Structure of oxidized poplar plastocyanin at 1.6 A resolution

The structure of poplar plastocyanin in the oxidized (CuII) state at pH 6.0 has been refined, using 1.6 A resolution counter data. The starting co-ordinates were obtained from the 2.7 A electron density map computed with phases derived by the multiple isomorphous replacement method. The model was refined successively by constrained real space, unrestrained reciprocal space, and restrained reciprocal space least-squares methods. The final residual R value is 0.17 for 8285 reflections (I greater than 2 sigma (I)). It is estimated that the root-mean-square standard deviation of the atomic positions is 0.1 A when averaged over all atoms, and 0.05 A for the Cu ligand atoms alone. The refined structure retains all the essential features of the 2.7 A model. The co-ordination geometry of the copper atom is confirmed as being distorted tetrahedral. The two Cu-N(His) bonds, 2.10 and 2.04 A, are within the range normally found in low molecular weight CuII complexes with Cu-N(imidazole) bonds. The Cu-S(Cys) bond, 2.13 A, is also normal, but the Cu-S(Met) bond, 2.90 A, is sufficiently long to raise important questions about its significance. The hydrogen-bonding and secondary structure can now be assigned confidently. Forty-four water molecules are included in the final model. Repetition of the refinement, using new data to 1.9 A resolution recorded from crystals at pH 4.2, has led to a residual R value of 0.16 for 6060 reflections (I greater than sigma (I)). There are few significant changes in the structure of poplar CuII-plastocyanin between pH 6.0 and pH 4.2. In particular, the geometry of the copper site is not affected. The observed changes in redox behaviour of plastocyanin at low pH are therefore unlikely to be connected with structural changes in the oxidized form of the protein. A number of features of the molecular structure appear to be directly related to the function of plastocyanin as an electron carrier in photosynthesis. Comparison between the known amino acid sequences of 67 plant plastocyanins reveals 52 conserved and 11 conservatively substituted residues in a total of 99. If three algal plastocyanin sequences are included in the comparison, there are still 26 conserved and 12 conservatively substituted residues. In many cases, the importance of these residues in determining the tertiary structure can be rationalized.     

Refined structure of dimeric diphtheria toxin at 2.0 A resolution.

The refined structure of dimeric diphtheria toxin (DT) at 2.0 A resolution, based on 37,727 unique reflections (F > 1 sigma (F)), yields a final R factor of 19.5% with a model obeying standard geometry. The refined model consists of 523 amino acid residues, 1 molecule of the bound dinucleotide inhibitor adenylyl 3'-5' uridine 3' monophosphate (ApUp), and 405 well-ordered water molecules. The 2.0-A refined model reveals that the binding motif for ApUp includes residues in the catalytic and receptor-binding domains and is different from the Rossmann dinucleotide-binding fold. ApUp is bound in part by a long loop (residues 34-52) that crosses the active site. Several residues in the active site were previously identified as NAD-binding residues. Glu 148, previously identified as playing a catalytic role in ADP-ribosylation of elongation factor 2 by DT, is about 5 A from uracil in ApUp. The trigger for insertion of the transmembrane domain of DT into the endosomal membrane at low pH may involve 3 intradomain and 4 interdomain salt bridges that will be weakened at low pH by protonation of their acidic residues. The refined model also reveals that each molecule in dimeric DT has an "open" structure unlike most globular proteins, which we call an open monomer. Two open monomers interact by "domain swapping" to form a compact, globular dimeric DT structure. The possibility that the open monomer resembles a membrane insertion intermediate is discussed.     

Refined crystal structure of dogfish M4 apo-lactate dehydrogenase

The crystal structure of M4 apo-lactate dehydrogenase from the spiny dogfish (Squalus acanthius) was initially refined by a constrained-restrained, and subsequently restrained, least-squares technique. The final structure contained 286 water molecules and two sulfate ions per subunit and gave an R-factor of 0.202 for difraction data between 8.0 and 2.0 A resolution. The upper limit for the co-ordinate accuracy of the atoms was estimated to be 0.25 A. The elements of secondary structure of the refined protein have not changed from those described previously, except for the appearance of a one-and-a-half turn 3(10) helix immediately after beta J. There is also a short segment of 3(10) helix between beta C and beta D in the part of the chain that connects the two beta alpha beta alpha beta units of the six-stranded parallel sheet (residues Tyr83 to Ala87). Examination of the interactions among the different elements of secondary structure by means of a surface accessibility algorithm supports the four structural clusters in the subunit. The first of the two sulfate ions is in the active site and occupies a cavity near the essential His195. Its nearest protein ligands are Arg171, Asp168 and Asn140. The second sulfate ion is located near the P-axis subunit interface. It is liganded by His188 and Arg173. These two residues are conserved in bacterial lactate dehydrogenase and form part of the fructose 1,6-bisphosphate effector binding site. Two other data sets in which one (collected at pH 7.8) or both (collected at pH 6.0) sulfate ions were replaced by citrate ions were also analyzed. Five cycles of refinement with respect to the pH 6.0 data (25 to 2.8 A resolution) resulted in an R value of 0.191. Only water molecules occupy the subunit boundary anion binding site at pH 7.8. The amino acid sequence was found to be in poor agreement with (2Fobs-Fcalc) electron density maps for the peptide between residues 207 and 211. The original sequence WNALKE was replaced by NVASIK. The essential His195 is hydrogen bonded to Asp168 on one side and Asn140 on the other. The latter residue is part of a turn that contains the only cis peptide bond of the structure at Pro141. The "flexible loop" (residues 97 to 123), which folds down over the active center in ternary complexes of the enzyme with substrate and coenzyme, has a well-defined structure. Analysis of the environment of Tyr237 suggests how its chemical modification inhibits the enzyme.     

Refinement of the crystal structure of ribonuclease S. Comparison with and between the various ribonuclease A structures.

Ribonuclease S (RNase-S) is a complex that consists of two proteolytic fragments of bovine pancreatic ribonuclease A (RNase-A): the S-peptide (residues 1-20) and S-protein (residues 21-124). We have refined the crystal structures of three RNase-S complexes. The first two contain the full-length 20-residue S-peptide and were studied at pHs of 4.75 and 5.5. The third one consists of a truncated form of S-peptide (residues 1-15) and was studied at pH 4.75 as the reference structure for a series of mutant peptide complexes to be reported separately. Excluding residues 16-23 which are either missing (in the S15 complex) or disordered (in both S20 complexes), all three structures refined at 1.6-A resolution are identical within the estimated errors in the coordinates (0.048 A for the backbone atoms). The R-values, residual error, range from 17.4% to 18.6%. The final model of S20, pH 4.75, includes 1 sulfate and 84 water molecules. The side chains of 11 residues were modeled in two discrete conformations. The final structures were independent of the particular RNase-A or RNase-S used as a starting model. An extensive comparison with refined crystal structures of RNase-A reveals that the core of the molecule which is held together with extensive hydrogen bonds is in identical pattern in all cases. However, the loop regions vary from one structure to another and are often characterized by high B-factors. The pattern of thermal parameters appears to be dependent on crystal packing and correlates well with the accessibility calculated in the crystal. Gln60 is a conserved residue in all sequences known to date for this class of ribonucleases. However, it is the only residue that is clearly defined in an unfavorable position (phi = -100 degrees, psi = -130 degrees) on the Ramachandran plot. The origin of the substantial differences between RNase-A and RNase-S in stability to both acid and temperature denaturation and in susceptibility to proteolysis at neutral pH is not obvious in our visual comparison of these two structures.     

The crystal structure of the ternary complex of staphylococcal nuclease, Ca2+, and the inhibitor pdTp, refined at 1.65 A

The structure of a complex of staphylococcal nuclease with Ca2+ and deoxythymidine 3',5'-bisphosphate (pdTp) has been refined by stereochemically restrained least-squares minimization to a crystallographic R value of 0.161 at 1.65 A resolution. The estimated root-mean-square (rms) error in the coordinates is 0.16 A. The final model comprises 1082 protein atoms, one calcium ion, the pdTp molecule, and 82 solvent water molecules; it displays an rms deviation from ideality of 0.017 A for bond distances and 1.8 degrees for bond angles. The mean distance between corresponding alpha carbons in the refined and unrefined structures is 0.6 A; we observe small but significant differences between the refined and unrefined models in the turn between residues 27 and 30, the loop between residues 44 and 50, the first helix, and the extended strand between residues 112 and 117 which forms part of the active site binding pocket. The details of the calcium liganding and solvent structure in the active site are clearly shown in the final electron density map. The structure of the catalytic site is consistent with the mechanism that has been proposed for this enzyme. However, we note that two lysines from a symmetry-related molecule in the crystal lattice may play an important role in determining the geometry of inhibitor binding, and that only one of the two required calcium ions is observed in the crystal structure; thus, caution is advised in extrapolating from the structure of the complex of enzyme and inhibitor to that of enzyme and substrate.     

Three-dimensional structure of endo-1,4-beta-xylanase II from Trichoderma reesei: two conformational states in the active site.

The three-dimensional structure of endo-1,4-beta-xylanase II (XYNII) from Trichoderma reesei has been determined by X-ray diffraction techniques and refined to a conventional R-factor of 18.3% at 1.8 A resolution. The 190 amino acid length protein was found to exist as a single domain where the main chain folds to form two mostly antiparallel beta-sheets, which are packed against each other in parallel. The beta-sheet structure is twisted, forming a large cleft on one side of the molecule. The structure of XYNII resembles that of Bacillus 1,3-1,4-beta-glucanase. The cleft is an obvious suggestion for an active site, which has putative binding sites for at least four xylose residues. The catalytic residues are apparently the two glutamic acid residues (Glu86 and Glu177) in the middle of the cleft. One structure was determined at pH 5.0, corresponding to the pH optimum of XYNII. The second structure was determined at pH 6.5, where enzyme activity is reduced considerably. A clear structural change was observed, especially in the position of the side chain of Glu177. The observed conformational change is probably important for the mechanism of catalysis in XYNII.     

Crystal structure of bovine beta-trypsin at 1.5 A resolution in a crystal form with low molecular packing density. Active site geometry, ion pairs and solvent structure

The crystal structure of bovine pancreatic beta-trypsin (BPT) has been determined from a novel orthorhombic crystal form which contains substantially more solvent (filling 57% of the volume of the unit cell) than previously determined orthorhombic (44%) and trigonal (37%) BPT structures. The native and benzamidine-inhibited crystal structures of BPT in ammonium sulphate at pH 5.3 have been determined for the new form by molecular replacement techniques. The structures have been refined at 1.5 A resolution with final R-values of 16.7% and 16.9%, respectively. Comparison with the previously refined old orthorhombic forms shows that the overall conformation of the protein backbone is highly conserved. A great number of previously undefined side-chains have been located in density. At the C terminus an extra ion pair involving lysines 87 and 107 has been revealed. A far more detailed picture of the ordered solvent structure has been derived. Thirty water clusters have been identified. A large water network extends from the calcium binding site to the activation area and the autolysis loop. There is evidence for a water channel reaching from the depth of the specificity pocket to the nearby protein surface which might be involved in the displacement of water molecules upon substrate binding. A sulphate anion which forms hydrogen bonds to the active site residues His57, Ser195 and Gly193 was for the first time positioned in clearly defined electron density. Interaction with the sulphate ion may explain the increase in the pKa value of His57 at high sulphate concentrations which was observed by nuclear magnetic resonance studies of a bacterial serine protease both in crystalline form and in solution. Thus, a His-Ser hydrogen bond will not exist in solvents containing sulphate at low pH (up to at least 6.8) where the imidazole of His57 is protonated. The new crystal form is of considerable interest for substrate binding studies. Wide solvent channels should allow diffusion of large substrates (comparable in size to, e.g. pancreatic trypsin inhibitor) into the enzyme crystal. The active site is accessible; intermolecular contact areas are further remote from the active site than in the old orthorhombic form.     

A semisynthetic bovine pancreatic ribonuclease containing a unique nitrotyrosine residue

A fully active semisynthetic ribonuclease, RNase 1-118:111-124, may be prepared by enzymatically removing six residues from the COOH terminus of the protein (positions 119-124) and then complementing the inactive RNase 1-118 with a chemically synthesized peptide containing the COOH-terminal 14 residues of the molecule (RNase 111-124) [M. C. Lin, B. Gutte, S. Moore, and R. B. Merrifield (1970) J. Biol. Chem. 245, 5169-5170]. Nitration of tyrosine-115 in the peptide followed by complex formation with RNase 1-118 affords a fully active enzyme containing a unique nitrotyrosine residue in a position which is known and which is very likely to be completely exterior to the active site region. The binding constant between the tetradecapeptide and RNase 1-118 (5 X 10(6) M-1 at pH 6.0) is not changed by the nitration. Crystals of the nitrated complex are isomorphous with those of RNase 1-118:111-124, for which a refined 1.8-A structure has recently been obtained.     

Structure of yeast triosephosphate isomerase at 1.9-A resolution

The structure of yeast triosephosphate isomerase (TIM) has been solved at 3.0-A resolution and refined at 1.9-A resolution to an R factor of 21.0%. The final model consists of all non-hydrogen atoms in the polypeptide chain and 119 water molecules, a number of which are found in the interior of the protein. The structure of the active site clearly indicates that the carboxylate of the catalytic base, Glu 165, is involved in a hydrogen-bonding interaction with the hydroxyl of Ser 96. In addition, the interactions of the other active site residues, Lys 12 and His 95, are also discussed. For the first time in any TIM structure, the "flexible loop" has well-defined density; the conformation of the loop in this structure is stabilized by a crystal contact. Analysis of the subunit interface of this dimeric enzyme hints at the source of the specificity of one subunit for another and allows us to estimate an association constant of 10(14)-10(16) M-1 for the two monomers. The analysis also suggests that the interface may be a particularly good target for drug design. The conserved positions (20%) among sequences from 13 sources ranging on the evolutionary scale from Escherichia coli to humans reveal the intense pressure to maintain the active site structure.     

The refined 2.0 A X-ray crystal structure of the complex formed between bovine beta-trypsin and CMTI-I, a trypsin inhibitor from squash seeds (Cucurbita maxima). Topological similarity of the squash seed inhibitors with the carboxypeptidase A inhibitor from potatoes

The stoichiometric complex formed between bovine beta-trypsin and the Cucurbita maxima trypsin inhibitor I (CMTI-I) was crystallized and its X-ray crystal structure determined using Patterson search techniques. Its structure has been crystallographically refined to a final R value of 0.152 (6.0-2.0 A). CMTI-I is of ellipsoidal shape; it lacks helices or beta-sheets, but consists of turns and connecting short polypeptide stretches. The disulfide pairing is CYS-3I-20I, Cys-10I-22I and Cys-16I-28I. According to the polypeptide fold and disulfide connectivity its structure resembles that of the carboxypeptidase A inhibitor from potatoes. Thirteen of the 29 inhibitor residues are in direct contact with trypsin; most of them are in the primary binding segment Val-2I (P4)-Glu-9I (P4') which contains the reactive site bond Arg-5I-Ile-6I and is in a conformation observed also for other serine proteinase inhibitors.     

Three-dimensional structure of a presynaptic neurotoxic phospholipase A2 from Daboia russelli pulchella at 2.4 A resolution

The phospholipase A(2 )from Daboia russelli pulchella (DPLA(2)) is the only known member of subclass II of group IIA. The three-dimensional structure of this presynaptic neurotoxic DPLA(2) enzyme has been determined at 2.4 A resolution. The structure was determined by the molecular replacement method using the model Crotalus atrox, and refined using X-PLOR to a final R-factor of 18.8 % for all data in the resolution range 20.0 A-2.4 A. The final refined model comprises 1888 atoms from two crystallographically independent protein molecules and 160 water oxygen atoms. The overall folding of DPLA(2), with three long helices and two short antiparallel beta-strands is grossly similar to those observed for other PLA(2)s. In the present structure, the calcium binding site is empty but the conformation of the calcium binding loop is similar to those observed in the calcium bound states. Two spatially adjacent regions of residues 55-61 (a typical beta-turn I) and 83-94 (a well defined loop) are remarkably different in conformation, electrostatic characteristics and inter-segmental interactions from those found in non-neurotoxic PLA(2)s. Yet another striking structural feature in DPLA(2 )pertains to the stretch of residues 53-77, which has a series of positively charged residues protruding outwardly. The above segment is presumed to be involved in the anticoagulant activity. A unique hydrophobic patch including residues Leu17, Ala18, Ile19, Pro20, Phe106 and Leu110 is found on the surface together with an equally emphatic region of -OH groups containing residues such as Ser21, Tyr22, Ser23, Ser24, Tyr25 and Tyr28. The interactions between two molecules of DPLA(2) in the asymmetric unit are remarkably different from those observed in the standard dimers and trimers of PLA(2)s, leaving the enzyme's active site fully exposed for enzyme-substrate reactions, it makes this structure one of the most favourable examples for structure-based drug design through soaking experiments.     

Refined structure of the pore-forming domain of colicin A at 2.4 A resolution.

The E1 subgroup (E1, A, B, IA, IB, K and N) of anti-bacterial toxins called colicins is known to form voltage-dependent channels in lipid bilayers. The crystal structure of the pore-forming domain of colicin A from Escherichia coli has been refined to the diffraction limit of the crystals at 2.4 A resolution by means of molecular dynamics and restrained least-squares methods to a conventional R-factor of 0.18 for all data between 6.0 and 2.4 A resolution. The polypeptide chain of 204 amino acid residues consists of ten alpha-helices organized in a three-layer structure. The helices range in length from 9 to 23 residues with an average length of 125 residues. The packing arrangement of the helices has been analysed; the packing is different from that observed in four-helix bundle proteins. The sites of 83 water molecules have been located and refined. Analysis of the structure provides insights into the mechanism of formation of a voltage-gated channel by the protein. Although it is proposed that substantial tertiary structural changes occur during membrane insertion, the secondary structural elements remain conserved. This idea has been proposed recently for a number of other protein-membrane events and thus may have more general applicability.     

The 2.2 A resolution crystal structure of influenza B neuraminidase and its complex with sialic acid.

Influenza virus neuraminidase catalyses the cleavage of terminal sialic acid, the viral receptor, from carbohydrate chains on glycoproteins and glycolipids. We present the crystal structure of the enzymatically active head of influenza B virus neuraminidase from the strain B/Beijing/1/87. The native structure has been refined to a crystallographic R-factor of 14.8% at 2.2 A resolution and its complex with sialic acid refined at 2.8 A resolution. The overall fold of the molecule is very similar to the already known structure of neuraminidase from influenza A virus, with which there is amino acid sequence homology of approximately 30%. Two calcium binding sites have been identified. One of them, previously undescribed, is located between the active site and a large surface antigenic loop. The calcium ion is octahedrally co-ordinated by five oxygen atoms from the protein and one water molecule. Sequence comparisons suggest that this calcium site should occur in all influenza A and B virus neuraminidases. Soaking of sialic acid into the crystals has enabled the mode of binding of the reaction product in the putative active site pocket to be revealed. All the large side groups of the sialic acid are equatorial and are specifically recognized by nine fully conserved active site residues. These in turn are stabilized by a second shell of 10 highly conserved residues principally by an extensive network of hydrogen bonds.     

Crystal structure analysis, refinement and enzymatic reaction mechanism of N-carbamoylsarcosine amidohydrolase from Arthrobacter sp. at 2.0 A resolution.

N-carbamoylsarcosine amidohydrolase from Arthrobacter sp., a tetramer of polypeptides with 264 amino acid residues each, has been crystallized and its structure solved and refined at 2.0 A resolution, to a crystallographic R-factor of 18.6%. The crystals employed in the analysis contain one tetramer of 116,000 M(r) in the asymmetric unit. The structure determination proceeded by multiple isomorphous replacement, followed by solvent-flattening and density averaging about the local diads within the tetramer. In the final refined model, the root-mean-square deviation from ideality is 0.01 A for bond distances and 2.7 degrees for bond angles. The asymmetric unit consists of 7853 protein atoms, 431 water molecules and four sulfate ions bound into the putative active site clefts in each subunit. One subunit contains a central six-stranded parallel beta-pleated sheet packed by helices on both sides. On one side, two helices face the solvent, while two of the helices on the other side are buried in the tight intersubunit contacts. The catalytic center of the enzyme, tentatively identified by inhibitor binding, is located at the interface between two subunits and involves residues from both. It is suggested that the nucleophilic group involved in hydrolysis of the substrate is the thiol group of Cys117 and a nucleophilic addition-elimination mechanism is proposed.     

Structure of a rhombohedral R6 insulin/phenol complex.

Hexameric insulin has been crystallized from different conditions in a variety of crystalline modifications. In the presence of approximately 1% phenol and at a pH of 8.5, a new rhombohedral form is produced, space group R3, a = 79.92A and c = 40.39A, in which the asymmetric unit consists of a dimer. The structure has been solved and refined, using data between 8.0 and 2.5A resolution, to a residual of 0.157. The two monomers in the asymmetric unit have nearly identical R conformations, that is, residues B1 through B8 are alpha-helical, producing a continuous alpha-helix from B1 through B19. A phenol molecule is hydrogen bonded to the carbonyl oxygen of A6 Cys of each monomer. Small differences in conformation and the final (2Fo-Fc) and difference electron density maps suggest that an additional phenol molecule is coordinated to one of the two zinc ions.     

Crystal structure of meso-2,3-butanediol dehydrogenase in a complex with NAD+ and inhibitor mercaptoethanol at 1.7 A resolution for understanding of chiral substrate recognition mechanisms

The crystal structure of a ternary complex of meso-2,3-butanediol dehydrogenase with NAD+ and a competitive inhibitor, mercaptoethanol, has been determined at 1.7 A resolution by means of molecular replacement and refined to a final R-factor of 0.194. The overall structure is similar to those of the other short chain dehydrogenase/reductase enzymes. The NAD+ binding site, and the positions of catalytic residues Ser139, Tyr152, and Lys156 are also conserved. The crystal structure revealed that mercaptoethanol bound specifically to meso-2,3-butanediol dehydrogenase. Two residues around the active site, Gln140 and Gly183, forming hydrogen bonds with the inhibitor, are important but not sufficient for distinguishing stereoisomerism of a chiral substrate.     

Structure of porin refined at 1.8 A resolution.

The crystal structure of porin from Rhodobacter capsulatus has been refined using the simulated annealing method. The final model consists of all 301 amino acid residues well obeying standard geometry, three calcium ions, 274 solvent molecules, three detergent molecules and one unknown ligand modeled as a detergent molecule. The final crystallographic R-factor is 18.6% based on 42,851 independent reflections in the resolution range 10 to 1.8 A. The model is described in detail.     

Enzyme adaptation to alkaline pH: atomic resolution (1.08 A) structure of phosphoserine aminotransferase from Bacillus alcalophilus

The crystal structure of the vitamin B(6)-dependent enzyme phosphoserine aminotransferase from the obligatory alkaliphile Bacillus alcalophilus has been determined at 1.08 A resolution. The model was refined to an R-factor of 11.7% (R(free) = 13.9%). The enzyme displays a narrow pH optimum of enzymatic activity at pH 9.0. The final structure was compared to the previously reported structure of the mesophilic phosphoserine aminotransferase from Escherichia coli and to that of phosphoserine aminotransferase from a facultative alkaliphile, Bacillus circulans subsp. alkalophilus. All three enzymes are homodimers with each monomer comprising a two-domain architecture. Despite the high structural similarity, the alkaliphilic representatives possess a set of distinctive structural features. Two residues directly interacting with pyridoxal-5'-phosphate are replaced, and an additional hydrogen bond to the O3' atom of the cofactor is present in alkaliphilic phosphoserine aminotransferases. The number of hydrogen bonds and hydrophobic interactions at the dimer interface is increased. Hydrophobic interactions between the two domains in the monomers are enhanced. Moreover, the number of negatively charged amino acid residues increases on the solvent-accessible molecular surface and fewer hydrophobic residues are exposed to the solvent. Further, the total amount of ion pairs and ion networks is significantly reduced in the Bacillus enzymes, while the total number of hydrogen bonds is increased. The mesophilic enzyme from Escherichia coli contains two additional beta-strands in a surface loop with a third beta-strand being shorter in the structure. The identified structural features are proposed to be possible factors implicated in the alkaline adaptation of phosphoserine aminotransferase.     

The 0.93A crystal structure of sphericase: a calcium-loaded serine protease from Bacillus sphaericus

We have previously isolated sphericase (Sph), an extracellular mesophilic serine protease produced by Bacillus sphaericus. The Sph amino acid sequence is highly homologous to two cold-adapted subtilisins from Antarctic bacilli S39 and S41 (76% and 74% identity, respectively). Sph is calcium-dependent, 310 amino acid residues long and has optimal activity at pH 10.0. S41 and S39 have not as yet been structurally analysed.In the present work, we determined the crystal structure of Sph by the Eu/multiwavelength anomalous diffraction method. The structure was extended to 0.93A resolution and refined to a crystallographic R-factor of 9.7%. The final model included all 310 amino acid residues, one disulfide bond, 679 water molecules and five calcium ions. Although Sph is a mesophilic subtilisin, its amino acid sequence is similar to that of the psychrophilic subtilisins, which suggests that the crystal structure of these subtilisins is very similar.The presence of five calcium ions bound to a subtilisin molecule, as found here for Sph, has not been reported for the subtilisin superfamily. None of these calcium-binding sites correlates with the well-known high-affinity calcium-binding site (site I or site A), and only one site has been described previously. This calcium-binding pattern suggests that a reduction in the flexibility of the surface loops of Sph by calcium binding may be responsible for its adaptation to mesophilic organisms.     

The refined crystal structure of ribonuclease A at 2.0 A resolution

This paper describes the structure of bovine pancreatic ribonuclease A, refined by a restrained parameter least squares procedure at 2.0 A resolution, and rebuilt using computer graphics. The final agreement factor (formula see text) is 0.159. The positions of the 951 main chain atoms have been determined with an estimated accuracy of 0.17 A. In addition, the model includes a phosphate group in the active site and 176 waters, many of them with partial occupancy. The bond lengths in the refined structure of RNase A differ from the ideal values by an overall root mean square deviation of 0.022 A; the corresponding value for angle distances is 0.06 A. The root mean square deviation of planar atoms from ideality is 0.017 A, and root mean square deviation of the peptide torsion angles from 180 degrees is 3.4 degrees. The model is in good agreement with the final difference Fourier maps. Two active site histidines, His 12 and His 119, form hydrogen bonds to the phosphate ion. His 119 is also hydrogen bonded to the carboxyl of ASp 121 and His 12 to the carbonyl of Thr 45. The structure of the RNase A is very similar to that of RNase S, particularly in the active site region. The root mean square discrepancy of all atoms from residues 1 to 16 and 24 to 123 is 1.06 A and the root mean square discrepancy for the active site region is 0.6 A.