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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.

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).

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 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.

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.