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The molecular structure of the photosynthetic reaction centre from Rhodopseudomonas viridis has been elucidated using X-ray crystallographic analysis. The central part of the complex consists of two subunits, L and M, each of which forms five membrane-spanning helices. We present the first description of the high-resolution structure of an integral membrane protein.

The atomic model of the photosynthetic reaction centre from the purple bacterium Rhodopseudomonas viridis has been refined to an R-value of 0.193 at 2.3 A resolution. The refined model contains 10,288 non-hydrogen atoms; 10,045 of these have well defined electron density. A Luzzati-plot indicates an average co-ordinate error of 0.26 A. During refinement, the positions of a partially ordered carotenoid, a unibiquinone in the partially occupied QB site, a detergent molecule, seven putative sulphate ions, and 201 water molecules were found. More than half of these waters are bound at interfaces between protein subunits and therefore contribute significantly to subunit interactions. Water molecules also play important structural and probably functional roles in the environment of some of the cofactors. Two water molecules form hydrogen bonds to the accessory bacteriochlorophylls and to the protein in the vicinity of the special pair of bacteriophylls, the primary electron donor. A group of about 10 water molecules is bound near the binding site of the secondary quinone QB. These waters are likely to participate in the transfer of protons to the doubly reduced QB.

The three-dimensional structure of antibody and its fragments has been the subject of several recent crystallographic studies. The determination of 6 A resolution of the structure of a myeloma protein [l] was followed by high resolution studies of a BenceJones dimer [2] and Fab fragments [3,4]. These latter studies [2-41 have demonstrated the independent folding of domains along the polypeptide chains [S] at least for the Fab fragment. We have recently reported the structure of a crystalline variable domain from a Bence-Jones protein REI [6] for which the complete amino acid sequence is known [ 71. In section 2 of this communication we outline some of the features of this structure. It seems likely that the observed structural homology between variable (V) and constant (C) domains in the Fab fragment [2,3] is extended to the Fc fragment. Studies of such homology can be made using the Rotation Function [B]. The results given in section 3 indicate a molecular symmetry for

The structure of the variable portions of a K-type Bence-Jones protein REI forming a dimer has been determined by X-ray diffraction to a resolution of 2.0 A. The structure has been refined using a constrained crystallographic refinement procedure. The final R value is 0.24 for 15000 significantly measured reflections; the estimated standard deviation of atomic positions is 0.09 A. A more objective assessment of the error in the atomic positions is possible by comparing the two independently refined monomers. The mean deviation of main-chain atoms of the two chains in internal segments in 0.22 A, of main-chain dihedral angles 6.3 degrees for these segments. The unrefined molecular structure of the VREI dimer has been published (Epp, O., Colman, P., Fehlhammer, H., Bode, W., Schiffer, M., Huber, R., and Palm, W. (1974), Eur. J. Biochem. 45, 513). Now a detailed analysis is presented in terms of hydrogen bonds and conformational angles. Secondary structural elements (antiparallel beta structure, reverse turns) are defined. A more precise atomic arrangement of the amino acid residues forming the contact region and the hapten binding site is given as well as the localization of solvent molecules. Two cis-prolines (Pro-8 and Pro-95) were detected. The intrachain disulfide bridge (Cys-23-Cys-88) occurs statistically in two alternative conformations. The structure suggests reasons for strong conservation of several amino acid residues. The knowledge of the refined molecular structure enables crystal structure analyses of related molecules to be made by Patterson search techniques. The calculated phases based on the refined structure are much improved compared to isomorphous phases. Therefore the effects of hapten binding on the molecular structure can be analyzed by the difference Fourier technique with more reliability. Hapten binding studies have been started.

X-ray analysis of three-dimensional crystals of the photosynthetic reaction center from the purple bacterium Rhodopseudomonas viridis led to an electron density distribution at 3 A resolution calculated with phases from multiple isomorphous replacement. The protein subunits of the complex were identified. An atomic model of the prosthetic groups of the reaction center complex (4 bacteriochlorophyll b, 2 bacteriopheophytin b, 1 non-heme iron, 1 menaquinone, 4 heme groups) was built. The arrangement of the ring systems of the bacteriochlorophyll b and bacteriopheophytin b molecules shows a local 2-fold rotation symmetry; two bacteriochlorophyll b form a closely associated, non-covalently linked dimer (“special pair”). A different local 2-fold symmetry axis is observed for the heme groups of the cytochrome part.

The three-dimensional structure of bovine erythrocyte glutathione peroxidase, a tetrameric enzyme containing 4 gram atoms of selenium per mole ( M r = 84,000), has been determined at 2.8 A resolution using the multiple isomorphous replacement method. By correlation calculations in Patterson space the tetramers were shown to exhibit molecular [222] symmetry, proving the monomers to be identical or at least very similar. The monomer consists of a single polypeptide chain of 178 amino acid residues. Its shape is nearly spherical with a radius of r ≈ 19 A . A tentative sequence corresponding to a partially refined model ( R = 0.38) is given. Each subunit is built up from a central core of two parallel and two anti-parallel strands of pleated sheet surrounded by four α-helices. One of the helices runs antiparallel to the neighbouring β-strands giving rise to a βαβ substructure, an architecture that has been found in several other proteins e.g. flavodoxin, thioredoxin, rhodanese and dehydrogenases. A comparison of the glutathione peroxidase subunit structure with thioredoxin-S 2 revealed large regions of structural resemblance. The central four-stranded β structure together with two parallel α-helices resembles nearly 80% of the thioredoxin fold. The active sites of glutathione peroxidase are located in flat depressions on the molecular surface. Probably each active centre is built up by segments from two subunits. The catalytically active selenocysteines were found at the N-terminal ends of long α-helices and are surrounded by an accumulation of aromatic side-chains. A difference Fourier map between oxidized and substrate-reduced glutathione peroxidase as well as heavy-atom binding led to the conclusion that the two-electron redox-cycle involves a reversible transition of the active-site selenium from a selenenic acid (RSeOH) to a seleninic acid (RSeOOH).

OMSVP3 and OMTKY3 (third domains of silver pheasant and turkey ovomucoid inhibitor) are Kazal-type serine proteinase inhibitors. They have been isomorphously crystallized in the monoclinic space group C2 with cell dimensions of a = 4.429 nm, b = 2.115 nm, c = 4.405 nm, beta = 107 degrees. The asymmetric unit contains one molecule corresponding to an extremely low volume per unit molecular mass of 0.0017 nm3/Da. Data collection was only possible for the OMSVP3 crystals. Orientation and position of the OMSVP3 molecules in the monoclinic unit cells were determined using Patterson search methods and the known structure of the third domain of Japanese quail ovomucoid (OMJPQ3) [Papamokos, E., Weber, E., Bode, W., Huber, R., Empie, M. W., Kato, I. and Laskowski, M., Jr (1982) J. Mol. Biol. 158, 515-537]. The OMSVP3 structure has been refined by restrained crystallographic refinement yielding a final R value of 0.199 for data to 0.15 nm resolution. Conformation and hydrogen-bonding pattern of OMSVP3 and OMJPQ3 are very similar. Large deviations occur at the NH2 terminus owing to different crystal packing, and at the C terminus of the central helix, representing an intrinsic property and resulting from amino acid substitutions far away from this site. The deviation of OMSVP3 from OMTKY3 complexed with the Streptomyces griseus protease B is very small [Fujinaga, M., Read, R. J., Sielecki, A., Ardelt, W., Laskowski, M., Jr and James, M. N. G. (1982) Proc. Natl Acad. Sci. USA, 79, 4868-4872].

Patterson search calculations using the three-dimensional structure of the α-amylase inhibitor from Streptomyces tendae obtained from experimental nuclear magnetic resonance (n.m.r.) data were performed to study the possibility of solving the phase problem in the X-ray diffraction method with protein structures determined by n.m.r. Using all heavy atoms (C, N, O, S) of the residues 5 to 73 in the best n.m.r. structure of the α-amylase inhibitor (520 out of the 558 heavy atoms in the complete polypeptide chain), the maximum of the rotation function corresponded to the correct solution obtained by the previous independent determination of the crystal structure. However, additional local maxima, which are not significantly lower than the global maximum, also showed up. Performing the Patterson search with a model containing the backbone atoms and the heavy atoms of only the interior side-chains (399 atoms), which are much better defined by the n.m.r. data, the correct maximum was significantly higher than all other maxima. A translation search for the best orientation of the latter model yielded the correct solution. The energy-restrained crystallographic refinement was performed with this model to an R-factor of 26%. This corresponds approximately to the R-factor calculated for the X-ray crystal structure previously determined using the isomorphous replacement technique, if the residues 1 to 4 and 74 and all localized solvent molecules were removed from this structure. During the refinement the root-mean-square deviation between the two structures decreased from 1·03 A to 0·26 A for the polypeptide backbone and from 1·64 A to 0·73 A for all heavy atoms. There are no major local conformational differences between the two structures, with the single exception of the side-chain of Gln52.

The structure of a χ-type Bence-Jones protein variable fragment Au has been determined by molecular replacement methods using the known structure of an other Bence-Jones variable fragment Rei (Epp et al., Eur. J. Biochem. 45, 513 (1974)). The crystallographic R factor is 0.31 for about 4000 significantly measured reflections between 6.8 to 2.5 a. The Au protein forms a dimer across a crystallographic two fold axis. The spatial relationship of the two monomers, the conformation of the backbones and of the internal residues is extremely similar to that found in Rei.

The atomic structure of the monomeric insect haemoglobin is closely similar to the structure of whale myoglobin, although only about 20% of the amino acids are identical in both protein molecules. A quantitative comparison of both structures, however, showed that in the vicinity of some amino acid substitutions considerable changes occur. The comparison also revealed unexpected long range structural changes induced by amino acid deletions. In particular a deletion of four amino acids in the A B corner shifts the whole helix B considerably. The general distribution of amino acids in erythrocruorin is analogous to that which has been found in other haemoglobin molecules. The large proportion of phenylalanines in the hydrophobic core of the molecule is remarkable. Also the haem pocket is lined with seven phenylalanine side chains. Four of these are unknown in vertebrate haemoglobins. The distal histidine E 7, which is invariant in all known vertebrate haemoglobins, is replaced by a glutamic acid, which has no haem contact, but protrudes into the surrounding solution. This proves that a histidine or another polar residue is not essential for the function of a haemoglobin molecule as a reversible oxygen carrier.

The insect haemoglobin erythrocruorin undergoes a reversible structural change between pH 7 and pH 4 in the crystalline state. The groups responsible for this structural change were identified.

The crystal structure analysis of a human Fc fragment was pursued to 3.5 A resolution and a complete model was built and refined into the isomorphous Fourier map. The CH2 and CH3 domains show the immunoglobulin fold, with CH3 being closely similar to CH1, but CH2 intermediate in structure between V and CH3. The carbohydrate is rigidly attached to CH2, covering the C face. CH3 dimerizes as CH1-CL, but CH2 has no contact to the second chain. Residues involved in the lateral CH3-CH3 and the longitudinal CH3-CH2 contact are conserved in Ig classes and sub-classes. In IgM and IgE the two C-terminal domains also show this characteristic distribution of contact residues.

The crystal structure of bovine erythrocyte glutathione peroxidase has been refined by a combined procedure of restrained crystallographic refinement and energy minimization at 0.20 nm resolution. The final R value at this resolution is 0.178. The r.m.s. deviation of main-chain atoms of the two independently refined monomers is 0.019 nm. The structure at 0.28 nm resolution, which has been determined by multiple isomorphous replacement, served as a starting model. The refined model allowed a detailed survey of the hydrogen-bonding pattern and of the subunit contact areas in the molecule. The model contains 165 solvent molecules per dimer, all taken as water molecules. The mobility of the structure was derived from the individual atomic temperature factors. The complete tetramer, including the active sites, seems to be rather rigid, except for narrow loops near to the N-terminal ends and some β turns exposed to solvent. The active centres of glutathione peroxidase are found in flat depressions on the molecular surface. The catalytically active selenocysteine residues could be located at the N-terminal ends of α helices forming βαβ substructures together with two adjacent parallel β strands. In the vicinity of the reactive group some aromatic amino acid side-chains could be localized. Especially Trp-148, which could be hydrogen bonded to SeCys-35, may play a functional role during catalysis. The results of substrate and inhibitor binding studies in solution and in the crystalline state could be interpreted by an apparent half-site reactivity of glutathione peroxidase. The enzyme seems to react in the sense of negative cooperativity with dimers being the functional units. Based on difference Fourier analyses of appropriate derivatives a reasonable model of glutathione binding is presented. Among the residues which could be of functional importance are Arg-40, Gln-130 and Arg-167, presumably forming salt bridges and a hydrogen bond to the glutathione molecule. In conclusion, a general picture of a minimal reaction mechanism, which is in good agreement with functional and structural data, is proposed. The main reaction of the catalytic cycle presumably shuttles between the selenolate and the selenenic acid state of SeCys-35.

An X-ray structure analysis of the photosynthetic reaction centre from the purple bacterium Rhodopseudomonas viridis provides structural details of the pigment-binding sites. The photosynthetic pigments are found in rather hydrophobic environments provided by the subunits L and M. In addition to apolar interactions, the bacteriochlorophylls of the primary electron donor (`special pair') and the bacteriopheophytins, but not the accessory bacteriochlorophylls, form hydrogen bonds with amino acid side chains of these protein subunits. The two branches of pigments which originate at the primary electron donor, and which mark possible electron pathways across the photosynthetic membrane, are in different environments and show different hydrogen bonding with the protein: this may help to understand why only one branch of pigments is active in the light-driven electron transfer. The primary electron acceptor, a menaquinone (QA), is in a pocket formed by the M subunit and interacts with it by hydrophobic contacts and hydrogen bonds. Competitive inhibitors of the secondary quinone QB (o-phenanthroline, the herbicide terbutryn) are bound into a pocket provided by the L subunit. Apart from numerous van der Waals interactions they also form hydrogen bonds to the protein.

The tetrameric selenoenzyme GSH peroxidase (M=84000) catalyzes the reduction of a variety of hydroperoxides including H2O2 and serves to protect biomembranes and other essential cellular components against oxidative challenge [1,2,3]. GSH serves as specific electron donor substrate and alimentary selenium provides the prosthetic group, a selenocysteine residue.

The environment of the haem group of the insect haemoglobin erythrocruorin is described as found from a 2.8 A resolution Fourier synthesis. It proved basically similar to the haem group environment of the mammalian haemoglobins. Some interesting amino acid replacements could be detected, the most important being the substitution of the invariant amino acid Leu F4 for an aromatic amino acid.

Patterson search calculations demonstrated the presence of a local pseudo 2-fold axis in the crystalline state of the monomeric Bence-Jones protein Rei. These local diads are also observed in a preliminary Fourier synthesis at 4 A resolution. They appear to relate the variable and constant halves of neighbouring molecules. The presence of local diads is evidence that the tertiary structures of the constant and variable halves of the Bence-Jones protein are similar.

The analysis of a monoclinic crystal modification of l-asparaginase from Escherichia coli supports the chemical and physio-chemical evidence for a tetrameric subunit structure. The pseudo-symmetries occurring in the crystal structure provide evidence that the molecules lie on positions with approximate point symmetry 222.

The structures of the deoxy and carbonmonoxy derivatives of the insect haemoglobin erythrocruorin have been determined by difference Fourier syntheses. In deoxyerythrocruorin the iron atom of the haem group was found to be five-coordinated. The carbonmonoxy- versus deoxy- difference map showed that the carbon monoxide molecule is inclined to the haem plane. Slight structural changes could be observed to take place on carbon monoxide binding. These structural changes are probably due to the difference in spin state between deoxy- and carbonmonoxy haemoglobin.