scholarly journals The SecB-like chaperone Rv1957 fromMycobacterium tuberculosis: crystallization and X-ray crystallographic analysis

2016 ◽  
Vol 72 (6) ◽  
pp. 457-461 ◽  
Author(s):  
Zuokun Lu ◽  
Han Wang ◽  
TingTing Yu
Keyword(s):  
Crystallographic Analysis ◽  
Unfolded Proteins ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
Sec Pathway ◽  
Secretion Pathway ◽  
High Concentrations

Protein export is important in all bacteria, and bacteria have evolved specialized export machineries to fulfil this task. InMycobacterium tuberculosis, the causative agent of tuberculosis, the general secretion pathway (Sec pathway) is conserved and is essential in performing the export of proteins. The bacterial Sec pathway post-translationally exports unfolded proteins out of the cytoplasm, and the core of the Sec pathway is composed of a heterotrimeric membrane-embedded channel, SecYEG, and two cytosolic components, SecA and SecB. SecB functions by stabilizing unfolded proteins, maintaining them in an export-competent state. Although SecB is mainly found in Proteobacteria, a SecB-like protein, Rv1957, that controls a stress-response toxin–antitoxin system, is found inM. tuberculosis. Rv1957 can also functionally replace theEscherichia coliSecB chaperone bothin vivoandin vitro. In this work, the production, crystallization and X-ray crystallographic analysis of Rv1957 are reported. Notably, diffraction-quality crystals were obtained only at high concentrations of dimethyl sulfoxide,i.e.about 12%(v/v). The crystals of Rv1957 belonged to space groupP212121, with unit-cell parametersa= 64.5,b= 92.0,c= 115.4 Å.

scholarly journals Characterization, crystallization and preliminary X-ray crystallographic analysis of the Uba5 fragment necessary for high-efficiency activation of Ufm1

2014 ◽  
Vol 70 (6) ◽  
pp. 765-768 ◽  
Author(s):  
Shutao Xie
Keyword(s):  
High Efficiency ◽  
Crystallographic Analysis ◽  
Terminal Region ◽  
Adenylation Domain ◽  
Asymmetric Unit ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
Multicellular Organisms

Uba5 is the smallest ubiquitin-like molecule-activating enzyme and contains an adenylation domain and a C-terminal region. This enzyme only exists in multicellular organisms. The mechanism through which the enzyme recognizes and activates ubiquitin-fold modifier 1 (Ufm1) remains unknown. In this study, Uba5 adenylation domains with different C-terminal region lengths were cloned, expressed and purified. The results of anin vitrotruncation assay suggest that Uba5 residues 57–363 comprise the minimal fragment required for the high-efficiency activation of Ufm1. Crystallization of Uba5 residues 57–363 was performed at 277 K using PEG 3350 as the precipitant, and crystals optimized by microseeding diffracted to 2.95 Å resolution, with unit-cell parametersa=b= 97.66,c= 144.83 Å, α = β = 90, γ = 120°. There is one molecule in the asymmetric unit; the Matthews coefficient and the solvent content were calculated to be 2.93 Å3 Da−1and 58.1%, respectively.

Sectioned rubisco crystals in TEM

1990 ◽  
Vol 48 (3) ◽  
pp. 664-665
Author(s):  
Gunnel Karlsson ◽  
Jan-Olov Bovin ◽  
Michael Bosma
Keyword(s):  
Plant Cell ◽  
Carbon Fixation ◽  
Unit Cell ◽  
Protein Crystals ◽  
Unit Cell Parameters ◽  
Cell Parameters ◽  
Photosynthetic Carbon Fixation ◽  
Photosynthetic Carbon

RuBisCO (D-ribulose-l,5-biphosphate carboxylase/oxygenase) is the most aboundant enzyme in the plant cell and it catalyses the key carboxylation reaction of photosynthetic carbon fixation, but also the competing oxygenase reaction of photorespiation. In vitro crystallized RuBisCO has been studied earlier but this investigation concerns in vivo existance of RuBisCO crystals in anthers and leaves ofsugarbeets. For the identification of in vivo protein crystals it is important to be able to determinethe unit cell of cytochemically identified crystals in the same image. In order to obtain the best combination of optimal contrast and resolution we have studied different staining and electron accelerating voltages. It is known that embedding and sectioning can cause deformation and obscure the unit cell parameters.

scholarly journals Purification, crystallization and preliminary crystallographic analysis of a 4-thiouridine synthetase–RNA complex

2013 ◽  
Vol 69 (4) ◽  
pp. 421-424 ◽  
Author(s):  
Peter-Thomas Naumann ◽  
Charles T. Lauhon ◽  
Ralf Ficner
Keyword(s):  
Diffraction Data ◽  
Thermotoga Maritima ◽  
Crystallographic Analysis ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
Orthorhombic Space Group ◽  
X Ray ◽  
Cell Parameters ◽  
Dependent Modification ◽  
Rna Complex

The sulfurtransferase 4-thiouridine synthetase (ThiI) is involved in the ATP-dependent modification of U8 in tRNA. ThiI fromThermotoga maritimawas cloned, overexpressed and purified. A complex comprising ThiI and a truncated tRNA was prepared and crystallized, and X-ray diffraction data were collected to a resolution of 3.5 Å. The crystals belonged to the orthorhombic space groupP212121, with unit-cell parametersa= 102.9,b= 112.8,c= 132.8 Å.

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 Expression, purification, crystallization and preliminary X-ray crystallographic analysis of Enpp6

2014 ◽  
Vol 70 (6) ◽  
pp. 794-799 ◽  
Author(s):  
Junko Morita ◽  
Kazuki Kato ◽  
Emiko Mihara ◽  
Ryuichiro Ishitani ◽  
Junichi Takagi ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Crystallographic Analysis ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
Data Set ◽  
X Ray ◽  
Cell Parameters ◽  
Choline Metabolism ◽  
Protein Molecules ◽  
Membrane Bound

Enpp (ectonucleotide phosphodiesterase/pyrophosphatase) 6 is a membrane-bound glycoprotein that hydrolyzes choline-containing compounds such as lysophosphatidylcholine and glycerophosphorylcholine, and presumably participates in choline metabolism. The catalytic domain of mouse Enpp6 was expressed in HEK293T cells, purified using the TARGET tag/P20.1-Sepharose system and crystallized. An X-ray diffraction data set was collected to 1.8 Å resolution. The crystal belonged to space groupP1, with unit-cell parametersa= 63.7,b= 68.8,c= 69.7 Å, α = 60.6, β = 87.0, γ = 68.1°. Assuming the presence of two protein molecules per asymmetric unit, the solvent content was estimated to be 49.5%.

scholarly journals Purification, crystallization and preliminary crystallographic analysis of histone lysine demethylase NO66 fromHomo sapiens

2012 ◽  
Vol 68 (7) ◽  
pp. 764-766 ◽  
Author(s):  
Xing Zhou ◽  
Yue Tao ◽  
Minhao Wu ◽  
Dandan Zhang ◽  
Jianye Zang
Keyword(s):  
Crystallographic Analysis ◽  
Diffusion Method ◽  
Amino Acid Residues ◽  
X Ray Diffraction ◽  
Cell Parameters ◽  
Histone Lysine Demethylase ◽  
Jmjc Domain ◽  
Lysine Demethylase

NO66 is a JmjC domain-containing histone demethylase with specificity towards histone H3 methylated on both Lys4 and Lys36in vitroandin vivo. A fragment of NO66 lacking the N-terminal 167 amino-acid residues was overexpressed inEscherichia coli, purified and crystallized using the sitting-drop vapour-diffusion method. X-ray diffraction data were collected to a resolution of 2.29 Å. NO66 crystallized in space groupP31orP32, with unit-cell parametersa= 89.35,b = 89.35,c= 304.86 Å, α = β = 90, γ = 120°, and the crystal is likely to contain four molecules in the asymmetric unit.

scholarly journals Purification, crystallization and X-ray crystallographic analysis of human RAB11(S20V), a constitutively active GTP-binding form

2015 ◽  
Vol 71 (10) ◽  
pp. 1247-1250 ◽  
Author(s):  
Chang Min Kim ◽  
Jae Young Choi ◽  
Jong Hwan Yoon ◽  
Hyun Ho Park
Keyword(s):  
Crystallographic Analysis ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
Gtp Hydrolysis ◽  
X Ray ◽  
Cell Parameters ◽  
Gtp Binding ◽  
His Tag ◽  
Hydrolysis Activity ◽  
Constitutively Active

RAB11, a member of the Ras superfamily of small G proteins, is involved in the regulation of vesicle trafficking during endosome recycling. Substitution of Ser20 by Val20 in Rab11 [RAB11(S20V)] inhibits its GTP hydrolysis activity and produces a constitutively active GTP-binding form. In this study, the RAB11(S20V) mutant was overexpressed inEscherichia coliwith an engineered C-terminal His tag. RAB11(S20V) was then purified to homogeneity and was crystallized at 293 K. X-ray diffraction data were collected to a resolution of 2.4 Å from a crystal belonging to space groupI4, with unit-cell parametersa = 74.11,b= 74.11,c= 149.44 Å. The asymmetric unit was estimated to contain two molecules of RAB11(S20V).

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

2014 ◽  
Vol 70 (9) ◽  
pp. 1240-1243
Author(s):  
Ji Young Son ◽  
Jee Un Lee ◽  
Ki-Young Yoo ◽  
Woori Shin ◽  
Dong-Won Im ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Catalytic Domain ◽  
Family Members ◽  
Large Family ◽  
Crystallographic Analysis ◽  
Related Protein ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
Related Proteins

Myotubularin-related proteins are a large family of phosphatases that have the catalytic activity of dephosphorylating the phospholipid molecules phosphatidylinositol 3-phosphate and phosphatidylinositol 3,5-bisphosphate. Each of the 14 family members contains a phosphatase catalytic domain, which is inactive in six family members owing to amino-acid changes in a key motif for the activity. All of the members also bear PH-GRAM domains, which have low homologies between them and have roles that are not yet clear. Here, the cloning, expression, purification and crystallization of human myotubularin-related protein 3 encompassing the PH-GRAM and the phosphatase catalytic domain are reported. Preliminary X-ray crystallographic analysis shows that the crystals diffracted to 3.30 Å resolution at a synchrotron X-ray source. The crystals belonged to space groupC2, with unit-cell parametersa= 323.3,b= 263.3,c= 149.4 Å, β = 109.7°.

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

2014 ◽  
Vol 70 (9) ◽  
pp. 1224-1227 ◽  
Author(s):  
Udaya Kumar Tiruttani Subhramanyam ◽  
Jan Kubicek ◽  
Ulf B. Eidhoff ◽  
Joerg Labahn
Keyword(s):  
Crystallographic Analysis ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
Data Set ◽  
X Ray ◽  
Cell Parameters ◽  
Intrinsically Disordered ◽  
Tumour Suppressor Function ◽  
C Terminus ◽  
Apoptotic Protein

Prostate apoptosis response-4 protein is an intrinsically disordered pro-apoptotic protein with tumour suppressor function. Par-4 is known for its selective induction of apoptosis in cancer cells only and its ability to interact with various apoptotic proteinsviaits C-terminus. Par-4, with its unique function and various interacting partners, has gained importance as a potential target for cancer therapy. The C-terminus of the rat homologue of Par-4 was crystallized and a 3.7 Å resolution X-ray diffraction data set was collected. Preliminary data analysis shows the space group to beP41212. The unit-cell parameters area=b= 115.351,c= 123.663 Å, α = β = γ = 90°.

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%.

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