Search

The BLANC program suite is a set of programs which can be used for macromolecular structure determination by X-ray crystallography. The suite is designed to provide experienced crystallographers and students with a number of simple tools, at the same time allowing the building and testing of new algorithms. Besides a set of small programs, the BLANC system introduces so-called superprograms which represent larger programs composed of several smaller ones. They utilize the so-called black-box principle, requiring minimum preparation or intervention from a user. The programs are written in standard Fortran77 and communicate with each other by the standard BLANC data files. The package has been ported to all the major platforms such as UNIX, VMS and DOS. A current version of the suite is distributed by anonymous ftp.

The crystal structure of chicken cytosolic aspartate aminotransferase (cAATase; EC 2.6.1.1) has been solved and refined at 1.9 A resolution. Orthorhombic crystals, space group P2(1)2(1)2(1), a = 56.4 A, b = 126.0 A and c = 142.3 A, were grown from polyethylene glycol solutions in the presence of maleate, a dicarboxylic inhibitor that forms a Michaelis-like complex. The pyridoxal form of the enzyme was used for crystallization. Diffraction data were collected using synchrotron radiation. The structure of the new orthorhombic crystal form was solved by molecular replacement using the partially refined 2.8 A resolution structure of the high-salt crystal form as a search model. The final value of the crystallographic R-factor after rigid body and restrained least-squares refinement is 0.175 with very good model geometry. The two 2-fold-related subunits of cAATase have distinct environments in the crystal lattice. Domain movement is strictly hindered by the lattice contacts in one subunit, while the second one possesses conformational freedom. Despite their different environments, both subunits were found in the closed conformation with one maleate molecule tightly bound in each active site. The present study allows a detailed comparison of the highly refined structures of the aspartate aminotransferase isozymes, and thus provide better insight into the role of conserved and variable residues in substrate recognition and catalysis.

The crystal structure of carboxypeptidase T from Thermoactinomyces vulgaris has been determined at 0.235-nm resolution by X-ray diffraction. Carboxypeptidase T is a remote homologue of mammalian Zn-carboxypeptidases. In spite of the low degree of amino acid sequence identity, the three-dimensional structure of carboxypeptidase T is very similar to that of pancreatic carboxypeptidases A and B. The core of the protein molecule is formed by an eight-stranded mixed beta sheet. The active site is located at the C-edge of the central (parallel) part of the beta sheet. The structural organization of the active centre appears to be essentially the same in the three carboxypeptidases. Amino acid residues directly involved in catalysis and binding of the C-terminal carboxyl of a substrate are strictly conserved. This suggests that the catalytic mechanism proposed for the pancreatic enzymes is applicable to carboxypeptidase T and to the whole family of Zn-carboxypeptidases. Comparison of the amino acid replacements at the primary specificity pocket of carboxypeptidases A, B and T provides an explanation of the unusual 'A+B' type of specificity of carboxypeptidase T. Four calcium-binding sites localized in the crystal structure of carboxypeptidase T could account for the high thermostability of the protein.

The tyrosine phenol lyase (EC 4.1.99.2) from Citrobacter intermedius has been crystallised in the apo form by vapour diffusion. The space group is P21212. The unit cell has dimensions a = 76.0 Å, b = 138.3 Å, c = 93.5 Å and it contains two subunits of the tetrameric molecule in the asymmetric unit, Diffraction data for the native enzyme and two heavy atom derivatives have been collected with synchrotron radiation and an image plate scanners. The structure has been solved at 2.7 Å resolution by isomorphous replacement with subsequent modification of the phases by averaging the density around the non-crystallographic symmetry axis. The electron density maps clearly show the relative orientation of the subunits and most of the trace of the polypeptide chain. Each subunit consists of two domains. The topology of the large domain appears to be similar to that of the aminotransferases.

A simplified multidimensional search was applied to determine the structure of deoxyhexamer CGCGCG in the crystal form belonging to the space group C222(1) (a = 45.6 A, b = 37.3 A, c = 70.3 A). This crystal form contains two Z-DNA duplexes per asymmetric unit in a similar orientation. The search consists of several main steps. As a first step, the analysis of packing modes is carried out and constraints for the position of duplexes in asymmetric unit are formulated. In order to choose true packing mode duplexes are represented as cylinders of a constant density and global search is carried out. Only reflections belonging to the plane in reciprocal space perpendicular to DNA axis were used in the calculations. An analytical representation of diffraction from a hollow cylinder is given which allows further refinement of corresponding parameters. During the global search the value of a linear correlation coefficient is estimated for each solution to pick up the best one. At the second step the "more local" search is carried out. The DNA model of a certain conformation (in our case Z-DNA) is taken and several parameters are being varied. These are: a) rotations of the independent duplexes around their axes (only two rotational parameters for two duplexes in our example) and b) a few positional parameters for which there is no corresponding constraint in the packing mode tested. In our case we had to vary only x1 and y2, where subindex corresponds to molecule number. The last stage concerned is quite similar to the multidimensional search realised in program ULTIMA. The only difference that the number of variable parameters is less in our search. This number for the case under consideration was 4 instead of 12 that would be necessary in a conventional multidimensional search.

The human progastricsin crystal structure has been solved by the molecular replacement method. The intensities of reflections from native progastricsin crystals were measured at the 4.0 A resolution by the omega-scan method with a Nicolet P3 diffractometer operated in automatic regime. To determine the orientation and position of progastricsin molecules in the unit cell, programme packages MERLOT and BRUTE were applied running on a MicroVAX-II computer. Prior to the translation search, several rotation function peaks were subjected to a rigid body refinement against the correlation coefficient between the observed and calculated structure factors. This approach clearly identified the correct orientation of the molecule. The solution obtained from the BRUTE translation function map was refined by the 6-dimensional correlation search and then by programme CORELS. The human progastricsin molecules packing in the crystal unit ell is described.

The ternary complex of NAD-dependent formate dehydrogenase (FDH) from the methylotrophic bacterium Pseudomonas sp. 101 (enzyme-NAD-azide) has been crystallised in the space group P2(1)2(1)2(1) with cell dimensions a = 11.60 nm, b = 11.33 nm, c = 6.34 nm. There is 1 dimeric molecule/asymmetric unit. An electron density map was calculated using phases from multiple isomorphous replacement at 0.30 nm resolution. Four heavy atom derivatives were used. The map was improved by solvent flattening and molecular averaging. The atomic model, including 2 x 393 amino acid residues, was refined by the CORELS and PROLSQ packages using data between 1.0 nm and 0.30 nm excluding structure factors less than 1 sigma. The current R factor is 27.1% and the root mean square deviation from ideal bond lengths is 4.2 pm. The FDH subunit is folded into a globular two-domain (coenzyme and catalytic) structure and the active centre and NAD binding site are situated at the domain interface. The beta sheet in the FDH coenzyme binding domain contains an additional beta strand compared to other dehydrogenases. The difference in quaternary structure between FDH and the other dehydrogenases means that FDH constitutes a new subfamily of NAD-dependent dehydrogenases: namely the P-oriented dimer. The FDH nucleotide binding region of the structure is aligned with the three dimensional structures of four other dehydrogenases and the conserved residues are discussed. The amino acid residues which contribute to the active centre and which make contact with NAD have been identified.

An X-ray crystallographic structure determination has been carried out on bovine lens leucine aminopeptidase at 4.0 A resolution by using a combination of isomorphous replacement and solvent flattening. The two heavy atom derivatives used were obtained by soaking crystals in ethyl mercury chloride, which bound at four sites, and phenyl mercury acetate, which bound at one site in the monomer. The electron density map reveals that the enzyme hexameric oligomer, arranged in 32 symmetry, has a triangular barrel appearance and dimensions, of height 88 A and maximal width 118 A in barrel equatorial plane. Each subunit in an elongated ellipsoid of approximate length 92 A. Subunits contacts have been described. From an analysis of the map each subunit appears to contain some 36% alpha-helix and is organized into two distinct globular domains. Direct location of zinc cluster and competitive inhibitor binding site are presented.

X-Ray structural data concerning the substrate binding site of cytosolic chicken aspartate aminotransferase (AspAT) are reported. The structure of the complex of AspAT with the substrate-like inhibitor maleate has been refined at 2.2 A resolution. The lengths of hydrogen bonds between a bound molecule of maleate and side chains of amino acid residues in the active site are presented as well as other interatomic distances in the substrate binding site. The data obtained for the cytosolic AspAT have been compared with those for the mitochondrial chicken AspAT. It has been inferred that differences in substrate specificity of the AspAT isoenzymes are determined by interactions involving amino acid residues which are situated in the immediate vicinity of the active site and influence ionization or orientation of functional groups interacting with substrate. An explanation is suggested for different rates of transamination of aromatic amino acids in the active sites of the cytosolic and mitochondrial isoenzymes.

A high resolution structure of hen egg-white lysozyme containing 36 ± I mol H2O per mol of protein has been obtained using triclinic (P1) crystals cross-linked with glutaraldehyde. Analysis of dehydration-induced structural changes has revealed displacement in relative position of domains and numerous small displacements in positions of individual atoms with r.m.s. deviation of main atoms 0.60 Å, and that of all atoms 0.97 Å. An increase in the average packing density of atoms in dry lysozyme by 4–6% seems to be the most probable reason for the loss of its activity and mobility.

Previous comparison of amino acid sequences and three-dimensional structures has shown that the microbial ribonucleases from an evolutionarily related family that is separate from that of the pancreatic ribonucleases (Hill et al., 1983). The microbial and pancreatic ribonucleases catalyse the same two step hydrolysis of RNA via a cyclic 2′3′ intermediate. However, the specificities are different; the microbial enzymes prefer a purine base on the 3′ side of the scissile phosphate, and in some cases are very guanine specific.

RNase Th1 is an alkaline guanyl-specific fungal ribonuclease. Primary structure of RNase Th1 has been determined (Table 1). The homology of RNase Th1 with other fungal RNases (T1, C2, Pb1) is about 70%.

Structural aspects of specificity and catalytic activity of enzymes are studied on various objects, in particular on ribonucleases. Remarkable progress has been achieved in understanding the pyrimidine specificity of RNAse A at molecular level. The tertiary structures of different microbial ribonucleases were solved at atomic resolution recently (Hill et al., 1983). These enzymes belong to the so-called guanyl-specific RNases such as the well-known RNAse T1, which degrades single-stranded RNA to give oligonucleotides with terminal 3′ guanosine monophosphate. In many of these, the cleavage occurs as a two-step mechanism via 2′, 3′ cyclic nucleotide intermediates. In order to reveal molecular bases for substrate specificity of RNAse T1 the structures of the RNAse T1-2′-or 3′-GMP complex were studied at 2 A resolution (Heinemann and Saenger, 1982; Sugio et al., 1985). Some important features of recognition patterns were obtained from these data. However, it was not possible to draw cleancut conclusions in respect to the molecular mechanism for substrate discrimination. For this reason we have undertaken a study of the crystal structure of RNAse Pb1 (from Penicillium brevicompactum), a close analogue of RNAse T1.

Ribonuclease C2 from Aspergillus clavatus is a guanyl-specific ribonuclease. The molecular weight of RNase C2 is about 11000. Its primary structure was determined (Bezborodova et al., 1983).