Robert Cudney

Protein and virus crystal growth on International Microgravity Laboratory-2

Two T = 1 and one T = 3 plant viruses, along with a protein, were crystallized in microgravity during the International Microgravity Laboratory-2 (IML-2) mission in July of 1994. The method used was liquid-liquid diffusion in the European Space Agencys Advanced Protein Crystallization Facility (APCF). Distinctive alterations in the habits of Turnip Yellow Mosaic Virus (TYMV) crystals and hexagonal canavalin crystals were observed. Crystals of cubic Satellite Tobacco Mosaic Virus (STMV) more than 30 times the volume of crystals grown in the laboratory were produced in microgravity. X-ray diffraction analysis demonstrated that both crystal forms of canavalin and the cubic STMV crystals diffracted to significantly higher resolution and had superior diffraction properties as judged by relative Wilson plots. It is postulated that the establishment of quasi-stable depletion zones around crystals growing in microgravity are responsible for self-regulated and more ordered growth.

Crystallographic characterization and molecular symmetry of edestin, a legumin from hemp

Edestin, a legumin class reserve protein from hemp seeds having six identical subunits was crystallized from ammonium phosphate at pH 5 and subsequently characterized by X-ray diffraction. The crystals are of space group R32 with a = 127 A and gamma = 116 degrees having an equivalent triply centered hexagonal cell of a = b = 215 A, c = 80 A. There is one hexameric protein in the rhombohedral unit cell, hence the subunits of the Edestin molecule must be arranged with 32 point group symmetry.

Crystallization of macromolecules in silica gels

Procedures are described for the crystallization of proteins, nucleic acids and viruses in a silica-gel matrix using otherwise standard reagents and conditions. Methods are given based on both vapor diffusion in a sitting drop and liquid-liquid diffusion. Using a variety of macromolecules our results suggest that the gel matrix suppresses nucleation, reduces the rate of growth, and generally leads to larger, higher-quality crystals of enhanced stability. Presumably these effects arise from the decreased mobility of the macromolecules and their flux at the crystal surface during growth.

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.

Structure of a crystal form of human methemoglobin indicative of fiber formation

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.

High-resolution structure of proteinase K cocrystallized with digalacturonic acid.

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.