Structure of Panulirus interruptus hemocyanin at 5 Å resolution

The hemocyanin from the spiny lobster Panulirus interruptus, a hexamer with a molecular weight of approximately 540,000, was crystallized in space group P2₁ with two molecules in the unit cell and cell dimensions $\text{a = 119.8}\text{A}\text{?}\text{, b = 193.1}\text{A}\text{?}\text{, c = 122.2}\text{A}\text{?}\text{and}\text{β = 118.1 °}$. With screened precession photographs a three-dimensional set of reflections was collected up to 10 Å resolution. Both the conventional and the fast rotation function programs were applied and gave results that were in excellent agreement with each other. The hemocyanin hexamer has 32 point group symmetry. Its 3-fold molecular axis runs approximately parallel to the crystallographic 2-fold screw axis.X-ray diffraction data to 5 Å resolution were collected by the oscillation method. Rotation function studies with data between 7 and 5 Å resolution confirmed the 10 Å studies and, furthermore, showed that the rotation axes relating subunits within one hexameric molecule can be distinguished from the rotation axes relating subunits belonging to different hexamers in the unit cell. The local 3-fold axis in the hexamer makes an angle of about 6 ° with the crystallographic 2-fold screw axis.For a mercury and a platinum derivative three-dimensional data sets were collected to 5 Å by the oscillation method. The difference Patterson of the platinum derivative could be solved. The eventual number of heavy-atom sites was 36 for the platinum derivative and 70 for the mercury derivative. From the well-occupied sites the point-group symmetry of the molecule could be established accurately. In addition, the centre of the hexamer could be located within 0.2 Å.Protein phases were obtained from isomorphous as well as anomalous differences. A “best” electron density map calculated with these phases showed the shape of the hexameric molecule as well as the boundaries of the six subunits. Correlation coefficients between the densities of the subunits showed little variation, suggesting a random distribution of the different subunit types (Van Eerd & Folkerts, 1981) over the six positions in the hexamer.The subunits are positioned at the corner of an antiprism. When viewed along the 3-fold axis the hexamer is roughly hexagonal in shape, with a diameter of approximately 120 Å. Viewed along one of the 2-fold axes the molecule is of rectangular shape with dimensions 95 Å × 120 Å. The subunit can be described as an ellipsoid of irregular shape with axes of 80 Å, 55 Å and 48 Å. Each subunit makes extensive contacts with three other subunits in the hexamer and, possibly, a much weaker contact with a fourth subunit.     

Structure of actinidin,after refinement at 1.7 Å resolution

The structure of the sulphydryl protease, actinidin, after refinement at 1.7 Å resolution, is described. The positions of most of the 1666 atoms have been determined with an accuracy better than 0.1 Å; only two residues (219 and 220) and the side-chain of a third (87) cannot be seen. In addition, the model contains 272 solvent molecules, all taken as water, except one which may be an ammonium ion. Atomic B values give a good indication of the mobility of different parts of the structure. Actinidin has a double domain structure, with one domain mostly helical in its secondary structure, and the other domain built around a twisted β-sheet. The geometry of hydrogen bonds in helices, β-structure and turns has been analysed. All are significantly non-linear, with the angle N-?…O ~160 °. Carbonyl groups are tilted outwards from the axis of each helix, the tilting apparently unaffected by whether or not additional hydrogen bonds are made (e.g. to water or side-chain atoms). Each domain is folded round a substantial core of non-polar side-chains, but the interface between domains is mostly polar. Interactions across this interface involve a network of eight buried water molecules, the buried carboxyl and amino groups of Glu35, Glu50, Lys181 and Lys17, other polar side-chains and a few hydrophobic groups. One other internal charged side-chain, that of Glu52, is adjacent to a buried solvent molecule, probably an ammonium ion. Other side-chain environments are described. One proline residue has a cis configuration. The sulphydryl group is oxidized, probably to SO₂^?, with one oxygen atom clearly visible but the other somewhat less certain. The active site geometry is otherwise compatible with the mechanism proposed by Drenth et al. (1975,1976) for papain. The positions of the 272 solvent molecules are described. The best-ordered water molecules are those that are internal (total of 17), in surface pockets, or in the intermolecular contact regions. These generally form three or four hydrogen bonds, two to proton acceptors and one or two to proton donors. Other water molecules make water bridges on the surface, sometimes covering the exposed edges of non-polar groups. Intermolecular contacts involve few protein atoms, but many water molecules.     

Crystal and molecular structure of the sulfhydryl protease calotropin DI at 3·2 Å resolution

The three-dimensional structure of the sulfhydryl protease calotropin DI from the madar plant, Calotropis gigantea, has been determined at 3·2 Å resolution using the multiple isomorphous replacement method with five heavy atom derivatives. A Fourier synthesis based on protein phases with a mean figure of merit of 0·857 was used for model building. The polypeptide backbone of calotropin DI is folded to form two distinct lobes, one of which is comprised mainly of α-helices, while the other is characterized by a system of all antiparallel pleated sheets. The overall molecular architecture closely resembles those found in the sulfhydryl proteases papain and actinidin.Despite the unknown amino acid sequence of calotropin DI a number of residues around its active center could be identified. These amino acid side-chains were found in a similar arrangement as the corresponding ones in papain and actinidin. The polypeptide chain between residues 1 and 18 of calotropin DI folds in a unique manner, providing a possible explanation for the unusual inability of calotropin DI to hydrolyze those synthetic substrates that papain and actinidin act upon.     

Refinement of human lysozyme at 1.5 Å resolution analysis of non-bonded and hydrogen-bond interactions

The structure of human lysozyme has been crystallographically refined at 1.5 Å resolution by difference map and restrained least-squares procedures to an R factor of 0.187. A comprehensive analysis of the non-bonded and hydrogen-bonded contacts in the lysozyme molecule, which were not restrained, revealed by the refinement has been carried out. The non-bonded CC contacts begin at ~3.45 Å, and the shorter contacts are dominated, as expected, by interactions between trigonal and tetrahedral carbon atoms. The CO contact distances have a “foot” at 3.05 Å. The CN distance plot shows a significant peak at 3.25 Å, which results from close contact between peptide NHs and carbonyl carbons involved in N_iC′_{i ? 2} interactions in α-helices and reverse turns. The distances involving sulphur atoms discriminate SC trigonal interactions at 3.4 to 3.6 Å from SC tetrahedral interactions at 3.7 Å. All these types of non-bonded interactions show minimum distances close to standard van der Waals' separations.Analysis of hydrogen-bond distances has been carried out by using standard geometry to place hydrogen atoms and measuring the XHO distances. On this basis, there are 130 intramolecular hydrogens: 111 NHO bonds, of which 69 are between main-chain atoms, 13 between side-chain atoms and 29 between mainchain and side-chain atoms. If a cluster of four well-defined internal water molecules is included in the protein structure, there is a total of 19 OHO hydrogen bonds. The mean NO, NHO distances and HN?O angles are 2.96 ± 0.17 Å, 2.05 ± 0.18 Å and 18.5 ± 9.6 °, and the mean OO, OHO distances and HÔO angles are 2.83 ± 0.19 Å, 1.98 ± 0.26 Å and 23.8 ± 13.4 °. The distances agree well with standard values, although the hydrogen bonds are consistently more non-linear than in equivalent small molecules. An analysis of the hydrogen-bond angles at the receptor atom indicates that the α-helix, β-sheet and reverse turn have characteristic angular values. A detailed analysis of the regularity of the α-helices and reverse turns shows small but consistent differences between the α-helices in lysozyme and the current standard model, which may now need revision. Of the 21 reverse turns that include a hydrogen bond, the conformations of 19 agree very closely with four of the five standard types. We conclude that the restrained least-squares method of refinement has been validated by these analyses.     

Structure of glycolate oxidase from spinach at a resolution of 5.5 Å

The crystal structure of glycolate oxidase from spinach has been determined to 5.5 Å resolution, using two isomorphous heavy-atom derivatives and their anomalous contributions. In the electron density map the boundaries of the octameric molecules are clearly seen. The subunit molecular weight is 37,000. Two protomers are in very close contact around one of the crystallographic 2-fold axes. Four such dimers are in contact around the 4-fold axis, so that the glycolate oxidase molecules are arranged as octamers with 422 symmetry in the crystal lattice. The roughly spherical octameric molecules have a diameter of approximately 100 Å. These octamers are arranged in a network, such that large solvent channels, approximately 60Å in diameter, pass right through the crystal lattice.The secondary structure of two-thirds of the subunit density has been interpreted in terms of eight consecutive β strand-α-helix units forming a cylinder very similar to the structure of triose phosphate isomerase. This interpretation is based on the very characteristic arrangement of the eight helices which form such a cylinder. The binding site of a substrate analogue, thioglycolate, has been localized in a deep cleft of the subunit at one end of the βα-barrel close to its axis.     

Structure of bovine pancreatic phospholipase A2 at 1.7 Å resolution

The crystal structure of bovine pancreatic phospholipase A2 has been refined to 1.7 Å resolution. The starting model for this refinement was the previously published structure at a resolution of 2.4 Å (Dijkstra et al., 1978). This model was adjusted to the multiple isomorphous replacement map with Diamond's real space refinement program (Diamond, 1971,1974) and subsequently refined using Agarwal's least-squares method (Agarwal, 1978). The final crystallographic R-factor is 17.1% and the estimated root-mean-square error in the positional parameters is 0.12 Å. The refined model allowed a detailed survey of the hydrogen-bonding pattern in the molecule. The essential calcium ion is located in the active site and is stabilized by one carboxyl group as well as by a peptide loop with many residues unvaried in all known phospholipase A2 sequences. Five of the oxygen ligands octahedrally surround the ion. The sixth octahedral position is shared between one of the carboxylate oxygens of Asp49 and a water molecule. The entrance to the active site is surrounded by residues involved in the binding of micelle substrates. The N-terminal region plays an important role here. Its α-NH₊³ group is buried and interacts with Gln4, the carbonyl oxygen of Asn71 and a fully enclosed water molecule, which provides a link between the N terminus and several active site residues. A total of 106 water molecules was located in the final structure, most of them in a two-layer shell around the protein molecule. The mobility in the structure was derived from the individual atomic temperature factors. Minimum mobility is found for the main chain atoms in the central part of the two long α-helices. The active site is rather rigid.     

Crystal structure analysis of sea lamprey hemoglobin at 2 Å resolution

The crystal structure of the predominant hemoglobin component of blood from the sea lamprey, Petromyzon marinus, has been determined by X-ray diffraction analysis. Crystals for this analysis were grown from cyanide methemoglobin V as crystal type D₂. These crystals are in space group P2₁2₁2₁ and have unit cell dimensions of $\text{a = 44.57}\text{A}\text{?}\text{, b = 96.62}\text{A}\text{?}\text{and}\text{c = 31.34}\text{A}\text{?}$. Isomorphous heavyatom derivatives were prepared by soaking crystals in solutions of Hg(CN)₂, K₂Hg(CNS)₄ and KAu(CN)₂. Diffracted intensities to as far as 2 Å spacings were measured on a diffractometer. Phases were found by means of the isomorphous replacements and anomalous scattering, with supplementary information provided by the tangent formula. An atomic model was fitted to the final electron density map in a Richards optical comparator. The lamprey hemoglobin molecule is generally similar in structure to other globins, but differs in many details. Each molecule is in contact with ten neighboring molecules in the crystal lattice. The nature of the binding of the heavy atoms to lamprey hemoglobin has been interpreted.     

Crystal Structure of Bovine Heart Cytochrome c Oxidase at 2.8 Å Resolution

Thirteen different polypeptide subunits, each in one copy, five phosphatidyl ethanolamines and three phosphatidyl glycerols, two hemes A, three Cu ions, one Mg ion, and one Zn ion are detectable in the crystal structure of bovine heart cytochrome c oxidase in the fully oxidized form at 2.8 Å resolution. A propionate of hems a, a peptide unit (–CO–NH–), and an imidazole bound to Cu_A are hydrogen-bonded sequentially, giving a facile electron transfer path from Cu_A to heme a. The O₂ binding and reduction site, heme a ₃, is 4.7 Å apart from Cu_B. Two possible proton transfer paths from the matrix side to the cytosolic side are located in subunit I, including hydrogen bonds and internal cavities likely to contain randomly oriented water molecules. Neither path includes the O₂ reduction site. The O₂ reduction site has a proton transfer path from the matrix side possibly for protons for producing water. The coordination geometry of Cu_B and the location of Tyr²⁴⁴ in subunit I at the end of the scalar proton path suggests a hydroperoxo species as the two electron reduced intermediate in the O₂ reduction process.     

Crystal structure of Turkey egg-white lysozyme: Results of the molecular replacement method at 5 Å resolution

Turkey egg-white lysozyme differs from hen egg-white lysozyme in its primary structure in 7 of the 129 residues. We have determined the rotational and translational parameters relating the known co-ordinates of hen egg-white lysozyme molecule to the turkey lysozyme. The rotational parameters were determined using the rotation function, the translational parameters were determined by placing the properly rotated molecule systematically at all positions within the unit cell and searching for those positions producing few intermolecular contacts between the α-carbon atoms of one molecule and all its neighbors. These parameters were refined by minimizing the conventional R factor between observed and calculated structure amplitudes. The final rotational and translational parameters give an R value of 46.7% for reflections with d spacings between 6 Å and 12 Å and have 7 intermolecular contacts closer than 5 Å between the a carbon atoms of one molecule and all its neighbors. An electron density map has been calculated at 5 Å resolution; the packing of the molecules in this form appears to present the entire length of the active cleft in the vicinity of the crystallographic 6-fold axis and does not appear to be blocked by neighboring molecules.     

Structure of the Drosophila apoptosome at 6.9 å resolution

The Drosophila Apaf-1 related killer forms an apoptosome in the intrinsic cell death pathway. In this study we show that Dark forms a single ring when initiator procaspases are bound. This Dark-Dronc complex cleaves DrICE efficiently; hence, a single ring represents the Drosophila apoptosome. We then determined the 3D structure of a double ring at ～6.9?? resolution and created a model of the apoptosome. Subunit interactions in the Dark complex are similar to those in Apaf-1 and CED-4 apoptosomes, but there are significant differences. In particular, Dark has "lost" a loop in the nucleotide-binding pocket, which opens a path for possible dATP exchange in the apoptosome. In addition, caspase recruitment domains (CARDs) form a crown on the central hub of the Dark apoptosome. This CARD geometry suggests that conformational changes will be required to form active Dark-Dronc complexes. When taken together, these data provide insights into apoptosome structure, function, and evolution.     

Structure of satellite tobacco necrosis virus after crystallographic refinement at 2.5 Å resolution

The structure of the protein subunit of satellite tobacco necrosis virus has been solved at 3.7 Å resolution. We have now crystallographically refined the original model and extended the resolution to 2.5 Å in order to get a model accurate enough to explain the details of the subunit interactions. The refinement was done with a novel method utilizing the icosahedral symmetry of the virus particle.The final model shows a complicated network of interactions, involving salt linkages, hydrogen bonds and hydrophobic contacts. In addition, we have located three different metal ion sites in the protein shell, linking the protein subunits together. These sites are probably occupied by calcium ions. One site is found in a general position near the icosahedral 3-fold axis of the virus. The ligands form an octahedral arrangement, with two main chain carbonyl oxygens (O-61 and O-64), one carboxylate oxygen (OD1 from Asp194) of the same subunit and a second carboxylate oxygen (OE1 of Glu25) from a 3-fold related subunit. Two water molecules complete the octahedral arrangement. A second site is on the icosahedral 3-fold axis and is liganded by the carboxylate oxygens of the 3-fold related Asp55 residues. The third metal ion site is found on the 5-fold axis, liganded by the five carbonyl oxygens of Thr138 and two water molecules.We are unable to locate the first 11 N-terminal amino acid residues, which point into the virus interior. No interpretable density for RNA has been found, indicating that the nucleic acid of the virus does not have a unique orientation in the crystal.     

Crystal structure of bullfrog M ferritin at 2.8 Å resolution: analysis of subunit interactions and the binuclear metal center

Ferritins concentrate and store iron as a mineral in all bacterial, plant, and animal cells. The two ferritin subunit types, H or M (fast) and L (slow), differ in rates of iron uptake and mineralization and assemble in vivo to form heteropolymeric protein shells made up of 24 subunits; H/L subunit ratios reflect cell specificity of H and L subunit gene expression. A diferric peroxo species that is the initial reaction product of Fe(II) in H-type ferritins, as well as in ribonucleotide reductase (R2) and methane monooxygenase hydroxylase (MMOH), has recently been characterized, exploiting the relatively high accumulation of the peroxo intermediate in frog H-subunit type recombinant ferritin with the M sequence. The stability of the diferric reaction centers in R2 and MMOH contrasts with the instability of diferric centers in ferritin, which are precursors of the ferric mineral. We have determined the crystal structure of the homopolymer of recombinant frog M ferritin in two crystal forms: P4(1)2(1)2, a = b = 170.0 A and c = 481.5 A; and P3(1)21, a = b = 210.8 A and c = 328.1 A. The structural model for the trigonal form was refined to a crystallographic R value of 19.0% (Rfree = 19.4%); the two structures have an r.m.s.d. of approximately 0.22 A for all C alpha atoms. Comparison with the previously determined crystal structure of frog L ferritin indicates that the subunit interface at the molecular twofold axes is most variable, which may relate to the presence of the ferroxidase site in H-type ferritin subunits. Two metal ions (Mg) from the crystallization buffer were found in the ferroxidase site of the M ferritin crystals and interact with Glu23, Glu58, His61, Glu103, Gln137 and, unique to the M subunit, Asp140. The data suggest that Gln137 and Asp140 are a vestige of the second GluxxHis site, resulting from single nucleotide mutations of Glu and His codons and giving rise to Ala140 or Ser140 present in other eukaryotic H-type ferritins, by additional single nucleotide mutations. The observation of the Gln137xxAsp140 site in the frog M ferritin accounts for both the instability of the diferric oxy complexes in ferritin compared to MMOH and R2 and the observed kinetic variability of the diferric peroxo species in different H-type ferritin sequences.     

Three-dimensional structure of deoxycholate-treated purple membrane at 6 Å resolution and molecular averaging of three crystal forms of bacteriorhodopsin

The three-dimensional structure of the deoxycholate-treated form of purple membrane has been determined to a resolution of about 6 Å. Using low temperature electron diffraction data, room temperature electron microscope images and improved methods of data analysis, higher resolution has been reached than was obtained using native membranes of the same size. Statistical analysis of the data shows that the new map is considerably better than earlier maps. The map indicates the probable sites for the lipid molecules that remain in the deoxycholate-treated membranes; some of these sites differ from those suggested by the projection map of Glaeser et al. (1985). Comparison of the bacteriorhodopsin structures now determined independently from three crystal forms shows that the monomer structure is independent of the detailed contacts with lipid molecules. The average of the three structures gives a picture with very little noise showing seven similar rod-like features which are clearly best interpreted as -helices; there is no indication that part of the structure is -sheet as suggested by Jap et al. (1983). Phases from the averaged structure at 6 Å resolution will enable better refinement of the parameters that will be required in the analysis of higher resolution images from tilted specimens needed to extend the projection map at 3.5 Å resolution (Henderson et al. 1986) to produce a three-dimensional atomic resolution map.     

Improving the accuracy of macromolecular structure refinement at 7 Å resolution

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Highlights? Improved models by DEN refinement at 7 Å ? Best method is DEN refinement with initial segmented rigid-body refinement ? R_free has predictive power at 7 Å     

Structure of a triclinic ternary complex of horse liver alcohol dehydrogenase at 2.9 Å resolution

The structure of a triclinic complex between liver alcohol dehydrogenase, reduced coenzyme NADH, and the inhibitor dimethylsulfoxide has been determined to 2.9 Å resolution using isomorphous replacement methods. The heavy-atom positions were derived by molecular replacement methods using phase angles derived from a model of the orthorhombic apoenzyme structure previously determined to 2.4 Å resolution. A model of the present holoenzyme molecule was built on a Vector General 3400 display system using the RING system of programs. This model gave a crystallographic R-value of 37.9%.There are extensive conformational differences between the protein molecules in the two forms. The conformational change involves a rotation of 7.5 ° of the catalytic domains relative to the coenzyme binding domains. A hinge region for this rotation is defined within a hydrophobic core between two helices. The internal structures of the domains are preserved with the exception of a movement of a small loop in the coenzyme binding domain. A cleft between the domains is closed by this coenzyme-induced conformational change, making the active site less accessible from solution and thus more hydrophobic.The two crystallographically independent subunits are very similar and bind both coenzyme and inhibitor in an identical way within the present limits of error. The coenzyme molecule is bound in an extended conformation with the two ends in hydrophobic crevices on opposite sides of the central pleated sheet of the coenzyme binding domain. There are hydrogen bonds to oxygen atoms of the ribose moities from Asp223, Lys228 and His51. The pyrophosphate group is in contact with the side-chains of Arg47 and Arg369.No new residues are brought into the active site compared to the apoenzyme structure. The active site zinc atom is close to the hinge region, where the smallest structural changes occur. Small differences in the co-ordination geometry of the ligands Cys46, His67 and Cysl74 are not excluded and may account for the ordered mechanism. The oxygen atom of the inhibitor dimethylsulfoxide is bound directly to zinc confirming the structural basis for the suggested mechanism of action based on studies of the apoenzyme structure.     

Crystallographic refinement and atomic models of the intact immunoglobulin molecule Kol and its antigen-binding fragment at 3.0 Å and 1.9 Å resolution

The crystal structures of the intact immunoglobulin G1, (λ) Kol and its Fab² fragment were crystallographically refined at 3.0 Å and 1.9 Å resolution, respectively. The methods used were real space refinement (RLSP) energy and residual refinement (EREF), phase combination, constrained rigid body refinement (CORELS) and difference and Fourier map inspection. The final R-values are 0.24 and 0.26. These analyses allowed the construction of atomic models of parts not seen in detail in the previous analyses at 5 Å and 3 Å resolution, respectively (Colman et al., 1976; Matsushima et al., 1978): i.e. the hinge segment, the hypervariable segments and their intimate interaction with the hinge segment of a crystallographically related molecule.The hinge segment forms a short poly-l-proline double helix from Cys527 to Cys530 (Eu numbering 226 to 230). The preceding segment forms an open turn of helix. This segment and the segment following the poly-l-proline part, which was found to be flexible in Fc fragment crystals (Deisenhofer et al., 1976) probably allow arm and stem movement of the antibody molecule. The combining site of Kol is compared with the combining site of Fab New (Saul et al., 1978). The narrow cleft formed by the hypervariable loops in Kol is filled with aromatic amino acid side-chains. In the crystal, the hypervariable loops contact the hinge and adjacent segments of a related molecule accompanied by a substantial loss in accessible surface area. This contact is preserved in Kol Fab crystals and presumably occurs in the Kol cryoprecipitate. A comparison of the quaternary structures of intact Kol and Fab New showed, in addition to the large change in elbow angle (Colman et al., 1976), changes in lateral domain association. These are discussed in the context of a possible signal transmission from the combining site to the distal end. An attempt was made to model build the IgG3 hinge segment, which is quadruplicated with respect to IgG1 (Michaelsen et al., 1977), on the basis of the Kol hinge structure. A polyproline double helix appeared to be the most plausible model. The Fc part was found to be disordered in intact Kol crystals (Colman et al., 1976). Refinement has reduced the electron density further in the crystal space, where the Fc parts must be located. Disorder, if static, must be fourfold or more in the crystalline state.Intensity measurements on Kol F(ab′)₂ and their comparison with intact Kol crystals provide evidence that the disorder is predominantly of a static nature.