X-ray grade crystals of the enzymatic fragment of diphtheria toxin

The enzymatic fragment of diphtheria toxin, fragment A (Mr = 21,167), complexed to the dinucleotide adenosine 3',5'-uridine (ApU), has been crystallized at two different values of pH by hanging drop vapor diffusion. Crystals grown at a pH value of 5.0 (from I) belong to the orthorhombic space group P2(1)2(1)2(1), with unit cell parameters a = 71.2 A, b = 73.0 A, c = 139.8 A and four protomers in the asymmetric unit. Crystals grown at a pH value of 8.1 (form II) belong to the monoclinic space group C2, with unit cell parameters a = 65.2 A, b = 85.6 A, c = 34.6 A, beta = 103.0 degrees and one protomer in the asymmetric unit. Both crystal forms diffract to 2.5 A resolution. The molecular structures of fragment A obtained from these two crystal forms may illuminate the pH-dependent transition of diphtheria toxin during membrane translocation.     

Molecular characterization of AmiC,a positive regulator in acetamidase operon of <Emphasis Type="Italic">Mycobacterium smegmatis</Emphasis>

Mycobacterium smegmatis, a rapidly growing non-pathogenic mycobacterium, is currently used as a model organism to study mycobacterial genetics. Acetamidase of M. smegmatis is the highly inducible enzyme of Mycobacteria, which utilizes several amide compounds as sole carbon and nitrogen sources. The acetamidase operon has a complex regulatory mechanism, which involves three regulatory proteins, four promoters, and three operator elements. In our previous study, we showed that over-expression of AmiA leads to a negative regulation of acetamidase by blocking the P2 promoter. In this study, we have identified a new positive regulatory protein, AmiC that interacts with AmiA through protein-protein interaction. Gel mobility shift assay showed that AmiC protein inhibits AmiA from binding to the P2 promoter. Interaction of AmiC with cis-acting elements identified its binding ability to multiple regulatory regions of the operon such as P3, OP3, and P1 promoter/operator. Consequently, the addition of inducer acetamide to AmiC complexe trips the complexes, causing AmiC to appear to be the sensory protein for the amides. Homology modeling and molecular docking studies suggest AmiC as a member of Periplasmic binding proteins, which preferentially bind to the inducers and not to the suppressor. Over-expression of AmiC leads to down-regulation of the negative regulator, amiA, and constitutive up-regulation of acetamidase. Based on these findings, we conclude that AmiC positively regulates the acetamidase operon.     

Crystallization and initial X-ray analysis of the C2-subunit of crustacyanin.

Crystals of the C2-subunit of crustacyanin have been grown from solutions containing ammonium sulphate and 2-methyl-2,4-pentanediol as co-precipitants. The crystals belong to space group P2(1)2(1)2(1) (a = 42.0 A, b = 80.9 A, c = 110.8 A) with two subunits per asymmetric unit and diffract beyond 2.2 A resolution.     

Preliminary crystallographic study of the alpha-D-galactose-specific lectin from jack fruit (Artocarpus integra) seeds

Crystals of the alpha-D-galactose-specific lectin from Jack fruit (Artocarpus integra) have been obtained from polyethylene glycol 400 solutions. The crystals are orthorhombic, space group P2(1)2(1)2 with a = 77.09 A, b = 123.3 A and c = 78.73 A (1 A = 0.1 nm) and have one 39,500 Mr tetramer per asymmetric unit. The crystals diffract to at least 2.8 A on precession photographs.     

Crystallization of the complex of actin with gelsolin segment 1.

Crystals of a 1:1 complex between human gelsolin segment 1 and actin have been grown from solutions containing polyethylene glycol 6000. The crystals are orthorhombic, space group P2(1)2(1)2(1); the axes are a = 57.4 A, b = 70.4 A, c = 184.5 A. They are moderately stable to X-rays and diffract to beyond 2.5 A. There is one molecule of complex in the asymmetric unit.     

Crystallization of alcohol oxidase from Pichia pastoris

Crystals of alcohol oxidase purified from Pichia pastoris were grown in microdialysis buttons in a solution of polyethylene glycol, sodium chloride and sodium azide. The crystals were stratified along the major axis and up to 3 mm in length. X-ray diffraction experiments indicated a space group of P2(1) and unit cell dimensions of a = 157.3 A, b = 171.5 A and c = 231.6 A. Crystals diffract to beyond 2.7 A and are suitable for X-ray structure analysis.     

Crystals of the NC1 domain of human type IV collagen

Crystals of the non-collagenous C-terminal region (NC1) of type IV collagen have been obtained from human placenta. These crystals diffract to 2.0 A, and belong to space group P22(1)2(1), with cell dimensions a = 81 A, b = 158 A, c = 138 A, alpha = beta = gamma = 90 degrees. The crystals contain one hexamer in the asymmetric unit; they are very stable with respect to X-rays.     

Crystals of protein L1 from the 50 S ribosomal subunit of Thermus thermophilus. Preliminary crystallographic data

Crystals have been obtained of protein L1 from the large ribosomal subunit of an extreme thermophile. Thermus thermophilus, using a mixed solution of ammonium sulphate/methane pentanediol. The crystals belong to the space group P2(1)2(1)2, with cell parameters a = 82.7 A, b = 63.4 A, c = 44.7 A. They diffract X-rays to 2.3 A resolution.     

Crystallization and preliminary X-ray analysis of two different forms of mitochondrial creatine kinase from chicken cardiac muscle

Crystals of mitochondrial creatine kinase isolated from chicken heart were grown by precipitation with polyethylene glycol 1000. The enzyme has been crystallized in the absence and presence of ATP in two different space groups. Crystals are tetragonal, with space group P42(1)2, a = b = 171 A, c = 150 A in the absence of ATP; and P422, a = b = 101 A, c = 114.4 A in the presence of ATP. We suggest that there is one octamer (346 kDa) per asymmetric unit without ATP and one dimer (86 kDa) per asymmetric unit with ATP. Using synchrotron radiation, the octameric form diffracts to at least 3 A resolution.     

Crystallization and preliminary X-ray diffraction studies of a deglycosylated glucose oxidase from Penicillium amagasakiense.

The dimeric glucose oxidase from Penicillium amagasakiense was deglycosylated, purified and crystallized as a complex with its coenzyme FAD. Deglycosylation and purification to isoelectric homogeneity were shown to be an important prerequisite step to obtain crystals suitable for X-ray investigations. Crystals of the deglycosylated enzyme were reproducibly grown using ammonium sulfate as precipitant at pH 7.4 to 7.5. Crystals diffract to at least 2.0 A resolution and belong to the orthorhombic space group P2(1)2(1)2(1), with refined lattice constants of a = 59.3 A, b = 136.3 A and c = 156.7 A. Assuming two monomers (approximately 135 kDa) per asymmetric unit the Vm value is 2.3 A3/Da.     

Preliminary investigation of the phage phi X174 crystal structure

Crystals of the single-stranded DNA bacteriophage phi X174 have been grown. They have a monoclinic unit cell with space group P2(1), unit cell dimensions of a = 306.0 (+/- 0.2) A, b = 361.1 (+/- 0.2) A, c = 299.7 (+/- 0.2 degrees) A, beta = 92.91 degrees (+/- 0.02 degrees) and diffract to at least 2.7 A resolution. There are two virus particles per unit cell. Packing considerations show that the mean diameter of the virus particles is 280 A. The virus separates into two bands in a sucrose gradient. The ratio between the absorbance at 260 nm and 280 nm is 1.45 to 1.65 for the faster and 1.15 to 1.35 for the slower bands, but both bands contain intact particles. Crystals derived from these bands are isomorphous and there is no detectable difference in their structure amplitudes.     

Peptidoglycan hydrolysis mediated by the amidase AmiC and its LytM activator NlpD is critical for cell separation and virulence in the phytopathogen Xanthomonas campestris

The essential stages of bacterial cell separation are described as the synthesis and hydrolysis of septal peptidoglycan (PG). The amidase, AmiC, which cleaves the peptide side‐chains linked to the glycan strands, contributes critically to this process and has been studied extensively in model strains of Escherichia coli. However, insights into the contribution of this protein to other processes in the bacterial cell have been limited. Xanthomonas campestris pv. campestris (Xcc) is a phytopathogen that causes black rot disease in many economically important plants. We investigated how AmiC and LytM family regulators, NlpD and EnvC, contribute to virulence and cell separation in this organism. Biochemical analyses of purified AmiC demonstrated that it could hydrolyse PG and its activity could be potentiated by the presence of the regulator NlpD. We also established that deletion of the genes encoding amiC1 or nlpD led to a reduction in virulence as well as effects on colony‐forming units and cell morphology. Moreover, further genetic and biochemical evidence showed that AmiC1 and NlpD affect the secretion of type III effector XC3176 and hypersensitive response (HR) induction in planta. These findings indicate that, in addition to their well‐studied role(s) in cell separation, AmiC and NlpD make an important contribution to the type III secretion (T3S) and virulence regulation in this important plant pathogen.     

Crystallization of arginine-, formylmethionine-tyrosine-, and glycine-transfer RNAs from Escherichia coli.

Crystals as large as 0.5 X 0.3 X 0.2 mm of purified arginine-transfer RNA from Escherichia coli have been prepared by a vapour diffusion method. X-ray diffraction photographs showed that the crystals gave reflections up to 3.7 A spacing. They have a trigonal space group P31 2 1 (OR P32 2 1) and cell-dimensions a=97.2, b=97.2, c=94.8A. Crystals of a mercury derivative of this transfer RNA have also been obtained, and an X-ray diffusion photography of one of them is presented. Formylmethionine-transfer RNA from E. coli was crystallized in various forms, and the appearance of the polymorphs was found to depend upon the amount of spermine in the solution from which the crystallization took place. Crystals of tyrosine-transfer RNA and glycine-transfer RNA have also been obtained.     

Crystallization and preliminary X-ray diffraction studies of the cobalamin-binding domain of methionine synthase from Escherichia coli.

Crystals of a cobalamin-binding domain (M(r) = 28,000) have been grown in polyethylene glycol 6000 at pH 7.5, starting from solutions of intact (M(r) = 133,000) cobalamin-dependent methionine synthase. The crystals are orthorhombic in space group P2(1)2(1)2(1), with cell dimensions a = 96.9 A, b = 55.4 A, c = 103.8 A. For two molecules per asymmetric unit, the calculated VM value is 2.45 A3/Da. A native data set has been collected to 3 A resolution.     

Crystallization and preliminary X-ray investigation of phosphoribosylaminoimidazolesuccinocarboxamide synthase from the yeast Saccharomyces cerevisiae.

Crystals of phosphoribosylaminoimidazolesuccinocarboxamide synthase (EC 6.3.2.6) from the yeast Saccharomyces cerevisiae were grown by the vapor diffusion hanging-drop technique, using ammonium sulfate as the precipitant. The crystals had dimensions up to 1.2 mm. X-ray diffraction experiments indicated a space group of P2(1)2(1)2(1) and unit cell parameters of a = 62.3 A, b = 63.5 A and c = 80.9 A, with one molecule in the asymmetric unit. Native data have been collected to 2.5 A resolution.     

Subcloning of a DNA fragment encoding a single cohesin domain of the Clostridium thermocellum cellulosome-integrating protein CipA: purification, crystallization, and preliminary diffraction analysis of the encoded polypeptide.

An Escherichia coli clone encoding a single cohesin domain of the cellulosome-integrating protein CipA from Clostridium thermocellum was constructed, and the corresponding polypeptide was purified, treated with papain, and crystallized from a PEG 8000 solution. Crystals exhibit orthorhombic symmetry, space group P2(1)2(1)2(1), with cell dimensions a = 37.7 A, b = 80.7 A, c = 93.3 A, and four or eight molecules in the unit cell. The crystals diffract X-rays to beyond 2 A resolution and are suitable for further crystallographic studies.     

Crystallization and preliminary x-ray investigation of the regulatory subunit of cAMP-dependent protein kinase

Crystals of type I cAMP-dependent protein kinase regulatory subunit have been grown from solutions of ammonium sulfate. The crystals are square bipyramids, space group P4(1)2(1)2 (P4(3)2(1)2), with a = b = 106.9 +/- 0.6 A and c = 212.4 +/- 1.0 A. There are two dimers of the regulatory subunit/crystallographic asymmetric unit. The crystals are stable for 3-4 days in the x-ray beam and diffract to at least 3.5-A resolution.     

LytM factor Alr3353 affects filament morphology and cell–cell communication in the multicellular cyanobacterium Anabaena sp. PCC 7120

Heterocyst‐forming cyanobacteria are organized as multicellular filaments of tightly interacting, functionally specialized cells. N₂‐fixing heterocysts differentiate from vegetative cells under nitrogen limitation in a semi‐regular pattern along the filament. Diazotrophic growth requires metabolite exchange between neighboring cells within the filament. This exchange occurs via cell–cell junction complexes that span the gap between the plasma membranes and thereby cross the septal peptidoglycan through an array of uniform nanopores formed by AmiC‐type cell wall hydrolases. We investigated how the lytic hydrolase AmiC1 (Alr0092) from Anabaena sp. PCC 7120, whose activity needs to be tightly controlled to avoid cell lysis, is regulated by the LytM factor Alr3353. Inactivation of alr3353 resulted in significantly fewer nanopores and as a consequence, a lower rate of fluorescent tracer exchange between cells. The mutant was not able to grow with N₂ as sole nitrogen source, although heterocysts were formed. Alr3353 localized mainly to fully developed intercellular septa of vegetative cells. The purified protein bound to peptidoglycan and enhanced the hydrolytic activity of AmiC1 in vitro. Our data show that the LytM factor Alr3353 regulates nanopore formation and cell–cell communication by directly interacting with AmiC1.