Microseed matrix screening for optimization in protein crystallization: what have we learned?

Microseed matrix screening is increasingly successful in the optimization of protein crystallization leads.

Chemical conversion of cisplatin and carboplatin with histidine in a model protein crystallized under sodium iodide conditions

Crystals of HEWL with cisplatin and HEWL with carboplatin grown in sodium iodide conditions both show a partial chemical transformation of cisplatin or carboplatin to a transiodoplatin (PtI₂X₂) form. The binding is only at the N^δ atom of His15. A further Pt species (PtI₃X) is also seen, in both cases bound in a crevice between symmetry-related protein molecules.

The binding of platinum hexahalides (Cl, Br and I) to hen egg-white lysozyme and the chemical transformation of the PtI₆ octahedral complex to a PtI₃ moiety bound to His15

The platinum hexahalides have an octahedral arrangement of six halogen atoms bound to a Pt centre, thus having an octahedral shape that could prove to be useful in interpreting poor electron-density maps. In a detailed characterization, PtI₆ chemically transformed to a square-planar PtI₃ complex bound to the N^δ atom of His15 of HEWL was also observed, which was not observed for PtBr₆ or PtCl₆.

Carboplatin binding to histidine

An X-ray crystal structure showing the binding of purely carboplatin to histidine in a model protein has finally been obtained. This required extensive crystallization trials and various novel crystal structure analyses.

Structural basis for the inhibition of poly(ADP-ribose) polymerases 1 and 2 by BMN 673, a potent inhibitor derived from di­hydropyridophthalazinone

BMN 673, a novel PARP1/2 inhibitor in clinical development with substantial tumor cytotoxicity, forms extensive hydrogen-bonding and π-stacking in the nicotinamide pocket, with its unique disubstituted scaffold extending towards the less conserved edges of the pocket. These interactions might provide structural insight into the ability of BMN 673 to both inhibit catalysis and affect DNA-binding activity.

Structure of nucleoside diphosphate kinase from pacific shrimp (Litopenaeus vannamei) in binary complexes with purine and pyrimidine nucleoside diphosphates

The crystal structure of shrimp nucleoside diphosphate kinase in binary complex with dCDP, dADP and ADP shows conserved contacts independent of the kind of nucleotide bound.

High-resolution crystal structures of alternate forms of the human CD44 hyaluronan-binding domain reveal a site for protein interaction

Two alternate crystal forms of the human CD44 hyaluronan-binding domain, one hexagonal and one monoclinic, have been determined at 1.60 and 1.08 Å resolution, respectively. A short peptide found in the monoclinic form may identify a site of CD44 for protein–protein interactions.

Structure of gluta­thione S-transferase 1 from the major human hookworm parasite Necator americanus (Na-GST-1) in complex with gluta­thione

The structure of Na-GST-1 in complex with GST is reported.

Structure of a DsbF homologue from Corynebacterium diphtheriae

In this study, the crystal structure of a DsbE or DsbF homologue protein from Corynebacterium diphtheriae has been determined, which revealed a thioredoxin-like domain with a typical CXXC active site.

Structure of Mycobacterium smegmatis Eis in complex with paromomycin

The mycobacterial Eis proteins are aminoglycoside acetyltransferases. Here, the crystal structure of M. smegmatis Eis in the paromomycin-bound form is reported at 3.3 Å resolution.

X-ray structures of Nfs2, the plastidial cysteine desulfurase from Arabidopsis thaliana

Cysteine desulfurases (CDs) are enzymes that provide sulfur for several biological processes, including the synthesis of iron–sulfur clusters. The difference in the capacity of the two existing groups of CDs to interact with various partners seems to depend on the flexibility of an extended lobe that harbours the catalytic cysteine.

Structure of Toxoplasma gondii fructose-1,6-bis­phosphate aldolase

The structure of T. gondii fructose-1,6-bisphosphate aldolase, a glycolytic enzyme and structural component of the invasion machinery, was determined to a resolution of 2.0 Å.

Structural analysis of the GH43 enzyme Xsa43E from Butyrivibrio proteoclasticus

The structure of Xsa43E possesses the characteristic five-bladed β-propeller domain seen in all GH43 enzymes. The functional characterization of Xsa43E shows it to be an arabinofuranosidase capable of cleaving arabinose side chains from short segments of xylan.

The structure of alanine racemase from Acinetobacter baumannii

The crystal structure of A. baumannii alanine racemase (Alr_Aba) from the highly antibiotic resistant NCTC13302 strain has been solved to 1.9 Å resolution. Comparison of (Alr_Aba) with alanine racemases from closely related bacteria demonstrates a conserved overall fold.

Crystallization, preliminary X-ray crystallographic and cryo-electron microscopy analysis of a bifunctional enzyme fucokinase/L-fucose-1-P-guanylyltransferase from Bacteroides fragilis

The expression, purification, crystallization, preliminary X-ray crystallographic and cryo-EM analysis of the bifunctional enzyme fucokinase/L-fucose-1-P-guanylyltransferase are reported. A diffraction data set was collected to 3.7 Å resolution from the full-length native protein crystal using synchrotron X-rays. The preliminary cryo-EM image showed two parallel disc-shaped objects with a diameter of 10 nm.

Crystallization of the C-terminal redox domain of the sulfur-assimilatory enzyme APR1 from Arabidopsis thaliana

The redox domain of APR1 from A. thaliana was crystallized in the tetragonal space group P4₃2₁2 or P4₁2₁2 and diffraction data were collected to 2.70 Å resolution.

Crystallization and preliminary X-ray analysis of the periplasmic domain of FliP, an integral membrane component of the bacterial flagellar type III protein-export apparatus

The periplasmic domain of FliP of the bacterial flagellar type III export apparatus has been expressed, purified and crystallized, and the crystals have been characterized by X-ray diffraction.

Crystallization and preliminary X-ray diffraction analysis of the periplasmic domain of the Escherichia coli aspartate receptor Tar and its complex with aspartate

The periplasmic domain of the E. coli aspartate receptor Tar was cloned, expressed, purified and crystallized with and without bound ligand. The crystals obtained diffracted to resolutions of 1.58 and 1.95 Å, respectively.

Cloning, expression, purification, crystallization and preliminary crystallographic analysis of the C-terminal domain of Par-4 (PAWR)

The cloning, overexpression, purification, crystallization and preliminary crystallographic analysis of the C-terminal domain of prostate apoptosis response-4 protein (Par-4) are presented.

Purification, crystallization and preliminary X-ray crystallographic analysis of glycosyltransferase-1 from Bacillus cereus

The purification, crystallization and preliminary X-ray crystallographic analysis of B. cereus glycosyltransferase-1 are reported.

Cloning, purification, crystallization and preliminary X-ray studies of a carbohydrate-binding module (CBM_E1) derived from sugarcane soil metagenome

Aiming at the identification of new carbohydrate-binding modules (CBMs), a sugarcane soil metagenomic library was analyzed and an uncharacterized CBM (CBM_E1) was identified. In this study, CBM_E1 was expressed, purified and crystallized and X-ray diffraction data were collected to 1.95 Å resolution.

Crystallization and preliminary X-ray crystallographic analysis of YfcM: an important factor for EF-P hydroxylation

E. coli YfcM was expressed, purified and crystallized. Crystals of YfcM were obtained by the in situ proteolysis crystallization method. Using these crystals, an X-ray diffraction data set was collected at 1.45 Å resolution.

Expression, purification, crystallization and preliminary crystallographic analysis of human myotubularin-related protein 3

Human myotubularin-related protein 3 was cloned, expressed, purified and crystallized. X-ray diffraction data were collected to 3.3 Å resolution.

Purification, characterization and preliminary X-ray crystallographic studies of monode­hydroascorbate reductase from Oryza sativa L. japonica

Monodehydroascorbate reductase (OsMDHAR) from O. sativa L. japonica was expressed as the active form using E. coli strain NiCo21 (DE3). X-ray diffraction data were collected to 1.9 Å resolution.

Expression, purification, crystallization and preliminary X-ray analysis of glucose-1-phosphate uridylyltransferase (GalU) from Erwinia amylovora

The glucose-1-phosphate uridylyltransferase from E. amylovora has been cloned, expressed, purified and crystallized. Data were collected to 2.46 Å resolution in space group P6₂ and the structure was solved by molecular replacement using the structure of the enzyme from E. coli.

Crystallization and preliminary X-ray analysis of the C-terminal fragment of Ski7 from Saccharomyces cerevisiae

C-terminal domains 2 and 3 of Ski7 from S. cerevisiae were purified and crystallized. The crystals diffracted to 2.0 Å resolution.

Purification, crystallization and preliminary crystallographic analysis of the 16S rRNA methyltransferase RsmI from Escherichia coli

Preliminary crystallographic analysis of the 16S rRNA methyltransferase RsmI from E. coli is reported.

Purification, crystallization and preliminary X-ray crystallographic analysis of TssL from Vibrio cholerae

The crystallization of TssL from V. cholerae is reported.

Complex assembly, crystallization and preliminary X-ray crystallographic analysis of the chicken CD8αα–BF2*0401 complex

A crystal of the chicken MHC class I molecule BF2*0401 in complex with chicken CD8αα (CD8αα–BF2*0401) diffracted to 2.8 Å resolution and belonged to space group P2₁, with unit-cell parameters a = 90.6, b = 90.8, c = 94.9 Å, β = 98°.

Crystallization and preliminary X-ray diffraction analysis of Xaa-Pro dipeptidase from Xanthomonas campestris

The expression, purification and crystallization of Xaa-Pro dipeptidase from X. campestris (GenBank accession No. NP_637763) and its preliminary X-ray diffraction analysis to 1.83 Å resolution are reported.

Recombinant expression, purification, crystallization and preliminary X-ray diffraction analysis of the C-terminal DUF490_963–1138 domain of TamB from Escherichia coli

The C-terminal DUF490_963–1138 domain of TamB (previously YtfN) from E. coli strain K-12 has been overexpressed, purifed and crystallized. Preliminary diffraction data have been collected to 2.1 Å resolution.

Cloning, purification and preliminary crystallographic analysis of the Helicobacter pylori pseudaminic acid biosynthesis N-acetyltransferase PseH

The N-acetyltransferase PseH from the H. pylori pseudaminic acid biosynthesis pathway has been purified and co-crystallized with acetyl-CoA by the hanging-drop vapour-diffusion method. A diffraction data set has been collected to 2.5 Å resolution.

Crystallization and preliminary X-ray crystallographic analysis of the PH-GRAM domain of human MTMR4

The PH-GRAM domain of human MTMR4 was cloned, expressed, purified and crystallized. X-ray diffraction data were collected to 3.20 Å resolution.

Crystallization and preliminary X-ray analysis of two variants of the Escherichia coli O157 ParE2–PaaA2 toxin–antitoxin complex

A new crystal form of the complex of E. coli O157 PaaA2 and ParE2 is described. The crystals belong to space group P4₁2₁2 (or P4₃2₁2) and diffract to 2.8 Å resolution. SeMet-derivatized crystals of the complex diffracting to the same resolution were successfully obtained. Finally, crystallization of the complex of a PaaA2 truncation and ParE2 is also described.

Expression, purification, crystallization and preliminary X-ray diffraction analysis of acylpeptide hydrolase from Deinococcus radiodurans

The expression, purification and crystallization of acylpeptide hydrolase from D. radiodurans (GenBank accession No. NP_293889) and its preliminary X-ray diffraction analysis to 2.6 Å resolution are reported.

Molecular replacement with a large number of molecules in the asymmetric unit

A survey of the Protein Data Bank for entries with a large number of molecules in the asymmetric unit was performed and the strategies used for molecular replacement are described.