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1.  Crystallization and preliminary crystallographic analysis of Mycoplasma arthritidis-derived mitogen complexed with peptide/MHC class II antigen 
Mycoplasma arthritidis-derived mitogen (MAM), a bacterial superantigen, has been crystallized in complex with its human receptor, major histocompatibility complex (MHC) class II antigen, by the hanging-drop vapor-diffusion method. Crystals were obtained under three conditions, with ammonium sulfate, phosphate salt and PEG 8000 as the precipitant. The crystals grown under these conditions all belong to space group I222, with the same unit-cell parameters: a = 137.4, b = 178.2, c = 179.6 Å. Diffraction data were collected to 3.3 and 3.4 Å resolution from crystals of native and selenomethionylated MAM–MHC complexes, respectively. Self- and cross-rotation function calculations suggest the presence of two complex molecules in the asymmetric unit, resulting in a VM of 4.0 and a solvent content of 69%. An interpretable electron-density map was produced using a combination of molecular replacement and SAD phasing.
PMCID: PMC3924564  PMID: 14747723
2.  Detection of L-Lactate In Polyethylene Glycol Solutions Confirms Identity of Active Site Ligand in Proline Dehydrogenase Structure 
Polyethylene glycol (PEG) is often used in protein crystallography as a low ionic strength precipitant for crystallization and a cryoprotectant for low temperature data collection. Prompted by the discovery of an apparent L-lactate molecule bound in the active site of the E. coli PutA proline dehydrogenase domain crystal structure, we measured the L-lactate concentration of several PEG solutions. Fifty percent (w/v) solutions of PEGs with molecular weight 3000, 4000, and 8000 contain millimolar levels of L-lactate. In contrast, L-lactate was not detected in solutions of PEG monomethyl ethers or PEG 3350. These results help explain why L-lactate was present in the proline dehydrogenase domain crystal structure. This work also has implications for the crystallization of enzymes that bind L-lactate.
PMCID: PMC3733669  PMID: 15103160
3.  Harvesting the high-hanging fruit: the structure of the YdeN gene product from Bacillus subtilis at 1.8 Å resolution 
High-throughput (HT) protein crystallography is severely impeded by the relatively low success rate of protein crystallization. Proteins whose structures are not solved in the HT pipeline owing to attrition in any phase of the project are referred to as the high-hanging fruit, in contrast to those proteins that yielded good-quality crystals and crystal structures, which are referred to as low-hanging fruit. It has previously been shown that proteins that do not crystallize in the wild-type form can have their surfaces engineered by site-directed mutagenesis in order to create patches of low conformational entropy that are conducive to forming intermolecular interactions. The application of this method to selected proteins from the Bacillus subtilis genome which failed to crystallize in the HT mode is now reported. In this paper, the crystal structure of the product of the YdeN gene is reported. Of three prepared double mutants, i.e. E124A/K127A, E167A/E169A and K88A/Q89A, the latter gave high-quality crystals and the crystal structure was solved by SAD at 1.8 Å resolution. The protein is a canonical α/β hydrolase, with an active site that is accessible to solvent.
PMCID: PMC2792027  PMID: 15159570
4.  Crystallization and preliminary analysis of active nitroalkane oxidase in three crystal forms 
Nitroalkane oxidase (NAO), a flavoprotein cloned and purified from Fusarium oxysporum, catalyzes the oxidation of neutral nitroalkanes to the corresponding aldehydes or ketones, with the production of H2O2 and nitrite. In this paper, the crystallization and preliminary X-ray data analysis of three crystal forms of active nitroalkane oxidase are described. The first crystal form belongs to a trigonal space group (either P3121 or P3221, with unit-cell parameters a = b = 103.8, c = 487.0 Å) and diffracts to at least 1.6 Å resolution. Several data sets were collected using 2θ and κ geometry in order to obtain a complete data set to 2.07 Å resolution. Solvent-content and Matthews coefficient analysis suggests that crystal form 1 contains two homotetramers per asymmetric unit. Crystal form 2 (P212121; a = 147.3, b = 153.5, c = 169.5 Å) and crystal form 3 (P31 or P32; a = b = 108.9, c = 342.5 Å) are obtained from slightly different conditions and also contain two homotetramers per asymmetric unit, but have different solvent contents. A three-wavelength MAD data set was collected from selenomethionine-enriched NAO (SeMet-NAO) in crystal form 3 and will be used for phasing.
PMCID: PMC1680162  PMID: 15272176

Results 1-4 (4)