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.     

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.     

X-ray analysis of the disk of tobacco mosaic virus protein. 3. A low resolution electron density map

The structure of the disk of tobacco mosaic virus protein at low resolution has been determined by X-ray crystal analysis. Signs for the three principal projections were found by isomorphous replacement, using a mercury derivative. The heavy-atom positions were located by interpretation of difference Patterson maps on the basis of the non-crystallographic 17-fold rotational symmetry of the disk, and of the packing of the disks determined in the preceding paper (Finch et al., 1974).The electron density in the corresponding three projections was computed to a resolution of 6 Å. From the projections in the [100] and [010] directions, which are at right-angles to the 17-fold rotation axis, a three-dimensional electron density map has been calculated making use of the non-crystallographic symmetry. The procedure is similar to the three-dimensional image reconstruction technique used in electron microscopy.The map indicates that the subunits in the two rings face the same way and, hence, that the disk is polar. There are differences between the subunits in the two rings at high radius, which are presumably a consequence of the pairing interaction responsible for stabilizing the two-layer polar structure. The map has been compared with a low-resolution map of the intact virus, and certain common features can be identified, notably the site of the nucleic acid.     

Projection structure and oligomeric state of the osmoregulated sodium/glycine betaine symporter BetP of Corynebacterium glutamicum

The high-affinity glycine betaine uptake system BetP, an osmosensing and osmoregulated sodium-coupled symporter from Corynebacterium glutamicum, was overexpressed in Escherichia coli with an N-terminal StrepII-tag, solubilized in beta-dodecylmaltoside and purified by streptactin affinity chromatography. Analytical ultracentrifugation indicated that BetP forms trimers in detergent solution. Detergent-solubilized BetP can be reconstituted into proteoliposomes without loss of function, suggesting that BetP is a trimer in the bacterial membrane. Reconstitution with E.coli polar lipids produced 2D crystals with unit cell parameters of 182A x 154A, gamma=90 degrees exhibiting p22(1)2(1) symmetry. Electron cryo-microscopy yielded a projection map at 7.5A. The unit cell contains four non-crystallographic trimers of BetP. Within each monomer, ten to 12 density peaks characteristic of transmembrane alpha-helices surround low-density regions that define potential transport pathways. Small but significant differences between the three monomers indicate that the trimer does not have exact 3-fold symmetry. The observed differences may be due to crystal packing, or they may reflect different functional states of the transporter, related to osmosensing and osmoregulation. The projection map of BetP shows no clear resemblance to other secondary transporters of known structure.     

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.     

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)     

Projection structure of channelrhodopsin-2 at 6 Å resolution by electron crystallography

Channelrhodopsin-2 (ChR2) is the prototype of a new class of light-gated ion channels that is finding widespread applications in optogenetics and biomedical research. We present a  6-Å projection map of ChR2, obtained by cryo-electron microscopy of two-dimensional crystals grown from pure, heterologously expressed protein. The map shows that ChR2 is the same dimer with non-crystallographic 2-fold symmetry in three different membrane crystals. This is consistent with biochemical analysis, which shows a stable dimer in detergent solution. Comparison to the projection map to bacteriorhodopsin indicates a similar structure of seven transmembrane alpha helices. Based on the projection map and sequence alignments, we built a homology model of ChR2 that potentially accounts for light-induced channel gating. Although a monomeric channel is not ruled out, comparison to other membrane channels and transporters suggests that the ChR2 channel is located at the dimer interface on the 2-fold axis, lined by transmembrane helices 3 and 4.     

Placement of the structural proteins in Sindbis virus

The structure of the lipid-enveloped Sindbis virus has been determined by fitting atomic resolution crystallographic structures of component proteins into an 11-A resolution cryoelectron microscopy map. The virus has T=4 quasisymmetry elements that are accurately maintained between the external glycoproteins, the transmembrane helical region, and the internal nucleocapsid core. The crystal structure of the E1 glycoprotein was fitted into the cryoelectron microscopy density, in part by using the known carbohydrate positions as restraints. A difference map showed that the E2 glycoprotein was shaped similarly to E1, suggesting a possible common evolutionary origin for these two glycoproteins. The structure shows that the E2 glycoprotein would have to move away from the center of the trimeric spike in order to expose enough viral membrane surface to permit fusion with the cellular membrane during the initial stages of host infection. The well-resolved E1-E2 transmembrane regions form alpha-helical coiled coils that were consistent with T=4 symmetry. The known structure of the capsid protein was fitted into the density corresponding to the nucleocapsid, revising the structure published earlier.     

X-ray structure of lipoamide dehydrogenase from Azotobacter vinelandii determined by a combination of molecular and isomorphous replacement techniques

The crystal structure of lipoamide dehydrogenase from Azotobacter vinelandii has been determined by a combination of molecular replacement and isomorphous replacement techniques yielding eventually a good-quality 2.8 A electron density map. Initially, the structure determination was attempted by molecular replacement procedures alone using a model of human glutathione reductase, which has 26% sequence identity with this bacterial dehydrogenase. The rotation function yielded the correct orientation of the model structure both when the glutathione reductase dimer and monomer were used as starting model. The translation function could not be solved, however. Consequently, data for two heavy-atom derivatives were collected using the Hamburg synchotron facilities. The derivatives had several sites in common, which was presumably a major reason why the electron density map obtained by isomorphous information alone was of poor quality. Application of solvent flattening procedures cleaned up the map considerably, however, showing clearly the outline of the lipoamide dehydrogenase dimer, which has a molecular weight of 100,000. Application of the "phased translation function", which combines the phase information of both isomorphous and molecular replacement, led to an unambiguous determination of the position of the model structure in the lipoamide dehydrogenase unit cell. The non-crystallographic 2-fold axis of the dimer was optimized by several cycles of constrained-restrained least-squares refinement and subsequently used for phase improvement by 2-fold density averaging. After ten cycles at 3.5 A, the resolution was gradually extended to 2.8 A in another 140 cycles. The 2.8 A electron density distribution obtained in this manner was of much improved quality and allowed building of an atomic model of A. vinelandii lipoamide dehydrogenase. It appears that in the orthorhombic crystals used each dimer is involved in contacts with eight surrounding dimers, leaving unexplained why the crystals are rather fragile. Contacts between subunits within one dimer, which are quite extensive, can be divided into two regions separated by a cavity. In one of the contact regions, the level of sequence identity with glutathione reductase is very low but it is quite high in the other. The folding of the polypeptide chain in each subunit is quite similar to that of glutathione reductase, as is the extended conformation of the co-enzyme FAD.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Structure of a ternary complex of an allosteric lactate dehydrogenase from Bacillus stearothermophilus at 2.5 A resolution.

We report the refined structure of a ternary complex of an allosterically activated lactate dehydrogenase, including the important active site loop. Eightfold non-crystallographic symmetry averaging was utilized to improve the density maps. Interactions between the protein and bound coenzyme and oxamate are described in relation to other studies using site-specific mutagenesis. Fructose 1,6-bisphosphate (FruP2) is bound to the enzyme across one of the 2-fold axes of the tetramer, with the two phosphate moieties interacting with two anion binding sites, one on each of two subunits, across this interface. However, because FruP2 binds at this special site, yet does not possess an internal 2-fold symmetry axis, the ligand is statistically disordered and binds to each site in two different orientations. Binding of FruP2 to the tetramer is signalled to the active site principally through two interactions with His188 and Arg173. His188 is connected to His195 (which binds the carbonyl group of the substrate) and Arg173 is connected to Arg171 (the residue that binds the carboxylate group of the substrate).     

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.     

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 ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum at 2.9 A resolution

The three-dimensional structure of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from Rhodospirillum rubrum has been determined at 2.9 Å resolution by X-ray crystallographic methods. The MIR-electron density map was substantially improved by two-fold non-crystallographic symmetry averaging. The polypeptide chains in the dimer were traced using a graphics display system with the help of the BONES option in FRODO. The dimer has approximate dimensions of 50 x 72 x 105 Å. The enzyme subunit is a typical two-domain protein. The smaller, N-terminal domain consists of 137 amino acid residues and forms a central, mixed five-stranded β-sheet with α-helices on both sides of the sheet. The larger C-terminal domain consists of 329 amino acid residues. This domain has an eight-stranded parallel α/β barrel structure as found in triosephosphate isomerase and a number of other functionally non-related proteins. The active site in Rubisco determined by difference Fourier techniques and fitting of active site residues to the electron density map, is located at the carboxy-end of the β-strands in the α/β barrel of the C-terminal domain. There are few domain–domain interactions within the subunit. The interactions at the interface between the two subunits of the dimer are tight and extensive. There are tight contacts between the two C-terminal domains, which build up the core of the molecule. There are also interactions between the N-terminal domain of one subunit and the C-terminal domain of the second subunit, close to the active site.     

Crystallization of an NADP+-dependent malic enzyme from rat liver

Crystals of a tetrameric NADP+-dependent malic enzyme from rat liver have been grown in the presence of NADP+ using the hanging-drop method of vapour diffusion with ammonium sulphate as the precipitant. Measurement of the crystal density and calculation of the values of Vm for different numbers of polypeptide chains in the unit cell indicate that the asymmetric unit of the crystal contains a complete tetramer, allowing the application of non-crystallographic symmetry to the determination of the molecular structure of this enzyme. This structure would provide only the second example for an enzyme involved in oxidative decarboxylation, the other being 6-phosphogluconate dehydrogenase. In addition, then, to providing an insight into the structure-function relationship in malic enzyme, the successful structure determination would permit valuable comparisons to be made between these two and other enzymes with this catalytic activity.     

Crystallographic analysis of acrosomal bundle from Limulus sperm

The acrosomal process of Limulus sperm contains a bundle of filaments composed of actin and a 102 kDa protein in a 1:1 molar ratio. The structure of the bundle in true discharge was investigated by electron cryomicroscopy, X-ray scattering and crystallographic image analysis. A bundle can be characterized as a quasi-crystal with continuously varying views along the bundle axis. Each segment of the bundle is found to obey the symmetry of space group P1, with a = b = 147 A, c = 762 A, alpha = 90 degrees, beta = 90.6 degrees, gamma = 120 degrees. A unit cell contains a helical repeat of the filament with a selection rule following that of an actin filament. A 24 A projection map based on the h0l view was reconstructed after averaging 5300 unit cells from six electron images. Filaments in this projection are well separated and clearly display a 21 screw symmetry. This screw symmetry results from the helical parameters of the bundle filament and is found to be a non-crystallographic symmetry element present in the unit cell. Our structural analysis has led to the proposal that the assembly of a stable bundle with a defined maximum diameter can be controlled by the crystallographic packing of the twisted filaments.     

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.     

2.9 A resolution structure of the N-terminal domain of a variant surface glycoprotein from Trypanosoma brucei

The variant surface glycoprotein (VSG) of Trypanosoma brucei forms a coat on the surface of the parasite; by the expression of a series of antigenically distinct VSGs in the surface coat the parasite escapes the host immune response. The 2.9 A resolution crystal structure of the N-terminal domain of one variant, MITat 1.2, has been determined. The structure was solved using data collected from two crystal forms. Initially a partial model was built into an electron density map based on multiple isomorphous replacement phases and improved by phase combination methods. Subsequently this model was used to obtain the molecular replacement solution for a second crystal form, providing starting phases which were refined using 2-fold non-crystallographic symmetry averaging. The current model includes 362 residues and has been refined using X-PLOR to an R value of 0.22 for data between 7 and 2.9 A. The molecule is a dimer, approximately 100 A long, having an asymmetrical cross section with maximum dimensions of approximately 40 A x 60 A. Two long, approximately 70 A, alpha-helices from each monomer pack together to form, with several other helices, a core helix bundle that extends nearly the full length of the molecule. The "top" of the protein, which in the surface coat may be exposed to the external environment, is formed from the ends of the two long helices, a short three-stranded beta-sheet, and a strand having irregular conformation that packs above these secondary structure elements. Two conserved disulfide bridges are in this part of the molecule. Several elements of the MITat 1.2 sequence, which contribute to the formation of the helix bundle structure, have been identified. These elements can be found in the sequences of several different VSGs, suggesting that to some extent the VSG structure is conserved in those variants.     

Low resolution crystal structures of Taka-amylase A and its complexes with inhibitors.

The molecular structure of Taka-amylase A, an alpha-amylase from Aspergillus oryzae, has been studied at 6 A resolution by X-ray diffraction analysis. The electron density map showed a non-crystallographic three-fold screw arrangement of the molecules in the crystal. The molecule is an ellipsoid with approximate dimensions of 80 x 45 x 35 A and contains a hollow which may correspond to the active center. The inhibitor molecules bind to Taka-amylase A at four different sites, one of which is located in the hollow of the enzyme. The probable position of a thiol group is discussed in connection with heavy atom binding.     

Structure determination of the constitutive 20S proteasome from bovine liver at 2.75 A resolution

The crystal structure of the 20S proteasome from bovine liver was determined by the molecular replacement method using the structure of the 20S proteasome from the yeast Sacccharomyces cerevisiae. The initial phases were refined by density modification coupled with non-crystallographic symmetry averaging. The structural model was refined with the program CNS. The final R-factor and R(free) were 0.25 and 0.29, respectively. The constitutive proteasome without any contamination by the immunoproteasome was identified in the crystal structure.