The molecular structure of insecticyanin from the tobacco hornworm Manduca sexta L. at 2.6 A resolution.

Insecticyanin, a blue biliprotein isolated from the tobacco hornworm Manduca sexta L., is involved in insect camouflage. Its three-dimensional structure has now been solved to 2.6 A resolution using the techniques of multiple isomorphous replacement, non-crystallographic symmetry averaging about a local 2-fold rotation axis and solvent flattening. All 189 amino acids have been fitted to the electron density map. The map clearly shows that insecticyanin is a tetramer with one of its molecular 2-fold axes coincident to a crystallographic dyad. The individual subunits have overall dimensions of 44 A X 37 A X 40 A and consist primarily of an eight-stranded anti-parallel beta-barrel flanked on one side by a 4.5-turn alpha-helix. Interestingly the overall three-dimensional fold of the insecticyanin subunit shows remarkable similarity to the structural motifs of bovine beta-lactoglobulin and the human serum retinol-binding protein. The electron density attributable to the chromophore is unambiguous and shows that it is indeed the gamma-isomer of biliverdin. The biliverdin lies towards the open end of the beta-barrel with its two propionate side chains pointing towards the solvent and it adopts a rather folded conformation, much like a heme.     

Using cryoEM Reconstruction and Phase Extension to Determine Crystal Structure of Bacteriophage ϕ6 Major Capsid Protein

Bacteriophage ?6 is a double-stranded RNA virus that has been extensively studied as a model organism. Here we describe structure determination of ?6 major capsid protein P1. The protein crystallized in base centered orthorhombic space group C222₁. Matthews’s coefficient indicated that the crystals contain from four to seven P1 subunits in the crystallographic asymmetric unit. The self-rotation function had shown presence of fivefold axes of non-crystallographic symmetry in the crystals. Thus, electron density map corresponding to a P1 pentamer was excised from a previously determined cryoEM reconstruction of the ?6 procapsid at 7 Å resolution and used as a model for molecular replacement. The phases for reflections at higher than 7 Å resolution were obtained by phase extension employing the fivefold non-crystallographic symmetry present in the crystal. The averaged 3.6 Å-resolution electron density map was of sufficient quality to allow model building.     

Electron microscopy of the stacked disk aggregate of tobacco mosaic virus protein. I. Three-dimensional image reconstruction

The three-dimensional structure of the stacked disk aggregate of tobacco mosaic virus protein has been determined from “phase plate” electron micrographs to an effective resolution of about 12 Å. It is a long rod comprised of paired rings of protein (disks), the subunits of which have different conformations according to which ring they belong. The two subunit conformations are such that the rings come close together within a disk near the outer surface of the particle, but between disks on the inside. This property, interpreted on the basis of a polar packing of the subunits, was established from an earlier, lower resolution, study by Finch &; Klug (1971). The present study shows, in addition, that the pairing is contributed mainly by axial distortions of the subunits in one of the rings, the axial distortions of the subunits in the other being largely replaced at lower radii by a tilt or twist and, at higher radii, by a slew. The subunits in the latter ring appear to have a conformation similar to that of the protein molecules in the virus.     

Three-dimensional structure of physalis mottle virus: implications for the viral assembly.

The structure of the T=3 single stranded RNA tymovirus, physalis mottle virus (PhMV), has been determined to 3.8 A resolution. PhMV crystals belong to the rhombohedral space group R 3, with one icosahedral particle in the unit cell leading to 20-fold non-crystallographic redundancy. Polyalanine coordinates of the related turnip yellow mosaic virus (TYMV) with which PhMV coat protein shares 32 % amino acid sequence identity were used for obtaining the initial phases. Extensive phase refinement by real space molecular replacement density averaging resulted in an electron density map that revealed density for most of the side-chains and for the 17 residues ordered in PhMV, but not seen in TYMV, at the N terminus of the A subunits. The core secondary and tertiary structures of the subunits have a topology consistent with the capsid proteins of other T=3 plant viruses. The N-terminal arms of the A subunits, which constitute 12 pentamers at the icosahedral 5-fold axes, have a conformation very different from the conformations observed in B and C subunits that constitute hexameric capsomers with near 6-fold symmetry at the icosahedral 3-fold axes. An analysis of the interfacial contacts between protein subunits indicates that the hexamers are held more strongly than pentamers and hexamer-hexamer contacts are more extensive than pentamer-hexamer contacts. These observations suggest a plausible mechanism for the formation of empty capsids, which might be initiated by a change in the conformation of the N-terminal arm of the A subunits. The structure also provides insights into immunological and mutagenesis results. Comparison of PhMV with the sobemovirus, sesbania mosaic virus reveals striking similarities in the overall tertiary fold of the coat protein although the capsid morphologies of these two viruses are very different.     

Structure determination of crystalline lobster D-glyceraldehyde-3-phosphate dehydrogenase

Single crystal X-ray data were collected on film for the holoenzyme of lobster d-glyceraldehyde-3-phosphate dehydrogenase to 3·0 Å resolution. Films of potassium tetraiodomercurate, K₂HgI₄, comprising a complete low resolution set, with some additional high resolution terms, were given to us by Drs H. C. Watson and L. J. Banaszak. A 3·0 Å high resolution data set was collected of a p-chloromercuri-phenylsulfonate derivative. All these films were processed on a computer controlled Optronics film scanner. The K₂HgI₄ derivative difference Patterson was initially interpreted in terms of four single sites, one for each polypeptide chain, consistent with the previously determined molecular 222 symmetry. Single isomorphous replacement phases were then sufficient to identify other heavy atom sites. Least-squares refined parameters were used to give multiple isomorphous replacement phases at low resolution, and single isomorphous replacement phases at high resolution. The resultant electron density map was oriented along the molecular 2-fold axes and then averaged over all four equivalent subunits. This process produced a much improved electron density map, which could easily be interpreted in terms of a single polypeptide chain per subunit consistent with the known amino acid sequence. The use of non-crystallographic symmetry to improve the electron density map is equivalent to the molecular replacement method. A comparison is also made with other dehydrogenases.     

Three-dimensional structure of ferricytochrome c' from Rhodospirillum rubrum at 2.8 A resolution.

The structure of ferricytochrome c' extracted from Rhodospirillum rubrum has been determined by the X-ray crystallographic method. Crystals in hexagonal space group P6(1), with unit-cell dimensions a = b = 51.72 A and c = 155.49 A, contain one dimer molecule composed of chemically identical polypeptide chains (monomer I and monomer II) per asymmetric unit. An electron density map has been calculated at a resolution of 2.8 A by the multiple isomorphous replacement method using four-circle diffractometer data from native crystals and two heavy-atom derivatives. The quality of the map was improved by averaging the electron density about the non-crystallographic 2-fold axis relating the two monomers. The initial three-dimensional model of monomer I was built on a computer graphics system and that of monomer II was derived from monomer I using the non-crystallographic symmetry matrices. The dimer structure has been refined using a combination of simulated annealing and conventional restrained least-squares crystallographic refinement. The current model includes 244 amino acid residues (122 x 2) and 2 hemes, with a root-mean-square deviation in bond lengths from ideal values of 0.022 A. The current crystallographic R-factor is 23.3% for 4,481 independent reflections [magnitude of Fo greater than or equal to sigma (F)] between 5.0 and 2.8 A resolution. The monomer molecule is structurally organized as an array of four nearly parallel alpha-helices which construct a left-twisted bundle. One end of the bundle, in which a covalently bound protoheme IX prosthetic group is incorporated, is more divergent than the other.(ABSTRACT TRUNCATED AT 250 WORDS)     

Crystal structure of muconate lactonizing enzyme at 6.5 A resolution

We have obtained crystals of Pseudomonas putida muconate lactonizing enzyme. They diffract to better than 2.4 A resolution and have two monomers in the asymmetric unit, related by a non-crystallographic 2-fold axis. The cell dimensions are 139.3 A X 139.3 A X 84.1 A, and the space group is I4. The electron density map at 6.5 A resolution shows that the enzyme is an octamer with D4 symmetry.     

Structure of yeast hexokinase. II. A 6 angstrom resolution electron density map showing molecular shape and heterologous interaction of subunits

An electron density map of yeast hexokinase has been calculated at 6 Å resolution using six heavy atom derivatives. The map shows each of the enzyme's two 51,000 molecular weight subunits to consist of two separate lobes connected by a narrow bridge of density. Furthermore, these two subunits are related to each other in the asymmetric unit of the crystal by a quasi-2-fold rather than a true 2-fold axis. That is, they are related by a rotation of 180 ° plus a relative translation of 3.6 Å along the symmetry axis. This gives rise to a heterologous subunit interaction and a possibility of non-identical structure and function for these chemically identical subunits. The molecule is quite asymmetric, having dimensions of 150 Å × 45 Å × 55 Å. Each subunit is about 80 Å × 40 Å × 50 Å.A portion of an electron density map at 3 Å resolution has been also calculated, based on phases from two heavy atom derivatives. Polypeptide backbone and side chains are visible in this map.     

Protein-RNA interactions during TMV assembly

A review of the structural studies of tobacco mosaic virus (TMV) is given. TMV is essentially a flat helical microcrystal with 16 1/3 subunits per turn. A single strand of RNA runs along the helix and is deeply embedded in the protein. The virus particles form oriented gels from which high-resolution X-ray fiber diffraction data can be obtained. This may be interpreted by the use of six heavy-atom derivatives to give an electron density map at 0.4 nm resolution from which the RNA configuration and the form of the inner part of the protein subunit may be determined. In addition, the protein subunits form a stable 17-fold two-layered disk which is involved in virus assembly and which crystallizes. By the use of noncrystallographic symmetry and a single heavy-atom derivative, it has been possible to solve the structure of the double disk to 0.28 nm resolution. In this structure one sees that an important structural role is played by four alpha-helices, one of which (the LR helix) appears to form the main binding site for the RNA. The main components of the binding site appear to be hydrophobic interactions with the bases, hydrogen bonds between aspartate groups and the sugars, and arginine salt bridges to the phosphate groups. The binding site is between two turns of the virus helix or between the turns of the double disk. In the disk, the region proximal to the RNA binding site is in a random coil until the RNA binds, whereupon the 24 residues involved build a well-defined structure, thereby encapsulating the RNA.     

Sequence information within the lamB genes in required for proper routing of the bacteriophage lambda receptor protein to the outer membrane of Escherichia coli K-12

The structure of Satellite tobacco necrosis virus (STNV) has been determined to 3.0 Å resolution by X-ray crystallography. Electron density maps were obtained with phases based on one heavy-atom derivative and several cycles of phase refinement using the 60-fold non-crystallographic symmetry in the particle. A model for one protein subunit was built using a computer graphics display. The subunit is constructed mainly of a β-roll structure forming two β-sheets, each of four antiparallel strands. The N-termini of the subunits form bundles of three α-helices extending into the RNA region of the virus at the 3-fold axis. The topology of the polypeptide chain is the same as, and the conformation clearly similar to, that of the shell domains of the Tomato bushy stunt virus (TBSV) and Southern bean mosaic virus (SBMV) protein subunits. The subunit packing in the T = 1 STNV structure is, however, significantly different from the packing of these T = 3 viruses: parts of some of the structural elements facing the RNA in TBSV and SBMV are utilized for subunit-subunit contacts in STNV. No RNA structure is obvious in the present icosahedrally averaged electron density maps. The protein surface facing the RNA contains mainly hydrophilic residues, especially lysine and arginine.     

Twinning in crystals of human skeletal muscle d-glyceraldehyde-3-phosphate dehydrogenase

The coenzyme-bound form of human skeletal muscle d-glyceraldehyde-3-phosphate dehydrogenase has been shown to crystallize in the space group C2 and not C222₁ as previously reported. The unit cell contains two tetrameric molecules with the dimer of molecular weight 72,000 as the crystallographic asymmetric unit. The recorded X-ray intensity distribution clearly indicates the presence of non-crystallographic 2-fold axes perpendicular to the crystallographic 2-fold axis showing that the subunits are arranged with near perfect 222 symmetry.Isomorphous derivatives of the enzyme have been prepared and the heavy atom positions defined in complete agreement with the C2 space group assignment. Further confirmation that the space group is C2 and not C222₁ comes from the 3.5 Å resolution electron density map of the human enzyme, which appears almost identical to that of the lobster holo-enzyme where no such space group ambiguity exists.     

Structure and assembly of turnip crinkle virus. I. X-ray crystallographic structure analysis at 3.2 A resolution

The structure of turnip crinkle virus has been determined at 3.2 A resolution, using the electron density of tomato bushy stunt virus as a starting point for phase refinement by non-crystallographic symmetry. The structures are very closely related, especially in the subunit arm and S domain, where only small insertions and deletions and small co-ordinate shifts relate one chain to another. The P domains, although quite similar in fold, are oriented somewhat differently with respect to the S domains. Understanding of the structure of turnip crinkle virus has been important for analyzing its assembly, as described in an accompanying paper.     

Refinement of the influenza virus hemagglutinin by simulated annealing

We have applied the method of simulated annealing to the refinement of the 3 A resolution crystal structure of the influenza virus hemagglutinin glycoprotein, using the program X-PLOR. Two different methods were introduced into X-PLOR to treat the non-crystallographic symmetry present in this and in other crystal structures. In the first, only the unique protomer atoms are refined; by application of the non-crystallographic symmetry operators to the protomer atoms, the X-ray structure factor derivatives are effectively averaged, and a non-bonded energy term models the interactions of the protomer with its neighbors in the oligomer without explicit refinement of the other protomers in the crystallographic asymmetric unit. In the second method, the entire asymmetric unit is refined, but an effective energy term is added to the empirical energy that restrains symmetry-related atomic positions to their average values after least-squares superposition. Several other modifications and additions were made to previously published X-PLOR protocols, including weighting of the X-ray terms, maintenance of the temperature of the molecular dynamics simulation, treatment of charged groups, changes in the values of certain empirical energy parameters, and the use of N-linked carbohydrate empirical energy parameters. The hemagglutinin refinement proceeded in several stages. An initial round of simulated annealing of the monomer was followed by rigid-body refinement of the 3-fold non-crystallographic symmetry axis position and a second round of monomer refinement. A third round was performed on the trimer using non-crystallographic symmetry restraints in all regions except those in lattice contacts showing obvious derivations from 3-fold symmetry. The refinement was completed with several rounds of conventional positional and isotropic temperature factor refinement needed to correct bad model geometry introduced by high-temperature molecular dynamics in regions of weak electron density. This structure was then used as the basis for refinement of three crystallographically isomorphous hemagglutinin structures, including complexes with the influenza virus receptor, sialic acid. Model geometry comparable to well-refined high-resolution structures was obtained with relatively little manual intervention, demonstrating the ability of simulated annealing refinement to produce highly idealized structures at moderate resolution.     

Low resolution crystal structure of muconolactone isomerase. A decamer with a 5-fold symmetry axis

Muconolactone isomerase from Pseudomonas putida crystallizes from sodium sulfate solution in space group P2(1) (a = 65.84 A, b = 105.70 A, c = 77.20 A, beta = 90.5 degrees) with ten 11,000 Mr subunits per asymmetric unit. The 7 A resolution crystal structure was solved by single isomorphous replacement followed by 10-fold symmetry averaging. The decameric enzyme has an uncommon non-crystallographic 5-fold symmetry axis and a large cavity in its center.     

Finding and using local symmetry in identifying lower domain movements in hexon subunits of the herpes simplex virus type 1 B capsid

A characteristic of virus assembly is the use of symmetry to construct a complex capsid from a limited number of different proteins. Many spherical viruses display not only icosahedral symmetry, but also local symmetries, which further increase the redundancy of their structural proteins. We have developed a computational procedure for evaluating the quality of these local symmetries that allows us to probe the extent of local structural variations among subunits. This type of analysis can also provide orientation parameters for carrying out non-icosahedral averaging of quasi-equivalent subunits during three-dimensional structural determination. We have used this procedure to analyze the three types of hexon (P, E and C) in the 8.5 A resolution map of the herpes simplex virus type 1 (HSV-1) B capsid, determined by electron cryomicroscopy. The comparison of the three hexons showed that they have good overall 6-fold symmetry and are almost identical throughout most of their lengths. The largest difference among the three lies near the inner surface in a region of about 34 A in thickness. In this region, the P hexon displays slightly lower 6-fold symmetry than the C and E hexons. More detailed analysis showed that parts of two of the P hexon subunits are displaced counterclockwise with respect to their expected 6-fold positions. The most highly displaced subunit interacts with a subunit from an adjacent P hexon (P'). Using the local 6-fold symmetry axis of the P hexon as a rotation axis, we examined the geometrical relationships among the local symmetry axes of the surrounding capsomeres. Deviations from exact symmetry are also found among these local symmetry axes. The relevance of these findings to the process of capsid assembly is considered.     

Three-dimensional structure of infectious bursal disease virus determined by electron cryomicroscopy.

Infectious bursal disease virus (IBDV), a member of the Birnaviridae group, is a commercially important pathogen of chickens. From electron micrographs of frozen, hydrated, unstained specimens, we have computed a three-dimensional map of IBDV at about 2 nm resolution. The map shows that the structure of the virus is based on a T=13 lattice and that the subunits are predominantly trimer clustered. The subunits close to the fivefold symmetry axes are at a larger radius than those close to the two- or threefold axes, giving the capsid a markedly nonspherical shape. The trimer units on the outer surface protrude from a continuous shell of density. On the inner surface, the trimers appear as Y-shaped units, but the set of units surrounding the fivefold axes appears to be missing. It is likely that the outer trimers correspond to the protein VP2, carrying the dominant neutralizing epitope, and the inner trimers correspond to protein VP3, which has a basic carboxy-terminal tail expected to interact with the packaged RNA.     

X-ray analysis of the disk of tobacco mosaic virus protein. II. The packing arrangement in the crystal

By a combined X-ray and electron microscopic analysis, the asymmetric unit of the crystal of tobacco mosaic virus protein has been identified with the disk aggregate, of molecular weight 600,000, composed of two rings each containing 17 subunits. The packing of the disks in the crystal has been determined, and consists of an approximately body-centred array of stacks comprising two disks each.     

The polypeptide chain fold in tyrosine phenol-lyase, a pyridoxal-5'-phosphate-dependent enzyme.

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 P2(1)2(1)2. The unit cell has dimensions a = 76.0 A, b = 138.3 A, c = 93.5 A 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 scanner. The structure has been solved at 2.7 A 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.     

Crystallographic analysis of ribulose 1,5-bisphosphate carboxylase from spinach at 2.4 A resolution. Subunit interactions and active site

The X-ray structure of the quaternary complex of ribulose 1,5-bisphosphate carboxylase/oxygenase from spinach with CO2, Mg2+ and a reaction-intermediate analogue (CABP) has been determined and refined at 2.4 A resolution. Cyclic non-crystallographic symmetry averaging around the molecular 4-fold axis and phase combination were used to improve the initial multiple isomorphous replacement phases. A model composed of one large subunit and one small subunit was built in the resulting electron density map, which was of excellent quality. Application of the local symmetry gave an initial model of the L8S8 molecule with a crystallographic R-value of 0.43. Refinement of this initial model was performed by a combination of conventional least-squares energy refinement and molecular dynamics simulation using the XPLOR program. Three rounds of refinement, interspersed with manual rebuilding at the graphics display, resulted in a model containing all of the 123 amino acid residues in the small subunit, and 467 of the 475 residues in the large subunit. The R-value for this model is 0.24, with relatively small deviations from ideal stereochemistry. Subunit interactions in the L8S8 molecule have been analysed and are described. The interface areas between the subunits are extensive, and bury almost half of the accessible surface areas of both the large and the small subunit. A number of conserved interaction areas that may be of functional significance have been identified and are described, and biochemical and mutagenesis data are discussed in the structural framework of the model.     

Structure of quinoprotein methylamine dehydrogenase at 2.25 A resolution.

The three-dimensional structure of quinoprotein methylamine dehydrogenase from Thiobacillus versutus has been determined at 2.25 A resolution by a combination of multiple isomorphous replacement, phase extension by solvent flattening and partial structure phasing using molecular dynamics refinement. In the resulting map, the polypeptide chain for both subunits could be followed and an X-ray sequence was established. The tetrameric enzyme, made up of two heavy (H) and two light (L) subunits, is a flat parallellepiped with overall dimensions of approximately 76 x 61 x 45 A. The H subunit, comprising 370 residues, is made up of two distinct segments: the first 31 residues form an extension which embraces one of the L subunits; the remaining residues are found in a disc-shaped domain. This domain is formed by a circular arrangement of seven topologically identical four-stranded antiparallel beta-sheets, with approximately 7-fold symmetry. In spite of distinct differences, this arrangement is reminiscent of the structure found in influenza virus neuraminidase. The L subunit consists of 121 residues, out of which 53 form a beta-sheet scaffold of a central three-stranded antiparallel sheet flanked by two shorter two-stranded antiparallel sheets. The remaining residues are found in segments of irregular structure. This subunit is stabilized by six disulphide bridges, plus two covalent bridges involving the quinone co-factor and residues 57 and 107 of this subunit. The active site is located in a channel at the interface region between the H and L subunits, and the electron density in this part of the molecule suggests that the co-factor of this enzyme is not pyrrolo quinoline quinone (PQQ) itself, but might be instead a precursor of PQQ.