Filamentous Bacterial viruses

The filamentous bacterial virus is a simple and well-characterized model system for studying how genetic information is transformed into molecular machines. The viral DNA is a single-stranded circle coding for about 10 proteins. The major viral coat protein is largely α-helical, with about 46 amino acid residues. Several thousand identical copies of this protein in a helical array form a hollow cylindrical tube 1–2μ long, of outer diameter 60 Å and inner diameter 20 Å, with the twisted circular DNA extending down the core of the tube. Before assembly, the viral coat protein spans the cell membrane, and assembly involves extrusion of the coat from the membrane. X-ray fibre diffraction patterns of the Pf 1 species of virus at 4°C, oriented in a strong magnetic field, give three-dimensional data to 4 Å resolution. An electron density map calculated from native virus and a single iodine derivative, using the maximum entropy technique, shows a helix pitch of 5.9 Å. This may indicate a stretched A-helix, or it may indicate a partially 3₁₀ helix conformation, resulting from the fact that the coat protein is an integral membrane protein before assembly, and is still in the hydrophobic environment of other coat proteins after assembly.     

Structure of polar pili from Pseudomonas aeruginosa strains K and O

The polar pili of Pseudomonas aeruginosa strains K and O are hollow cylinders with 52 Å outer diameter and 12 Å inner diameter. There is a girdle of low electron density (interpreted as due to a local concentration of hydrophobic amino acid side-chains) centred at 31 Å diameter. Similar X-ray diffraction patterns are obtained from oriented fibres of the two types of pili, to a resolution of 7 Å in the equatorial direction and 4 Å in the meridional direction. The two types of pilin protein subunits have a similar molecular weight, and their sequences contain a number of homologous regions. They form a helical array with 4.06 to 4.08 units per turn of a basic helix that has a pitch of 40.8 Å for strain K pili and 41.3 Å for strain O pili at 75% relative humidity. A method is described for distinguishing between very similar diffraction patterns.There is strong intensity at 10 Å near the equator and at 5 Å near the meridian on the diffraction patterns. This intensity distribution is characteristic of α-helical rods running roughly in the direction of the fibre axis. The orientation of these rods was established by the fit between the transform of an α-helical polyalanine model and the strong near-equatorial layer-line.     

Filamentous bacteriophage Pf1 structure determined at 7 Å resolution by refinement of models for the α-helical subunit

The molecular structure of filamentous bacteriophage Pf1 has been determined to 7 Å resolution by analysis of X-ray diffraction data from partially oriented fibers of virus particles. The continuous intensity distribution along layer-lines was measured by numerically separating contributions from overlapping layer-lines. The data were phased by an iterative refinement technique that used the known spatial extent and high α-helical content of the virus particle to refine a structural model. This refinement converges to a unique structural solution that is consistent with the X-ray data and with information derived from physical and chemical studies. The coat protein consists of two α-helical segments: one, almost parallel to the particle axis, is centered at a radius of about 15 Å; the other, at about 25 Å radius, is tilted by about 25 ° to the particle axis. This structure is consistent with every generalization about α-helical packing. The inner and outer segments interlock along most of their length with a crossing angle of 20.5 °. The inner α-helical segments also interact with symmetry-related copies of themselves, as do the outer segments. The double layer of tightly packed, intricately interlocked α-helices forms a stable, 20 Å thick protein coat around the viral DNA.     

An x-ray scattering investigation of broad bean mottle virus in solutions of various electron densities

Low-angle X-ray scattering data to a resolution of 30 Å are presented for broad bean mottle virus suspended in buffer and in solutions of higher electron density produced by the addition of sucrose or the trisaccharide melezitose. Comparison of the scattered intensity distributions with those of simple model particles are made and radial electron density distributions are obtained. The results indicate that in buffer the virus particle has a radius of gyration of 117 Å, a mean outer radius of about 147 Å, and a nearly hollow core of about 60 Å radius. The scattering data for the virus in sugar solutions supports these results and indicates that much of the region within the virus open to water is also open to penetration by the sugar molecules. Melezitose can penetrate about 60% of the volume of the virus open to water while sucrose can penetrate nearly 90%. The region of the virus within 90 Å from the center is more easily penetrated by these sugars than the region from 90 Å to the surface. It is concluded that the virus at this resolution appears as a hollow, approximately spherically symmetric object with a high density and probably well organized RNA region enclosed by a protein shell into which some of the RNA penetrates.     

Neutron crystallographic studies of T4 lysozyme at cryogenic temperature

Bacteriophage T4 lysozyme (T4L) has been used as a paradigm for seminal biophysical studies on protein structure, dynamics, and stability. Approximately 700 mutants of this protein and their respective complexes have been characterized by X‐ray crystallography; however, despite the high resolution diffraction limits attained in several studies, no hydrogen atoms were reported being visualized in the electron density maps. To address this, a 2.2 Å‐resolution neutron data set was collected at 80 K from a crystal of perdeuterated T4L pseudo‐wild type. We describe a near complete atomic structure of T4L, which includes the positions of 1737 hydrogen atoms determined by neutron crystallography. The cryogenic neutron model reveals explicit detail of the hydrogen bonding interactions in the protein, in addition to the protonation states of several important residues.     

Water structure in a protein crystal: rubredoxin at 1.2 A resolution

The model for rubredoxin based on X-ray diffraction data has been extensively refined with a 1.2 Å resolution data set. Water oxygen atoms were deleted from the model if B exceeded 50 Ų and occupancy was less than 0.3 eÅ^?3. The final water model consists of 127 sites with B values ranging from 15 to $\text{6}\text{?}$0 Ų and occupancies from unity down to 0.3, the most tightly bound water oxygen atoms being hydrogen bonded to two or more main-chain nitrogen or oxygen atoms. The water forms extensive hydrogen bond networks bridging the crevices on the molecular surfaces and between adjacent molecules. The minimum distances of the water sites from the protein surface are distributed about two distinct maxima, the major one at 2.5 to 3 Å and a minor one at 4 to 4.5 Å. Beyond $\text{5}\text{?}$ to 6 Å from the protein surface, the discrete water merges into the aqueous continuum.     

Preliminary X-ray diffraction analysis of crystals of tomato aspermy virus (TAV)

Tomato aspermy virus (TAV) is a member of the T = 3 cucumovirus group, and the chrysanthemum strain (C-TAV) has been crystallized in a form suitable for X-ray structural analysis. The crystals, which grow in 14–17% ethanol at pH 8.5, are of orthorhombic space group I222 with unit cell dimensions of a = 295.1 Å, b = 320.5 Å, and c = 383.6 Å. There are two T = 3 virus particles in the unit cell, which means that they must be centered at 0,0,0 and 1/2, 1/2, 1/2 with icosahedral 222 symmetry elements coincident with crystallographic symmetry operators. The asymmetric unit of the crystals, therefore, contains one quarter of a virus particle, or 45 capsid subunits. Native diffraction data to 4 Å resolution have been collected using synchrotron radiation, though data appear to be present beyond that resolution. © 1995 Wiley-Liss, Inc.     

An investigation of the structure of Alfalfa mosaic virus by small-angle neutron scattering

Small-angle neutron scattering experiments have been performed on the tubular bottom component of Alfalfa mosaic virus (AMV) and the “30 S” particle (a quasispherical reassembled AMV coat protein particle) with the aim of determining the internal structure of the virus. Scattering curves were obtained out to a resolution of $\text{1}\text{50}\text{A}\text{?}{}^{\mathrm{?1}}$ at a number of H₂O/²H₂O ratios and were analysed using a model fitting technique. This involves calculating the scattering intensity due to a parameterised distribution of scattering density representing the particle and comparing this to the experimental data after taking into account the effect of instrumental smearing. The use of the contrast variation method enables the internal consistency of the model to be well tested.Three models are used in an attempt to explain the scattering curve of the 30 S particle. A single homogeneous shell is shown to be inadequate and two other models introducing the presumed T = 1 icosahedral symmetry of the particle are presented and discussed. The most satisfactory of these consists of 60 spherical monomers of radius 19 Å symmetrically placed in pairs about the 2-fold icosahedral positions.The analysis of the bottom component data has yielded a low resolution model for the virus, which is shown to be consistent with its composition as given by earlier physico-chemical measurements. In the model the RNA is uniformly packed throughout the interior of the capsid (which is cylindrical with hemispherical ends) out to a radius of about 65 Å and with a packing fraction of 20%. Within the limitations of an homogeneous shell model, the protein capsid has an outer radius of 94 Å and thickness of 23 Å, but arguments are presented based on the marked lattice structure of the cylindrical capsid and the analysis of the scattering data of the 30 S particle, that this model underestimates the thickness of the protein shell and that it in fact makes contact with the RNA at about 65 Å.     

X-ray diffraction and electron microscope studies on the structure of bacterial F pili.

F pili are hollow cylinders with 80 Å outer diameter and 20 Å inner diameter. Both X-ray fibre diffraction and optical diffraction of electron micrographs show a strong layer-line corresponding to a spacing of 32 Å, to which a J₄ Bessel function is assigned on the basis of the optical diffraction. X-ray diffraction patterns show near-meridional intensity on a layer-line corresponding to a spacing of 12.8 Å, to which a J₁ Bessel function is assigned. Mass per length measurements on unstained specimens in the scanning transmission electron microscope give 3000 daltons/Å, indicating that the 11,200 dalton pilin subunits are 3.7 Å apart along the axial direction of the pili. These observations show that the pilus structure can be represented as four coaxial helices of pitch 128 Å with the pilin subunits elongated and overlapping along the line of these helices. Each of these helices of subunits is translated axially with respect to its neighbour, to give a basic helix of 3.6 units per turn of 12.8 Å pitch. Radial electron density calculations indicate a 50 Å diameter girdle of hydrophobic amino acids between the inner and outer diameters of the protein shell. A molecular model of the structure at low resolution is presented.     

Yeast tRNAAsp-Aspartyl-tRNA Synthetase Complex: Low Resolution Crystal Structure

Abstract

Yeast aspartyl-tRNA synthetase, a dimer of molecular weight 125000, and two molecules of its cognate tRNA (M_r = 24160) cocrystallize in the cubic space group 1432 (a = 354 Å). The crystal structure was solved to low resolution using neutron and X-ray diffraction data. Neutron single crystal diffraction data were collected in five solvents differing by their D₂O content in order to use the contrast variation method to distinguish between the protein and tRNA The synthetase was first located at 40 Å resolution using the 65% D₂O neutron data (tRNA matched). tRNA molecules were found at 20 Å resolution using both neutron and X-ray data. The resulting model was refined against 10 Å resolution X-ray data, using density modification and least-squares refinement of the tRNA positions. The crystal structure, solved without a priori phase knowledge, was confirmed later by isomorphous replacement. The molecular model of the complex is in good agreement with results obtained in solution by probing the protected part of the tRNA by chemical reagents.     

The positions of the N-terminus and residue 68 in tobacco mosaic virus

Specific chemical modifications of the tobacco mosaic virus coat protein lead to new heavy-atom derivatives. They can be used for the determination of phases in the isomorphous replacement method, but more important they are necessary as markers if one wants to trace the polypeptide chain through an electron density map of limited resolution (10 Å). In addition to the positions of two residues known from previous work, two more residues out of the 158 have now been located in three dimensions. The N-terminus is at the outside of the particle (r = 88 Å), and Lys-68 lies at a radius of 72 Å.     

Rapid increase of near atomic resolution virus capsid structures determined by cryo-electron microscopy

The recent technological advances in electron microscopes, detectors, as well as image processing and reconstruction software have brought single particle cryo-electron microscopy (cryo-EM) into prominence for determining structures of bio-molecules at near atomic resolution. This has been particularly true for virus capsids, ribosomes, and other large assemblies, which have been the ideal specimens for structural studies by cryo-EM approaches. An analysis of time series metadata of virus structures on the methods of structure determination, resolution of the structures, and size of the virus particles revealed a rapid increase in the virus structures determined by cryo-EM at near atomic resolution since 2010. In addition, the data highlight the median resolution (～3.0?Å) and size (～310.0?Å in diameter) of the virus particles determined by X-ray crystallography while no such limits exist for cryo-EM structures, which have a median diameter of 508?Å. Notably, cryo-EM virus structures in the last four years have a median resolution of 3.9?Å. Taken together with minimal sample requirements, not needing diffraction quality crystals, and being able to achieve similar resolutions of the crystal structures makes cryo-EM the method of choice for current and future virus capsid structure determinations.     

Neocarzinostatin: an antitumor protein a preliminary x-ray diffraction study.

The antitumor protein, neocarzinostatin, has been crystallized and examined by X-ray diffraction. Crystals of this globular protein are of space group P2₁2₁2₁ with cell parameters a = 27.4Å, b = 33.9Åand c = 102.0Å. There is one molecule of approximately 27Ådiameter per asymmetric unit. Crystals soaked in a K₂HgI₄ solution give diffraction patterns significantly different from native crystal diffraction patterns.     

Polymorphism in B-DNA: X-ray diffraction studies on Li-DNA fibres

From X-ray diffraction studies it is generally believed that B-DNA has the structural parameters n = 10 and h = 3.4 Å. However, for the first time we report that polymorphism in the B-form can be observed in DNA fibres. This was achieved by the precise control of salt and humidity in fibres and by the application of the precession method of X-ray diffraction to DNA fibres. The significant result obtained is that n = 10 is not observed for crystalline fibre patterns. In fact, n = 10 and h = 3.4 Å are not found to occur simultaneously. Instead, a range of values, n = 9.6–10.0 and h = 3.35 Å–3.41 Å is observed.     

Neutron diffraction studies on crystals of nucleosome cores using contrast variation

Neutron diffraction data have been collected from crystals of intact nucleosome core particles to a resolution of 25 Å. By varying the proportion of D₂O, the scattering of the mother liquor relative to the protein and DNA can be altered. At 39% D₂O, the solvent scattering matches that of the protein and so only the DNA is scattering, and similarly at 65% D₂0 only the protein scatters. Using this approach the neutron scattering of the two components and of the complete particle (0% D₂O) have been measured. The data corresponding to the principal projections are consistent with a model in which 1.8 turns of a DNA superhelix of pitch 27·5 Å and radius 42 Å are wound around a protein core.     

The reaction center profile structure derived from neutron diffraction

Both reaction center protein from the photosynthetic bacteria Rhodopseudomonas sphaeroides and egg phosphatidylcholine can be deuterium labelled; the reaction center protein can be incorporated into the phosphatidylcholine bilayers forming a homogeneous population of unilamellar vesicles. The lipid profile and the reaction center profile within these reconstituted membrane profiles were directly determined to 32 Å resolution using lamellar neutron diffraction from oriented membrane multilayers containing either deuterated or protonated reaction centers, and either deuterated or protonated phosphatidylcholine. The 32 Å resolution reaction center profile shows that the protein spans the membranes, and has an asymmetric mass distribution along the perpendicular to the membrane plane. These results were combined with previously described X-ray diffraction results in order to extend the resolution of the derived reaction center profile to 9 Å.     

Ferrochelatase activity in wild-type and mutant strains of Spirillum itersonii. Solubilization with chaotropic reagents

New low-angle X-ray diffraction data have been obtained from nerve myelin after rehydration. The X-ray patterns show the first six orders of diffraction of a lamellar repeat unit of about 100 Å. Direct methods of structure analysis have been used to determine uniquely the phases of the first three orders of diffraction. The electron density profile of rehydrated nerve myelin has been obtained on an absolute electron density scale and is compared with the electron density profile of normal nerve myelin at the same resolution of 16–17 Å. Possible electron-density profiles of rehydrated nerve myelin at a resolution of 8 Å are shown.     

5.5 Å crystallographic structure of penicillin β-lactamase and radius of gyration in solution

An electron density map of crystalline R-TEM Escherichia coli β-lactamase (penicillinase) has been calculated from X-ray diffraction data at 5.5 Å resolution with protein phases based on Friedel mates from a high-quality samarium derivative. The mean figure of merit for 854 independent reflections is 0.75. The monomeric molecule is slightly ellipsoidal and contains one and possibly two regions of α-helix which are 25 Å long. The Crystallographic search for the substrate binding site has so far been inconclusive. The radius of gyration of the enzyme in solution at pH 7 is 17.1 ± 1.0 Å from small-angle X-ray scattering measurements. This compares with 18.6 å calculated from the low-resolution electron density map of the molecule in the crystal.     

Preliminary X-ray diffraction analysis of crystals of turnip yellow mosaic virus (TYMV)

Turnip yellow mosaic virus (TYMV) was purified from Chinese cabbage and crystallized in a form that permits high resolution structural analysis using X-ray diffraction. The crystals have a hexagonal bipyramidal morphology and often achieve dimensions of 1.0 × 1.0 × 0.5 mm. The crystals appear to be of hexagonal space group P6₂22 with a = b = 525 Å, c=315 Å, but we cannot strictly rule out the possibility that the space group is P622. They appear different than any crystals of TYMV previously reported. There are three T = 3 virus particles in the unit cell, which implies that one quarter of the particle, or 45 protein subunits, comprises the asymmetric unit of the crystal. Native data have been collected using synchrotron radiation to a resolution of 3.2 Å. © 1995 Wiley-Liss, Inc.     

The size of the bacteriophage T4 head in solution with comments about the dimension of virus particles as visualized by electron microscopy

We report X-ray diffraction results permitting calculation of the radius of the bacteriophage T4 head in solution. Isometric headed mutant particles, consisting of the two hemispherical caps of wild-type T4, were found to be spheres of radius 425 Å. Giant headed particles, an amplification of the extra capsomeres which give the T4 head its prolate shape, were found to be cylinders of radius 427 Å. We use these and other small-angle X-ray diffraction results in a quantitative discussion of the distortion artefacts caused by a number of electron microscope specimen preparation techniques.