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Two crystal forms (P6(3) and R3) of human annexin V have been crystallographically refined at 2.3 A and 2.0 A resolution to R-values of 0.184 and 0.174, respectively, applying very tight stereochemical restraints with deviations from ideal geometry of 0.01 A and 2 degrees. The three independent molecules (2 in P6(3), 1 in R3) are similar, with deviations in C alpha positions of 0.6 A. The polypeptide chain of 320 amino acid residues is folded into a planar cyclic arrangement of four repeats. The repeats have similar structures of five alpha-helical segments wound into a right-handed compact superhelix. Three calcium ion sites in repeats I, II and IV and two lanthanum ion sites in repeat I have been found in the R3 crystals. They are located at the convex face of the molecule opposite the N terminus. Repeat III has a different conformation at this site and no calcium bound. The calcium sites are similar to the phospholipase A2 calcium-binding site, suggesting analogy also in phospholipid interaction. The center of the molecule is formed by a channel of polar charged residues, which also harbors a chain of ordered water molecules conserved in the different crystal forms. Comparison with amino acid sequences of other annexins shows a high degree of similarity between them. Long insertions are found only at the N termini. Most conserved are the residues forming the metal-binding sites and the polar channel. Annexins V and VII form voltage-gated calcium ion channels when bound to membranes in vitro. We suggest that annexins bind with their convex face to membranes, causing local disorder and permeability of the phospholipid bilayers. Annexins are Janus-faced proteins that face phospholipid and water and mediate calcium transport.

Crystals of the non-collagenous C-terminal region (NC1) of type IV collagen have been obtained from human placenta. These crystals diffract to 2.0 A, and belong to space group P22(1)2(1), with cell dimensions a = 81 A, b = 158 A, c = 138 A, alpha = beta = gamma = 90 degrees. The crystals contain one hexamer in the asymmetric unit; they are very stable with respect to X-rays.

The crystal structure of the light-harvesting protein-pigment complex C-phycocyanin from the cyanobacterium Agmenellum quadruplicatum has been determined by Patterson search techniques on the basis of the molecular model of C-phycocyanin from Mastigocladus laminosus. The crystal unit cell (space group P321) contains three (αβ)6 hexamers centred on the crystallographic triads. The hexamer at the origin of the unit cell exhibits crystallographic 32 point symmetry. The other two hexamers (independent of the former) show crystallographic 3-fold and local 2-fold symmetry. The 3-fold redundancy of the asymmetric unit of the crystal cell was used in the refinement process, which proceeded by cyclic averaging, model building and energy-restrained crystallographic refinement. Refinement was terminated with a conventional crystallographic R-value of 0·20 with data to 2·5 A resolution. The two independent hexamers of the unit cell are identical within the limits of error at all levels of aggregation. Two trimers, which closely resemble the M. laminosus C-phycocyanin, are aggregated head-to-head to form the hexamer. Both trimers fit complementarily and are held together by polar and ionic interactions. Conservation of the amino acid residues involved in protein-chromophore and intermonomer interactions suggests common structural features for all biliproteins. Most probably, the hexameric aggregation form present in the crystals is closely related to the discs of native phycobilisome rods. All tetrapyrrole chromophores are extended but with different geometries enforced by different protein surroundings. In particular, interactions of the propionic side-chains with arginine residues and of the pyrrole nitrogen atoms with aspartate residues define configuration and conformation of the chromophores. Relative chromophore distances and orientations have been determined and a preferential pathway for the energy transfer suggested. Accordingly, within a hexamer the absorbed energy is funnelled to chromophore B84 and then transduced via B84 chromophores along the phycobilisome rods.

Heavy riboflavin synthase of Bacillus subtilis is a bifunctional enzyme complex that catalyzes the formation of riboflavin from 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione and 3,4-dihydroxy-2-butanone 4-phosphate via 6,7-dimethyl-8-ribityllumazine. The 1,000,000 M r complex consists of 60 β-subunits ( M r = 16,200), which form a T = 1 icosahedral capsid enclosing three α-subunits ( M r = 23,500) in the central core. Crystallographic intensity data of native crystals and three heavy-atom derivatives were obtained using synchrotron radiation. Given the local particle symmetries, the heavy-atom positions were determined by vector search procedures applied to the corresponding 4 A (1 A = 0·1 nm) difference Patterson maps. The atomic structure of the β-subunit could be deduced from electron density maps, which were obtained by icosahedral symmetry averaging in real space and phase extension to 3·3 A resolution. The R -factor of the β-subunit model is 39·9%. It should be noted that the present electron density map contains no information on the α-subunit structure. The main chain of a β-subunit folds into a four-stranded parallel β-sheet structure flanked on both sides by two α-helical segments. Thus the observed pattern resembles to some extent the flavodoxin folding, although no sequence homology could be detected. The β-subunit fold shows no similarity with the subunit structures of icosahedral plant or animal viruses that have been reported. The intersubunit contacts and structural features of β-subunit dimers, trimers and pentamers occurring in the icosahedral β 60 capsid are analyzed and discussed with emphasis on the stability of these substructures. In the β-subunit pentamer, the N-terminal segment of each monomer is attached to the neighboring monomer and comprises the fifth strand of a parallel sheet. Along the 5-fold axis of each pentamer, a channel is formed by α-helix α 3 and its four symmetry-related neighbors. These helices are twisted into a superhelical motif, forming a pore structure with a diameter of roughly 9 A and an approximate length of 30 A. These pores may play a role in substrate and product exchange between the active centers in the particle core and the solvent space. The β 60 capsid is characterized by inner and outer radii of 39 A and 78·5 A, and an asymmetric distribution of charges on the inner and outer capsid wall. The binding of the substrate-analogous ligand 5-nitroso-6-ribityl-amino-2,4-(1H,3H)-pyrimidinedione was analyzed by difference Fourier methods and model building. Geometric features as well as possible functional properties of the substrate binding sites at the pentamer interfaces are described. Ligand binding at the pentamer interface regions increases the stability of the β 60 capsid considerably and influences the reassembly of isolated β-subunits.

The bilin binding protein (BBP) from the insect Pieris brassicae has been analysed for amino acid sequence, spectral properties and three-dimensional structure. The crystal structure that had been determined by isomorphous replacement has been refined at 2.0 A (1 A = 0.1 nm) resolution to an R-value of 0.20. The asymmetric unit contains four independent subunits of BBP. The co-ordinate differences are 0.25 A, in accord with the estimated error in co-ordinates. The polypeptide chain fold is characterized by an eight-stranded barrel. The connecting loops splay out at the upper end of the barrel and open it, whilst the lower end is closed. The overall shape resembles a calyx. The biliverdin IX gamma chromophore is located in a central cleft at the upper end of the barrel. The bilatriene moiety is in cyclic helical geometry with configuration Z,Z,Z and conformation syn,syn,syn. The geometry is in accord with the spectral properties and permits a correlation between sign of the circular dichroism bands and sense of the bilatriene helices. The fold of BBP is related to retinol binding protein (RBP), as had been recognized in the preliminary analysis, although the amino acid sequences of RBP and BBP show only 10% homology. There are large differences in the loops at the upper end of the barrel, whilst the segments of the centre and the lower end of the barrel superimpose closely. The ligands of BBP and RBP, biliverdin and retinol, respectively, are also similarly located.