Routine single particle CryoEM sample and grid characterization by tomography.

Single particle cryo-electron microscopy (cryoEM) is often performed under the assumption that particles are not adsorbed to the air-water interfaces and in thin, vitreous ice. In this study, we performed fiducial-less tomography on over 50 different cryoEM grid/sample preparations to determine the particle distribution within the ice and the overall geometry of the ice in grid holes. Surprisingly, by studying particles in holes in 3D from over 1000 tomograms, we have determined that the vast majority of particles (approximately 90%) are adsorbed to an air-water interface. The implications of this observation are wide-ranging, with potential ramifications regarding protein denaturation, conformational change, and preferred orientation. We also show that fiducial-less cryo-electron tomography on single particle grids may be used to determine ice thickness, optimal single particle collection areas and strategies, particle heterogeneity, and de novo models for template picking and single particle alignment.

Glycerol dehydrogenase. structure, specificity, and mechanism of a family III polyol dehydrogenase.

BACKGROUND: Bacillus stearothermophilus glycerol dehydrogenase (GlyDH) (glycerol:NAD(+) 2-oxidoreductase, EC 1.1.1.6) catalyzes the oxidation of glycerol to dihydroxyacetone (1,3-dihydroxypropanone) with concomitant reduction of NAD(+) to NADH. Analysis of the sequence of this enzyme indicates that it is a member of the so-called iron-containing alcohol dehydrogenase family. Despite this sequence similarity, GlyDH shows a strict dependence on zinc for activity. On the basis of this, we propose to rename this group the family III metal-dependent polyol dehydrogenases. To date, no structural data have been reported for any enzyme in this group. RESULTS: The crystal structure of B. stearothermophilus glycerol dehydrogenase has been determined at 1.7 A resolution to provide structural insights into the mechanistic features of this family. The enzyme has 370 amino acid residues, has a molecular mass of 39.5 kDa, and is a homooctamer in solution. CONCLUSIONS: Analysis of the crystal structures of the free enzyme and of the binary complexes with NAD(+) and glycerol show that the active site of GlyDH lies in the cleft between the enzyme's two domains, with the catalytic zinc ion playing a role in stabilizing an alkoxide intermediate. In addition, the specificity of this enzyme for a range of diols can be understood, as both hydroxyls of the glycerol form ligands to the enzyme-bound Zn(2+) ion at the active site. The structure further reveals a previously unsuspected similarity to dehydroquinate synthase, an enzyme whose more complex chemistry shares a common chemical step with that catalyzed by glycerol dehydrogenase, providing a striking example of divergent evolution. Finally, the structure suggests that the NAD(+) binding domain of GlyDH may be related to that of the classical Rossmann fold by switching the sequence order of the two mononucleotide binding folds that make up this domain.

Purification, crystallization and quaternary structure analysis of a glycerol dehydrogenase S305C mutant from Bacillus stearothermophilus.

Bacillus stearothermophilus glycerol dehydrogenase (GlyDH) is a 39.5 kDa molecular weight metalloenzyme which catalyzes the oxidation of glycerol to dihydroxyacetone with the concomitant reduction of NAD(+) to NADH. Despite its classification as a member of the 'iron-containing' polyol dehydrogenase family, studies on recombinant B. stearothermophilus GlyDH have shown this enzyme to be Zn(2+)-dependent. Crystals of a S305C GlyDH mutant were obtained by the hanging-drop vapour-diffusion method, using ammonium sulfate and PEG 400 as precipitating agents, in the presence and absence of NAD(+). The crystals belong to space group I422, with approximate unit-cell parameters a = b = 105, c = 149 A and one subunit in the asymmetric unit, corresponding to a packing density of 2.6 A(3) Da(-1). The crystals diffract X-rays to at least 1.8 A resolution on a synchrotron-radiation source. Determination of the structure will provide insights into the key determinations of catalytic activity of this class of enzymes, for which no structures are currently available.