Search

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 290 K 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 1.4 Å resolution and independent refinements of the STMV model were carried out versus the previous 1.8 Å resolution data representing merged data from 21 crystals (271,689 unique reflections), data consisting of corresponding reflections to 1.8 Å resolution from the cooled crystals and 1.4 Å 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.

Human methemoglobin was crystallized in a unique unit cell and its structure was solved by molecular replacement. The hexagonal unit cell has unit-cell parameters a = b = 54.6, c = 677.4 Å, with symmetry consistent with space group P6₁22. The unit cell has the second highest aspect ratio of all unit cells contained in the PDB. The 12 molecules in the unit cell describe a right-handed helical filament having no polarity, which is different from the filament composed of HbS fibers, which is the only other well characterized fiber of human hemoglobin. The filaments reported here can be related to canonical sickle-cell hemoglobin filaments and to an alternative sickle-cell filament deduced from fiber diffraction by slight modifications of intermolecular contacts.

Bovine pancreatic ribonuclease A (RNase A) was crystallized from a mixture of small molecules containing basic fuchsin, tobramycin and uridine 5'-monophosphate (U5P). Solution of the crystal structure revealed that the enzyme was selectively bound to U5P, with the pyrimidine ring of U5P residing in the pyrimidine-binding site at Thr45. The structure was refined to an R factor of 0.197 and an R(free) of 0.253.

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.

Proteinase K, a subtilisin-like fungal protease, was crystallized from a cocktail of small molecules containing digalacturonic acid (DGA). The crystal structure was determined to 1.32 A resolution and refined to an R factor of 0.158. The final model contained, beside the protein, two calcium ions, 379 water molecules, a molecule of DGA and a partially occupied HEPES molecule. The DGA molecule has one sugar moiety disposed exactly on a crystallographic twofold axis; the second ring was not observed. The DGA molecule is bound to two protein molecules across the twofold axis through hydrogen-bonding networks involving Ser150 and water molecules. One of the calcium-ion sites has not been reported previously. This study further illustrates the involvement of small molecules in the crystallization of macromolecules through their ability to form intermolecular lattice interactions.

We are developing an alternate strategy for the crystallization of macromolecules that does not, like current methods, depend on the optimization of traditional variables such as pH and precipitant concentration, but is based on the hypothesis that many conventional small molecules might establish stabilizing, intermolecular, non covalent crosslinks in crystals, and thereby promote lattice formation. To test the hypothesis, we carried out preliminary experiments encompassing 18,240 crystallization trials using 81 different proteins, and 200 chemical compounds. Statistical analysis of the results demonstrated the validity of the idea. In addition, we conducted X-ray diffraction analyses of some of the crystals grown in the experiments. These clearly showed incorporation of conventional molecules into the protein crystal lattices, and further validated the underlying hypothesis. We are currently extending the investigations to include a broader and more diverse set of proteins, an expanded search of conventional and biologically active small molecules, and a wider range of precipitants. The strategy proposed here is essentially orthogonal to current approaches and has an objective of doubling the success rate of today.

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.

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.

C(H)2-domain-deleted CC49 (HuCC49DeltaCH2), a recombinant humanized antibody that recognizes the TAG-72 antigen expressed on a variety of human carcinomas, is secreted from cultured cells as a mixture of two homodimeric isoforms. Isoform A contains two covalent interchain disulfide bonds at heavy chain positions 239 and 242, while isoform B fails to develop any interchain disulfide bonds but has 239-242 intrachain disulfide bonds instead. Form A is currently in preclinical development as a therapeutic agent for treating colorectal carcinoma, though form B shows equal efficacy. HuCC49DeltaCH2 form B can be crystallized from sodium formate only in the presence of detergents. X-ray diffraction data were collected on a single cryo-cooled crystal grown with Triton X-100 and the structure was solved by molecular replacement. The model has refined to R=0.246 (R(free)=0.297) for 2.8A data. The antibodies pack in the crystal around crystallographic 2-fold axes as tetramers with approximate 222 symmetry. Atomic force microscopy studies show that this tetrameric structure is the crystal building block and also exists free in the mother liquor. The tetramer is composed of two rings, back-to-back, with a thickness of approximately 83A. Each ring is composed of two antibodies with the complementarity-determining regions (CDR) of the two Fabs of one antibody interacting with the CDR regions of the second antibody in a head-to-head fashion. These rings are approximately 167A long and 112A wide. The C(H)3 domain is inverted with respect to the Fabs when compared to the usual orientation found in conventional antibodies. The polypeptides joining the C(H)3 domains to the Fab portions of the antibody are not seen and are almost certainly disordered. The antigen combining site of HuCC49DeltaCH2 is very similar, but not identical, in topology and charge distribution to that of antibody B72.3, which binds a similar epitope on TAG-72. The combining site consists of a deep cleft, heavily lined with aromatic amino acid side-chains but bounded by numerous charged groups.

X-ray crystallographic analyses of pig pancreatic [alpha]-amylase which contain a limit dextrin of six glucose residues, oligosaccharide, and [alpha]-cyclodextrin is reported. A comprehensive integration of the complexes provides a pathway to construct a model for the binding of polysaccharides which have the helical character found in natural substrates such as starch and glycogen.