John Day

The refined structure of canavalin from jack bean in two crystal forms at 2.1 and 2.0 Å resolution

The structure of canavalin was refined to 2.1 and 2.0 A resolution in cubic and hexagonal crystals of space group P2(1)3 and P6(3), respectively. The threefold molecular symmetry is expressed in the symmetry of both crystals, where each identical subunit is an asymmetric unit. The canavalin subunit consists of two very similar domains, each comprised of a core subdomain having Swiss-roll topology with a loop subdomain that contains helices. The refined canavalin models resolved the discrepancy in amino-acid registers of the secondary-structural elements compared with phaseolin. The presence of strand Z in both domains of canavalin was confirmed and a new helix in the loop between strands A and B of each domain was observed. The models were analyzed in terms of the duplicated vicilin domains. Three strictly conserved residues, two glycines and a proline, were identified. The similarity between entire vicilin molecules is greater than that between separate domains of canavalin and phaseolin. Homology modeling of the sucrose-binding protein (SBP) from soybean showed a plausible trimeric assembly of subunits similar to that of vicilins.

STRUCTURE OF ORTHORHOMBIC CRYSTALS OF BEEF LIVER CATALASE

The growth mechanisms and physical properties of the orthorhombic crystal form of beef liver catalase were investigated using in situ atomic force microscopy (AFM). It was observed that the crystals grow in the <001> direction by an unusual progression of sequential two-dimensional nuclei of half unit-cell layers corresponding to the bottoms and tops of unit cells. These were easily discriminated by their alternating asymmetric shapes and their strong growth-rate anisotropy. This pattern has not previously been observed with other macromolecular crystals. Orthorhombic beef liver catalase crystals exhibit an extremely high defect density and incorporate great numbers of misoriented microcrystals, revealed intact by etching experiments, which may explain their marginal diffraction properties. To facilitate interpretation of AFM results in terms of intermolecular interactions, the structure of the orthorhombic crystals, having an entire tetramer of the enzyme as the asymmetric unit, was solved by molecular replacement using a model derived from a trigonal crystal form. It was subsequently refined by conventional techniques. Although the packing of molecules in the two unit cells was substantially different, with very few exceptions no significant differences in the molecular structures were observed. In addition, no statistically significant deviation from ideal 222 molecular symmetry appeared within the tetramer. The packing of molecules in the crystal revealed by X-ray analysis explained in a satisfying way the process of crystal growth revealed by AFM.

Biophysical Studies on the RNA Cores of Satellite Tobacco Mosaic Virus

Satellite tobacco mosaic virus (STMV) was probed using a variety of proteases. Consequences of the degradation were analyzed using gel electrophoresis, quasi-elastic light scattering (QELS), and atomic force microscopy (AFM). Proteolysis rates of 30 minutes for complete degradation of the protein capsid, up to many hours, were investigated. With each protease, degradation of virions 17 nm in diameter was shown by QELS to result in particles of 10 nm diameter, which is that of the RNA core observed in the virion by x-ray diffraction analysis. This was verified by direct visualization with atomic force microscopy. Using QELS, it was further shown that freshly prepared RNA cores remain as individual, stable, 10-nm condensed particles for 12 to 24 h. Clusters of particles then formed, followed by very large aggregates of 500 to 1000 nm diameter. AFM showed that the aggregates were composed of groups of the condensed RNA cores and were not due to unfolding of the nucleic acid. No unfolding of the core particles into extended conformation was seen by AFM until the samples were heated well beyond 90 degrees C. Mass spectrometry of RNA core particles revealed the presence of a major polypeptide whose amino acid sequence corresponded to residues 2 through 25 of the coat protein. Amino acids 13 through 25 were previously observed to be in direct contact with the RNA and are presumably protected from protease digestion. Low resolution difference Fourier analyses indicated the courses of the remainders of the amino terminal strands (amino acids 2-12) in intact virions. Any individual strand appears to have several choices of path, which accounts for the observed disorder at high resolution. These positively charged strands, serving as virtual polyamines, engage the helical segments of RNA. The intimate association of amino acid residues 2 through 25 with RNA likely contributes to the stability of the condensed conformation of the nucleic acid cores.

X-ray diffraction and atomic force microscopy analysis of twinned crystals: rhombohedral canavalin

The structure of canavalin, the vicilin-class storage protein from jack bean, was refined to 1.7 A resolution in a highly twinned rhombohedral crystal of space group R3 and unit-cell parameters a = b = c = 83.0 A, alpha = beta = gamma = 111.1 degrees. The resulting R and R(free) were 0.176 and 0.245, respectively. The orthorhombic crystal structure (space group C222(1), unit-cell parameters a = 136.5, b = 150.3, c = 133.4 A) was also refined with threefold non-crystallographic symmetry restraints. R and R(free) were 0.181 and 0.226, respectively, for 2.6 A resolution data. No significant difference in the protein structure was seen between these two crystal forms, nor between these two and the hexagonal and cubic crystal forms reported elsewhere [Ko et al. (1993), Acta Cryst. D49, 478-489; Ko et al. (1993), Plant Physiol. 101, 729-744]. A phosphate ion was identified in the lumen of the C-terminal beta-barrel. Lattice interactions showed that the trimeric molecule could be well accommodated in both top-up and bottom-up orientations in a rhombohedral unit cell of the R3 crystal and explained the presence of a high twin fraction. The large inter-trimer stacking interface of the C222(1) crystal may account for its relative stability. Atomic force microscopy (AFM) investigations of the growth of three crystal forms of canavalin indicate the rhombohedral form to be unique. Unlike the other two crystal forms, it contains at least an order of magnitude more screw dislocations and stacking faults than any other macromolecular crystal yet studied, and it alone grows principally by generation of steps from the screw dislocations. The unusually high occurrence of the screw dislocations and stacking faults is attributed to mechanical stress produced by the alternate molecular orientations in the rhombohedral crystals and their organization into discrete domains or blocks. At boundaries of alternate domains, lattice strain is relieved by the formation of the screw dislocations.

A novel strategy for the crystallization of proteins: X-ray diffraction validation.

Recently, the hypothesis was advanced that protein crystallization could be driven by the inclusion of small molecules rich in hydrogen-bonding, hydrophobic and electrostatic bonding possibilities. Conventional organic and biologically active molecules would promote lattice formation by their mediation of intermolecular interactions in crystals. The results of an extensive series of crystallization experiments strongly supported the idea. Here, difference Fourier X-ray diffraction analyses of nine crystals grown in the experiments are presented, which convincingly demonstrate the validity of the hypothesis and illustrate some of the ways in which small molecules can participate in lattice interactions.

Structure of pig heart citrate synthase at 1.78 A resolution.

Pig heart citrate synthase was crystallized from a small-molecule cocktail containing cystamine dihydrochloride, aspartame and benzamidine hydrochloride. The structure was refined to an R factor of 0.179 (R(free) = 0.222) using synchrotron data to a resolution of 1.78 A. The model includes the full-length protein, a chloride ion, a sulfate ion, 305 water molecules and an unexpected moiety attached through a disulfide linkage to Cys184, which was modeled as a half-cystamine molecule generated by disulfide exchange with the cystamine in the small-molecule cocktail.

Determination of three crystal structures of canavalin by molecular replacement.

Canavalin, the major reserve protein of the jack bean, was obtained in four different crystal forms. From the structure determined by multiple isomorphous replacement in a hexagonal unit cell, the structures of three other crystals were determined by molecular replacement. In two cases, the rhombohedral and cubic crystals, placement was facilitated by coincidence of threefold molecular symmetry with crystallographic operators. In the orthorhombic crystal the canavalin trimer was the asymmetric unit. The rhombohedral, orthorhombic and cubic crystal structures were subsequently refined using a combination of several approaches with resulting R factors of 0.194, 0.185 and 0.211 at resolutions of 2.6, 2.6 and 2.3 A, respectively. Variation in the conformation of the molecule from crystal to crystal was small with an r.m.s. deviation in Calpha positions of 0.89 A. Packing is quite different among crystal forms but lattice interactions appear to play little role in the conformation of the molecule. Greatest variations in mean position are for those residues that also exhibit the greatest thermal motion. Crystal contacts in all crystals are mediated almost exclusively by hydrophilic side chains, and three to six intermolecular salt bridges per protein subunit are present in each case.

Satellite tobacco mosaic virus refined to 1.4 Å resolution.

Satellite tobacco mosaic virus (STMV) is among the smallest viruses, having 60 identical subunits arranged with T = 1 icosahedral symmetry. Its crystal structure was solved at 290u2005K and was refined using, in part, crystals grown in microgravity. Electron-density maps revealed nearly 57% of the genomic ssRNA. Using six flash-cooled crystals, diffraction data were recorded to 1.4u2005Å resolution and independent refinements of the STMV model were carried out versus the previous 1.8u2005Å resolution data representing merged data from 21 crystals (271,689 unique reflections), data consisting of corresponding reflections to 1.8u2005Å resolution from the cooled crystals and 1.4u2005Å resolution data from the cooled crystals comprised of 570,721 unique reflections. Models were independently refined with full NCS constraints using the program CNS and in restrained mode using the programs CNS, REFMAC5 and SHELX-97, with the latter two procedures including anisotropic temperature factors. Significant additional structural detail emerged from the analyses, including a unique cation and anion arrangement on fivefold axes and a precise assessment of icosahedral symmetry exactness in the crystal lattice. STMV represents the highest resolution native virus structure currently known by a substantial margin, and it permits the evaluation of a precise atomic model of a spherical virus at near-atomic resolution for the first time.

Combined use of AFM and X-ray diffraction to analyze crystals of an engineered, domain-deleted antibody.

A genetically engineered humanized C(H)2-domain-deleted monoclonal antibody lacking any interchain-hinge disulfide bonds has been crystallized in the presence of detergent in a form suitable for X-ray diffraction analysis. The crystals were grown from 4 M formate along with Triton X-100 and had P2(1)2(1)2 space-group symmetry, with unit-cell parameters a = 83, b = 224, c = 167 A. The crystals diffract to beyond 2.8 A resolution. A disordered crystal form of larger size and more attractive habit was also grown from 4 M formate, but in the presence of the Anapoe series of detergents. Preliminary X-ray data, in conjunction with atomic force microscopy images, are consistent with asymmetric units consisting of two intact antibodies forming a circular dimeric ring. The crystallizing unit, which must contain a twofold axis, is a toroidal assembly of four antibodies (two dimeric rings). Competition between dimers and tetramers to enter the lattice, along with a unique kind of planar defect of packing, may be responsible for the unusually high defect density and the disorder of the X-ray diffraction pattern exhibited by the second crystal form. An approach to crystallizing proteins showing phase separation, particularly intact antibodies, that uses a preliminary detergent test set is described.

A new crystal form of bovine pancreatic RNase a in complex with 2′-deoxyguanosine-5′-monophosphate

The structure of bovine pancreatic RNase A has been determined in complex with 2-deoxyguanosine-5-monophosphate (dGMP) at 1.33 A resolution at room temperature in a previously unreported unit cell belonging to space group P3(1). There are two molecules of nucleotide per enzyme molecule, one of which lies in the active-site cleft in the productive binding mode, whilst the guanine base of the other dGMP occupies the pyrimidine-specific binding site in a nonproductive mode such that it forms hydrogen bonds to the phosphate group of the first dGMP. This is the first RNase A structure containing a guanine base in the B2 binding site. Each dGMP molecule is involved in intermolecular interactions with adjacent RNase A molecules in the lattice and the two nucleotides appear to direct the formation of the crystal lattice. Because GMP may be produced during degradation of RNA, this association could represent an inhibitor complex and thereby affect the observed enzyme kinetics.