scholarly journals Crystallization of nepenthesin I using a low-pH crystallization screen

2016 ◽  
Vol 72 (1) ◽  
pp. 24-28 ◽  
Author(s):  
Karla Fejfarová ◽  
Alan Kádek ◽  
Hynek Mrázek ◽  
Jiří Hausner ◽  
Vyacheslav Tretyachenko ◽  
...  
Keyword(s):  
Low Ph ◽  
Asymmetric Unit ◽  
Unit Cell Parameters ◽  
Aspartic Proteases ◽  
Solvent Content ◽  
X Ray ◽  
Cell Parameters ◽  
Crystallization Experiment ◽  
Crystallization Conditions ◽  
Pepstatin A

Nepenthesins are aspartic proteases secreted by carnivorous pitcher plants of the genusNepenthes. They significantly differ in sequence from other plant aspartic proteases. This difference, which provides more cysteine residues in the structure of nepenthesins, may contribute to their unique stability profile. Recombinantly produced nepenthesin 1 (rNep1) fromN. gracilisin complex with pepstatin A was crystallized under two different crystallization conditions using a newly formulated low-pH crystallization screen. The diffraction data were processed to 2.9 and 2.8 Å resolution, respectively. The crystals belonged to space groupP212121, with unit-cell parametersa= 86.63,b= 95.90,c= 105.40 Å, α = β = γ = 90° anda= 86.28,b= 97.22,c= 103.78 Å, α = β = γ = 90°, respectively. Matthews coefficient and solvent-content calculations suggest the presence of two molecules of rNep1 in the asymmetric unit. Here, the details of the crystallization experiment and analysis of the X-ray data are reported.

scholarly journals Crystallization and X-ray diffraction analysis of native and selenomethionine-substituted PhyH-DI fromBacillussp. HJB17

2017 ◽  
Vol 73 (11) ◽  
pp. 607-611
Author(s):  
Fang Lu ◽  
Bei Zhang ◽  
Yong Liu ◽  
Ying Song ◽  
Gangxing Guo ◽  
...  
Keyword(s):  
Unit Cell ◽  
Diffusion Method ◽  
Data Sets ◽  
Asymmetric Unit ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
Solvent Content ◽  
Space Group ◽  
X Ray ◽  
Cell Parameters

Phytases are phosphatases that hydrolyze phytates to less phosphorylatedmyo-inositol derivatives and inorganic phosphate. β-Propeller phytases, which are very diverse phytases with improved thermostability that are active at neutral and alkaline pH and have absolute substrate specificity, are ideal substitutes for other commercial phytases. PhyH-DI, a β-propeller phytase fromBacillussp. HJB17, was found to act synergistically with other single-domain phytases and can increase their efficiency in the hydrolysis of phytate. Crystals of native and selenomethionine-substituted PhyH-DI were obtained using the vapour-diffusion method in a condition consisting of 0.2 Msodium chloride, 0.1 MTris pH 8.5, 25%(w/v) PEG 3350 at 289 K. X-ray diffraction data were collected to 3.00 and 2.70 Å resolution, respectively, at 100 K. Native PhyH-DI crystals belonged to space groupC121, with unit-cell parametersa = 156.84,b = 45.54,c = 97.64 Å, α = 90.00, β = 125.86, γ = 90.00°. The asymmetric unit contained two molecules of PhyH-DI, with a corresponding Matthews coefficient of 2.17 Å3 Da−1and a solvent content of 43.26%. Crystals of selenomethionine-substituted PhyH-DI belonged to space groupC2221, with unit-cell parametersa = 94.71,b= 97.03,c= 69.16 Å, α = β = γ = 90.00°. The asymmetric unit contained one molecule of the protein, with a corresponding Matthews coefficient of 2.44 Å3 Da−1and a solvent content of 49.64%. Initial phases for PhyH-DI were obtained from SeMet SAD data sets. These data will be useful for further studies of the structure–function relationship of PhyH-DI.

scholarly journals Crystallization and preliminary X-ray crystallographic analysis of human myotubularin-related protein 1

2015 ◽  
Vol 71 (3) ◽  
pp. 261-265 ◽  
Author(s):  
Seoung Min Bong ◽  
Seung Won Yang ◽  
Ji-Woong Choi ◽  
Seung Jun Kim ◽  
Byung Il Lee
Keyword(s):  
Escherichia Coli ◽  
Polyethylene Glycol ◽  
Synchrotron Radiation ◽  
Crystallographic Analysis ◽  
Asymmetric Unit ◽  
Related Protein ◽  
Unit Cell Parameters ◽  
Solvent Content ◽  
X Ray ◽  
Cell Parameters

Myotubularin-related protein 1 is a phosphatase that dephosphorylates phospholipids such as phosphatidylinositol 3-phosphate or phosphatidylinositol 3,5-bisphosphate. In this study, human MTMR1 was overexpressed inEscherichia coli, purified and crystallized at 277 K using polyethylene glycol 20 000 as a precipitant. Diffraction data were collected to 2.0 Å resolution using synchrotron radiation. The crystals belonged to space groupP1, with unit-cell parametersa= 67.219,b= 96.587,c= 97.581 Å, α = 87.597, β = 86.072, γ = 77.327°. Assuming the presence of four molecules in the asymmetric unit, the calculated Matthews coefficient value was 2.61 Å3 Da−1and the corresponding solvent content was 52.9%.

scholarly journals Purification, crystallization and preliminary X-ray crystallographic analysis of the UDP-N-acetylmuramoyl-tripeptide-D-alanyl-D-alanine ligase (MurF) fromAcinetobacter baumannii

2014 ◽  
Vol 70 (7) ◽  
pp. 976-978 ◽  
Author(s):  
Young Jun An ◽  
Chang-Sook Jeong ◽  
Jeong Hee Yu ◽  
Kyung Min Chung ◽  
Sun-Shin Cha
Keyword(s):  
Multidrug Resistant ◽  
Crystallographic Analysis ◽  
Asymmetric Unit ◽  
Unit Cell Parameters ◽  
Solvent Content ◽  
X Ray ◽  
Cell Parameters ◽  
Peptidoglycan Biosynthesis ◽  
Crystallization Method ◽  
Global Spread

The emergence and global spread of multidrug-resistantAcinetobacter baumanniistrains are major threats to public health. Inhibition of peptidoglycan biosynthesis is an effective strategy for the development of antibiotics. The ATP-dependent UDP-N-acetylmuramoyl-tripeptide-D-alanyl-D-alanine ligase (MurF) that is responsible for the last step of peptidoglycan biosynthesis is a validated target for the development of antibiotics. Crystals ofA. baumanniiMurF in complex with ATP were grown by the microbatch crystallization method at 295 K. The crystals belonged to space groupP3221, with unit-cell parametersa=b= 85.42,c= 129.86 Å. Assuming the presence of one molecule in the asymmetric unit, the solvent content was estimated to be about 54.32%.

scholarly journals Crystallization and preliminary X-ray analysis of the NAD+-reducing [NiFe] hydrogenase fromHydrogenophilus thermoluteolusTH-1

2015 ◽  
Vol 71 (1) ◽  
pp. 96-99 ◽  
Author(s):  
Midori Taketa ◽  
Hanae Nakagawa ◽  
Mao Habukawa ◽  
Hisao Osuka ◽  
Kiyohito Kihira ◽  
...  
Keyword(s):  
Diffusion Method ◽  
Asymmetric Unit ◽  
Dispersion Method ◽  
Unit Cell Parameters ◽  
Solvent Content ◽  
X Ray ◽  
Cell Parameters ◽  
Vapour Diffusion ◽  
Anomalous Signal ◽  
Th 1

NAD+-reducing [NiFe] hydrogenases catalyze the oxidoreduction of dihydrogen concomitant with the interconversion of NAD+and NADH. Here, the isolation, purification and crystallization of the NAD+-reducing [NiFe] hydrogenase fromHydrogenophilus thermoluteolusTH-1 are reported. Crystals of the NAD+-reducing [NiFe] hydrogenase were obtained within one week from a solution containing polyethylene glycol using the sitting-drop vapour-diffusion method and micro-seeding. The crystal diffracted to 2.58 Å resolution and belonged to space groupC2, with unit-cell parametersa= 131.43,b= 189.71,c= 124.59 Å, β = 109.42°. Assuming the presence of two NAD+-reducing [NiFe] hydrogenase molecules in the asymmetric unit,VMwas calculated to be 2.2 Å3 Da−1, which corresponds to a solvent content of 43%. Initial phases were determined by the single-wavelength anomalous dispersion method using the anomalous signal from the Fe atoms.

scholarly journals Purification, crystallization and preliminary X-ray data collection of the N-terminal domain of the 26S proteasome regulatory subunit p27 and its complex with the ATPase domain of Rpt5 fromMus musculus

2014 ◽  
Vol 70 (5) ◽  
pp. 611-615
Author(s):  
Wentao Diao ◽  
Xue Yang ◽  
Hao Zhou
Keyword(s):  
Unit Cell ◽  
26S Proteasome ◽  
Regulatory Subunit ◽  
Atpase Domain ◽  
Asymmetric Unit ◽  
Unit Cell Parameters ◽  
Solvent Content ◽  
Space Group ◽  
X Ray ◽  
Cell Parameters

The protein 26S proteasome regulatory subunit p27 is one of the four chaperones that help in the assembly of the 19S regulatory particle (RP) of the 26S proteasome. In the present work, the N-terminus of p27 (residues 1–128) fromMus musculuswas cloned, expressed, purified and crystallized alone and in complex with the C-terminal ATPase domain of Rpt5 (residues 173–442). The crystals of p27(1–128)diffracted to 1.7 Å resolution and belonged to space groupP212121, with unit-cell parametersa= 26.79,b= 30.39,c= 145.06 Å. Resolution-dependent Matthews coefficient probability analysis suggested the presence of only one molecule per asymmetric unit, with 40.5% solvent content and aVMvalue of 2.02 Å3 Da−1. The crystal of the p27(1–128)–Rpt5(173–442)complex diffracted to 4 Å resolution and belonged to space groupP222, with unit-cell parametersa= 75.93,b= 76.08,c= 336.85 Å. The presence of four heterodimers in the asymmetric unit with 53.2% solvent content and aVMvalue of 2.63 Å3 Da−1or five heterodimers in the asymmetric unit with 41.5% solvent content and aVMvalue of 2.10 Å3 Da−1is assumed.

scholarly journals Crystallization and X-ray analysis ofD-threonine aldolase fromChlamydomonas reinhardtii

2017 ◽  
Vol 73 (2) ◽  
pp. 86-89
Author(s):  
Yuki Hirato ◽  
Masaru Goto ◽  
Mayumi Tokuhisa ◽  
Minoru Tanigawa ◽  
Katsushi Nishimura
Keyword(s):  
Diffusion Method ◽  
Asymmetric Unit ◽  
Hanging Drop ◽  
Unit Cell Parameters ◽  
Solvent Content ◽  
X Ray ◽  
Cell Parameters ◽  
Threonine Aldolase ◽  
Vapour Diffusion ◽  
Resolution Analysis

D-Threonine aldolase from the green algaChlamydomonas reinhardtii(CrDTA) catalyzes the interconversion of several β-hydroxy-D-amino acids (e.g.D-threonine) and glycine plus the corresponding aldehydes. Recombinant CrDTA was overexpressed inEscherichia coliand purified to homogeneity; it was subsequently crystallized using the hanging-drop vapour-diffusion method at 295 K. Data were collected and processed at 1.85 Å resolution. Analysis of the diffraction pattern showed that the crystal belonged to space groupP1, with unit-cell parametersa= 64.79,b= 74.10,c= 89.94 Å, α = 77.07, β = 69.34, γ = 71.93°. The asymmetric unit contained four molecules of CrDTA. The Matthews coefficient was calculated to be 2.12 Å3 Da−1and the solvent content was 41.9%.

scholarly journals Expression, crystallization and preliminary X-ray crystallographic analysis ofD-alanine-D-alanine ligase from OXA-23-producingAcinetobacter baumanniiK0420859

2014 ◽  
Vol 70 (4) ◽  
pp. 505-508 ◽  
Author(s):  
Kim-Hung Huynh ◽  
Huyen-Thi Tran ◽  
Tan-Viet Pham ◽  
Ho-Phuong-Thuy Ngo ◽  
Sun-Shin Cha ◽  
...  
Keyword(s):  
Crystallographic Analysis ◽  
Antibacterial Drug ◽  
Asymmetric Unit ◽  
Unit Cell Parameters ◽  
Orthorhombic Space Group ◽  
Solvent Content ◽  
X Ray ◽  
Cell Parameters ◽  
Novel Target ◽  
Tract Infections

Acinetobacter baumanniicauses bacteraemia, pneumonia, other respiratory-tract and urinary-tract infections in humans. OXA-23 carbapenemase-producingA. baumanniiK0420859 (A. baumanniiOXA-23) is resistant to carbapenem, a common antibacterial drug. To develop an efficient and novel antibacterial drug againstA. baumanniiOXA-23, D-alanine-D-alanine ligase, which is essential in bacterial cell-wall synthesis, is of interest. Here, the D-alanine-D-alanine ligase (AbDdl) gene fromA. baumanniiOXA-23 was cloned and expressed, and theAbDdl protein was purified and crystallized; this enzyme can be used as a novel target for an antibacterial drug againstA. baumanniiOXA-23. TheAbDdl crystal diffracted to a resolution of 2.8 Å and belonged to the orthorhombic space groupP212121, with unit-cell parametersa= 113.4,b= 116.7,c= 176.5 Å, a correspondingVMof 2.8 Å3 Da−1and a solvent content of 56.3%, and six protomers in the asymmetric unit.

Crystallization and preliminary X-ray analysis of a complex between the Bowman–Birk trypsin inhibitor from barley and porcine pancreatic trypsin

1999 ◽  
Vol 55 (6) ◽  
pp. 1244-1246 ◽  
Author(s):  
Young Sil Kim ◽  
Hyun Kyu Song ◽  
Se Won Suh
Keyword(s):  
Polyethylene Glycol ◽  
Trypsin Inhibitor ◽  
Unit Cell ◽  
X Rays ◽  
Asymmetric Unit ◽  
Unit Cell Parameters ◽  
Orthorhombic Space Group ◽  
Solvent Content ◽  
X Ray ◽  
Cell Parameters

A 1:2 complex between the Bowman–Birk trypsin inhibitor from barley seeds and porcine pancreatic trypsin has been crystallized at 291 K using polyethylene glycol as precipitant. The crystals belong to the orthorhombic space group P212121, with unit-cell parameters a = 67.10, b = 88.38 and c = 203.65 Å. The asymmetric unit contains two monomers of the complex, with a corresponding Vm of 2.41 Å3 Da−1 and a solvent content of 49%. Native data to 2.2 Å resolution have been collected at 100 K using synchrotron X-rays.

scholarly journals Crystallographic analysis of FAD-dependent glucose dehydrogenase

2015 ◽  
Vol 71 (8) ◽  
pp. 1017-1019 ◽  
Author(s):  
Hirofumi Komori ◽  
Koji Inaka ◽  
Naoki Furubayashi ◽  
Michinari Honda ◽  
Yoshiki Higuchi
Keyword(s):  
Aspergillus Terreus ◽  
Glucose Dehydrogenase ◽  
Crystallographic Analysis ◽  
Diffusion Method ◽  
Asymmetric Unit ◽  
Unit Cell Parameters ◽  
Data Set ◽  
Solvent Content ◽  
X Ray ◽  
Cell Parameters

An FAD-dependent glucose dehydrogenase (GDH) fromAspergillus terreuswas purified and crystallized at 293 K using the sitting-drop vapour-diffusion method. A data set was collected to a resolution of 1.6 Å from a single crystal at 100 K using a rotating-anode X-ray source. The crystal belonged to space groupP21, with unit-cell parametersa= 56.56,b= 135.74,c= 74.13 Å, β = 90.37°. The asymmetric unit contained two molecules of GDH. The Matthews coefficient was calculated to be 2.2 Å3 Da−1and the solvent content was estimated to be 44%.

Crystallization and preliminary X-ray studies of metallothionein II from rabbit liver

1999 ◽  
Vol 55 (6) ◽  
pp. 1242-1243 ◽  
Author(s):  
Yu An ◽  
Genpei Li ◽  
Binggen Ru
Keyword(s):  
Sodium Citrate ◽  
Unit Cell ◽  
Rabbit Liver ◽  
Asymmetric Unit ◽  
Unit Cell Parameters ◽  
Citrate Buffer ◽  
Solvent Content ◽  
X Ray ◽  
Cell Parameters ◽  
Sodium Citrate Buffer

Metallothionein II (Mw = 6.8 kDa), induced by cadmium and purified from rabbit liver, has been crystallized in space group P6222 or P6422. The unit-cell parameters were a = b = 113.4, c = 219.1 Å, α = β = 90.0, γ = 120.0° when crystallized from sodium citrate buffer and a = b = 113.4, c = 219.6 Å, α = β = 90.0, γ = 120.0° when crystallized from Tris–HCl buffer. There are 12 molecules per asymmetric unit and the solvent content is about 57%.

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