen preparation, a more stable cryo-electron microscope with better alignment and the addition of an image-processing step, which gives weights to local areas of the image according to the local strength of the periodic component of the image. These improvements have enabled the calculation of a directly phased projection map at 2.8 A resolution.

Measurement and evaluation of electron diffraction patterns from two-dimensional crystals

Abstract Electron diffraction patterns from two different crystal forms of purple membrane have been obtained. In both cases, the diffraction spots extend to beyond 3.0 A resolution. Using specimens tilted at angles up to 60°, the three-dimensional intensities were recorded as a large number of two-dimensional patterns. We describe here the procedures used to record and digitise the patterns, and subsequently to measure and combine the individual spot intensities to produce a complete merged, refined and evaluated three-dimensional diffraction pattern. Approximately 100 patterns were required for adequate sampling in each crystal form.

Conformational changes in the multidrug transporter EmrE associated with substrate binding

EmrE is a bacterial multidrug transporter of the small multidrug resistance family, which extrudes large hydrophobic cations such as tetraphenylphosphonium (TPP(+)) out of the cell by a proton antiport mechanism. Binding measurements were performed on purified EmrE solubilized in dodecylmaltoside to determine the stoichiometry of TPP(+) binding; the data showed that one TPP(+) molecule bound per EmrE dimer. Reconstitution of purified EmrE at low lipid:protein ratios in either the presence or the absence of TPP(+) produced well ordered two-dimensional crystals. Electron cryo-microscopy was used to collect images of frozen hydrated EmrE crystals and projection maps were determined by image processing to 7A resolution. An average native EmrE projection structure was calculated from the c222 and p222(1) crystals, which was subsequently subtracted from the average of two independent p2 projection maps of EmrE with TPP(+) bound. The interpretation of the difference density image most consistent with biochemical data suggested that TPP(+) bound at the monomer-monomer interface in the centre of the EmrE dimer, and resulted in the movement of at least one transmembrane alpha-helix.