A simplified counter diffusion method combined with a 1D simulation program for optimizing crystallization conditions

Tanaka, Hiroaki; Inaka, Koji; Sugiyama, Shigeru; Takahashi, Sachiko; Сано, С.; Satô, Masaru; Yoshitomi, Susumu

doi:10.1107/s0909049503023446

Cited by 66 publications

(52 citation statements)

References 4 publications

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“…9 solution of Emerald BioSystems Wizard I random sparse matrix crystallization screen kit containing 1.0 M (NH 4 ) 2 HPO 4 and 0.1 M sodium acetate buffer, pH 4.5. The crystals suitable for X-ray analysis were obtained using the sitting-drop vapor-diffusion or counter-diffusion [19, 20] crystallization methods. The sitting drops were prepared by mixing 3 μl of the enzyme solution with an equal volume of reservoir solution containing 0.8–1.3 M (NH 4 ) 2 HPO 4 and 0.1 M sodium citrate buffer, pH 4.5–5.5, and equilibrated at 20°C with 0.5 ml of the reservoir solution.…”

Section: Methodsmentioning

confidence: 99%

Crystal Structure of Chitinase ChiW from Paenibacillus sp. str. FPU-7 Reveals a Novel Type of Bacterial Cell-Surface-Expressed Multi-Modular Enzyme Machinery

et al. 2016

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The Gram-positive bacterium Paenibacillus sp. str. FPU-7 effectively hydrolyzes chitin by using a number of chitinases. A unique chitinase with two catalytic domains, ChiW, is expressed on the cell surface of this bacterium and has high activity towards various chitins, even crystalline chitin. Here, the crystal structure of ChiW at 2.1 Å resolution is presented and describes how the enzyme degrades chitin on the bacterial cell surface. The crystal structure revealed a unique multi-modular architecture composed of six domains to function efficiently on the cell surface: a right-handed β-helix domain (carbohydrate-binding module family 54, CBM-54), a Gly-Ser-rich loop, 1st immunoglobulin-like (Ig-like) fold domain, 1st β/α-barrel catalytic domain (glycoside hydrolase family 18, GH-18), 2nd Ig-like fold domain and 2nd β/α-barrel catalytic domain (GH-18). The structure of the CBM-54, flexibly linked to the catalytic region of ChiW, is described here for the first time. It is similar to those of carbohydrate lyases but displayed no detectable carbohydrate degradation activities. The CBM-54 of ChiW bound to cell wall polysaccharides, such as chin, chitosan, β-1,3-glucan, xylan and cellulose. The structural and biochemical data obtained here also indicated that the enzyme has deep and short active site clefts with endo-acting character. The affinity of CBM-54 towards cell wall polysaccharides and the degradation pattern of the catalytic domains may help to efficiently decompose the cell wall chitin through the contact surface. Furthermore, we clarify that other Gram-positive bacteria possess similar cell-surface-expressed multi-modular enzymes for cell wall polysaccharide degradation.

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Section: Methodsmentioning

confidence: 99%

Crystal Structure of Chitinase ChiW from Paenibacillus sp. str. FPU-7 Reveals a Novel Type of Bacterial Cell-Surface-Expressed Multi-Modular Enzyme Machinery

et al. 2016

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“…Crystals of AlDHPyr1147 as isolated (Holo-1) were grown by the modified capillary counterdiffusion technique [27, 28] in microgravity within six weeks. Crystals of the apo form of AlDHPyr1147 (apo), the complex with NADP+ (Holo-2), and the ternary complex with NADP+ and the substrate were obtained by the hanging-drop vapor-diffusion method in Linbro plates (Hampton Research) at 20°C.…”

Section: Methodsmentioning

confidence: 99%

NADP-Dependent Aldehyde Dehydrogenase from ArchaeonPyrobaculum sp.1860: Structural and Functional Features

Bezsudnova

Petrova

Artemova

et al. 2016

Archaea

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We present the functional and structural characterization of the first archaeal thermostable NADP-dependent aldehyde dehydrogenase AlDHPyr1147. In vitro, AlDHPyr1147 catalyzes the irreversible oxidation of short aliphatic aldehydes at 60–85°С, and the affinity of AlDHPyr1147 to the NADP+ at 60°С is comparable to that for mesophilic analogues at 25°С. We determined the structures of the apo form of AlDHPyr1147 (3.04 Å resolution), three binary complexes with the coenzyme (1.90, 2.06, and 2.19 Å), and the ternary complex with the coenzyme and isobutyraldehyde as a substrate (2.66 Å). The nicotinamide moiety of the coenzyme is disordered in two binary complexes, while it is ordered in the ternary complex, as well as in the binary complex obtained after additional soaking with the substrate. AlDHPyr1147 structures demonstrate the strengthening of the dimeric contact (as compared with the analogues) and the concerted conformational flexibility of catalytic Cys287 and Glu253, as well as Leu254 and the nicotinamide moiety of the coenzyme. A comparison of the active sites of AlDHPyr1147 and dehydrogenases characterized earlier suggests that proton relay systems, which were previously proposed for dehydrogenases of this family, are blocked in AlDHPyr1147, and the proton release in the latter can occur through the substrate channel.

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“…The crystallization of the enzyme and its complexes was performed in micro gravity by the counter diffusion method according to the procedure described earlier [3]. The crystals were grown from protein solutions in a 10 mM HEPES buffer, pH 8.0, containing 0.15 M NaCl and 1 mM dithiothreitol (DTT) with the use of MPD and cobalt hexamine as precipitants [1].…”

Section: Methodsmentioning

confidence: 99%

Three-dimensional structure of phosphopantetheine adenylyltransferase from Mycobacterium tuberculosis in the apo form and in complexes with coenzyme A and dephosphocoenzyme A

et al. 2012

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Crystals of phosphopantetheine adenylyltransferase (PPAT) from Mycobacterium tuberculosis in the apo form and in complexes with coenzyme A (PPAT/CoA) and dephosphocoenzyme A (PPAT/dPCoA) were grown in microgravity by the capillary counter diffusion method. The structures of PPAT Mt in the apo form and in complexes with ligands were solved based on the X ray diffraction data collected from the grown crystals. The crystal structures were refined at 1.76, 1.59, and 1.59 Å resolution to Rf factors of 0.175, 0.159, and 0.157 and Rfree of 0.224, 0.208, and 0.206 for PPAT, PPAT/CoA, and PPAT/dPCoA, respectively. The atomic coordinates of the structures were deposited in the Protein Data Bank (PDB ID: 3RFF, 3RHS, and 3RBA). In these structures, the ligand binding sites were determined, the environment of these sites was characterized, and the conformational changes accompanying the ligand binding were analyzed.

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A simplified counter diffusion method combined with a 1D simulation program for optimizing crystallization conditions

Cited by 66 publications

References 4 publications

Crystal Structure of Chitinase ChiW from Paenibacillus sp. str. FPU-7 Reveals a Novel Type of Bacterial Cell-Surface-Expressed Multi-Modular Enzyme Machinery

Crystal Structure of Chitinase ChiW from Paenibacillus sp. str. FPU-7 Reveals a Novel Type of Bacterial Cell-Surface-Expressed Multi-Modular Enzyme Machinery

NADP-Dependent Aldehyde Dehydrogenase from ArchaeonPyrobaculum sp.1860: Structural and Functional Features

Three-dimensional structure of phosphopantetheine adenylyltransferase from Mycobacterium tuberculosis in the apo form and in complexes with coenzyme A and dephosphocoenzyme A

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