Cloning, expression, crystallization and preliminary X-ray crystallographic analysis of a human condensin SMC2 hinge domain with short coiled coils

In higher eukaryotes, the condensin complex, which mainly consists of two structural maintenance of chromosomes (SMC) subunits, SMC2 (CAP-E) and SMC4 (CAP-C), plays a critical role in the formation of higher order chromosome structures during mitosis. Biochemical and electron-microscopic studies have revealed that the SMC2 and SMC4 subunits dimerize through the interaction of their hinge domains, forming a characteristic V-shaped heterodimer. However, the details of their function are still not fully understood owing to a lack of structural information at the atomic level. In this study, the human SMC2 hinge domain with short coiled coils was cloned, expressed, purified and crystallized in the orthorhombic space groupC222 in native and SeMet-derivatized forms. Because of the poor diffraction properties of these crystals, the mutant Leu68→SeMet was designed and crystallized in order to obtain the experimental phases. The SeMet-derivatized crystals of the mutant belonged to space groupP3212, with unit-cell parametersa=b= 128.8,c = 91.4 Å. The diffraction data obtained from a crystal that diffracted to 2.4 Å resolution were suitable for SAD phasing.

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A proposed carbon-utilization and virulence protein A, CuvA (Rv1422), from Mycobacterium tuberculosis H37Rv: crystallization, X-ray diffraction analysis and ligand binding

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x20008626 ◽

2020 ◽

Vol 76 (7) ◽

pp. 314-319

Author(s):

Yoon Chae Jeong ◽

Ki Seog Lee

Keyword(s):

Mycobacterium Tuberculosis ◽

Environmental Changes ◽

Structural Information ◽

Critical Role ◽

Nutrient Utilization ◽

Diffusion Method ◽

Uridine Diphosphate ◽

Carbon Utilization ◽

Orthorhombic Space Group ◽

Cell Parameters

Mycobacterium tuberculosis possesses the ability to undergo physiological adaptations in order to persist during the prolonged course of infection despite the active immune response of the host and in order to overcome multiple environmental changes. Previous studies have proposed that M. tuberculosis CuvA (Rv1422; MtCuvA) might play a critical role in the adaptation of the bacterium to environmental changes, such as nutrient utilization and alteration of the growth rate. However, the detailed function of MtCuvA still remains unclear owing to a lack of structural information. To better understand its role in host adaptation, MtCuvA was purified to homogeneity and was crystallized for the first time using the hanging-drop vapor-diffusion method. The crystal of MtCuvA diffracted to a resolution of 2.1 Å and belonged to the orthorhombic space group P212121, with unit-cell parameters a = 47.27, b = 170.93, c = 178.10 Å. The calculated Matthews coefficient (V M) was 2.4 Å3 Da−1, with a solvent content of 48.02%, and thus four molecules appeared to be present in the asymmetric unit. Moreover, it is reported that MtCuvA can bind to the cell-wall precursor components uridine diphosphate (UDP)-glucose and UDP-N-acetylglucosamine.

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Two lithium tricyanomethanide compounds: syntheses and single-crystal structure determination of LiK[C(CN)3]2 and Li[C(CN)3]·½ (H3C)2CO

Zeitschrift für Naturforschung B ◽

10.1515/znb-2014-0250 ◽

2015 ◽

Vol 70 (3) ◽

pp. 191-196 ◽

Cited By ~ 1

Author(s):

Olaf Reckeweg ◽

Francis J. DiSalvo

Keyword(s):

Crystal Structure ◽

Single Crystal ◽

Single Crystals ◽

Structure Determination ◽

Monoclinic Space Group ◽

Orthorhombic Space Group ◽

Space Group ◽

Cell Parameters ◽

New Compounds

AbstractThe new compounds LiK[C(CN)3]2 and Li[C(CN)3]·½ (H3C)2CO were synthesized and their crystal structures were determined. Li[C(CN)3]·½ (H3C)2CO crystallizes in the orthorhombic space group Ima2 (no. 46) with the cell parameters a=794.97(14), b=1165.1(2) and c=1485.4(3) pm, while LiK[C(CN)3]2 adopts the monoclinic space group P21/c (no. 14) with the cell parameters a=1265.7(2), b=1068.0(2) and c=778.36(12) pm and the angle β=95.775(7)°. Single crystals of K[C(CN)3] were also acquired, and the crystal structure was refined more precisely than before corroborating earlier results.

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New crystal structures of adenylate kinase fromStreptococcus pneumoniaeD39 in two conformations

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x14020718 ◽

2014 ◽

Vol 70 (11) ◽

pp. 1468-1471

Author(s):

Trung Thanh Thach ◽

Sangho Lee

Keyword(s):

Crystal Structures ◽

Adenylate Kinase ◽

Bound States ◽

Critical Role ◽

Unit Cell ◽

Unit Cell Parameters ◽

Space Group ◽

Cell Parameters ◽

Ligand Free ◽

Adenylate Kinases

Adenylate kinases (AdKs; EC 2.7.3.4) play a critical role in intercellular homeostasis by the interconversion of ATP and AMP to two ADP molecules. Crystal structures of adenylate kinase fromStreptococcus pneumoniaeD39 (SpAdK) have recently been determined using ligand-free and inhibitor-bound crystals belonging to space groupsP21andP1, respectively. Here, new crystal structures of SpAdK in ligand-free and inhibitor-bound states determined at 1.96 and 1.65 Å resolution, respectively, are reported. The new ligand-free crystal belonged to space groupC2, with unit-cell parametersa= 73.5,b= 54.3,c= 62.7 Å, β = 118.8°. The new ligand-free structure revealed an open conformation that differed from the previously determined conformation, with an r.m.s.d on Cαatoms of 1.4 Å. The new crystal of the complex with the two-substrate-mimicking inhibitorP1,P5-bis(adenosine-5′-)pentaphosphate (Ap5A) belonged to space groupP1, with unit-cell parametersa= 53.9,b= 62.3,c= 63.0 Å, α = 101.9, β = 112.6, γ = 89.9°. Despite belonging to the same space group as the previously reported crystal, the new Ap5A-bound crystal contains four molecules in the asymmetric unit, compared with two in the previous crystal, and shows slightly different lattice contacts. These results demonstrate that SpAdK can crystallize promiscuously in different forms and that the open structure is flexible in conformation.

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Crystallization and preliminary X-ray crystallographic analysis of PBPD2 fromListeria monocytogenes

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x14005470 ◽

2014 ◽

Vol 70 (4) ◽

pp. 535-537 ◽

Cited By ~ 1

Author(s):

Hyung Jin Cha ◽

Jae-Hee Jeong ◽

Yeon-Gil Kim

Keyword(s):

Biosynthetic Pathway ◽

Crystallographic Analysis ◽

Diffusion Method ◽

Low Molecular Weight ◽

Molecular Replacement ◽

Orthorhombic Space Group ◽

Penicillin Binding Proteins ◽

Cell Parameters ◽

Replacement Method ◽

Molecular Replacement Method

Penicillin-binding proteins (PBPs), which mediate the peptidoglycan biosynthetic pathway in the bacterial cell wall, have been intensively investigated as a target for the design of antibiotics. In this study, PBPD2, a low-molecular-weight PBP encoded bylmo2812fromListeria monocytogenes, was overexpressed inEscherichia coli, purified and crystallized at 295 K using the sitting-drop vapour-diffusion method. The crystal belonged to the primitive orthorhombic space groupP212121, with unit-cell parametersa= 37.7,b= 74.7,c= 75.1 Å, and diffracted to 1.55 Å resolution. There was one molecule in the asymmetric unit. The preliminary structure was determined by the molecular-replacement method.

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High-resolution structure of an α-spectrin SH3-domain mutant with a redesigned hydrophobic core

Acta Crystallographica Section F Structural Biology and Crystallization Communications ◽

10.1107/s1744309110030095 ◽

2010 ◽

Vol 66 (9) ◽

pp. 1023-1027 ◽

Cited By ~ 3

Author(s):

Ana Cámara-Artigas ◽

Monserrat Andújar-Sánchez ◽

Emilia Ortiz-Salmerón ◽

Celia Cuadri ◽

Eva S. Cobos ◽

...

Keyword(s):

Structural Information ◽

Sh3 Domain ◽

Hydrophobic Core ◽

Orthorhombic Space Group ◽

Cell Parameters ◽

High Resolution Structure ◽

Model Protein ◽

Distal Loop ◽

Modular Domain ◽

The Stability

The α-spectrin SH3 domain (Spc-SH3) is a small modular domain which has been broadly used as a model protein in folding studies and these studies have sometimes been supported by structural information obtained from the coordinates of Spc-SH3 mutants. The structure of B5/D48G, a multiple mutant designed to improve the hydrophobic core and as a consequence the protein stability, has been solved at 1 Å resolution. The crystals belonged to the orthorhombic space groupP212121, with unit-cell parametersa= 24.79,b= 37.23,c= 62.95 Å. This mutant also bears a D48G substitution in the distal loop and this mutation has also been reported to increase the stability of the protein by itself. The structure of the B5/D48G mutant shows a highly packed hydrophobic core and a more ordered distal loop compared with previous Spc-SH3 structures.

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YopT domain of the PfhB2 toxin from Pasteurella multocida: protein expression, characterization, crystallization and crystallographic analysis

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x18000857 ◽

2018 ◽

Vol 74 (3) ◽

pp. 128-134

Author(s):

Sanjeev Kumar ◽

Victoria Hedrick ◽

Seema Mattoo

Keyword(s):

Pasteurella Multocida ◽

Opportunistic Infections ◽

Structural Information ◽

Sequence Similarity ◽

Catalytic Triad ◽

Crystallographic Analysis ◽

High Sequence Similarity ◽

Data Set ◽

Cell Parameters ◽

Tract Infections

Pasteurella multocida causes respiratory-tract infections in a broad range of animals, as well as opportunistic infections in humans. P. multocida secretes a multidomain toxin called PfhB2, which contains a YopT-like cysteine protease domain at its C-terminus. The YopT domain of PfhB2 contains a well conserved Cys–His–Asp catalytic triad that defines YopT family members, and shares high sequence similarity with the prototype YopT from Yersinia sp. To date, only one crystal structure of a YopT family member has been reported; however, additional structural information is needed to help characterize the varied substrate specificity and enzymatic action of this large protease family. Here, a catalytically inactive C3733S mutant of PfhB2 YopT that provides enhanced protein stability was used with the aim of gaining structural insight into the diversity within the YopT protein family. To this end, the C3733S mutant of PfhB2 YopT has been successfully cloned, overexpressed, purified and crystallized. Diffraction data sets were collected from native crystals to 3.5 Å resolution and a single-wavelength anomalous data set was collected from an iodide-derivative crystal to 3.2 Å resolution. Data pertaining to crystals belonging to space group P31, with unit-cell parameters a = 136.9, b = 136.9, c = 74.7 Å for the native crystals and a = 139.2, b = 139.2, c = 74.7 Å for the iodide-derivative crystals, are discussed.

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Crystal Structures and Stability of Two Bipyridyl Complexes of Metal Chloroacetates

Zeitschrift für Naturforschung B ◽

10.1515/znb-2007-1007 ◽

2007 ◽

Vol 62 (10) ◽

pp. 1271-1276 ◽

Cited By ~ 4

Author(s):

Liang Chen ◽

Xian-Wen Wanga ◽

Jing-Zhong Chen ◽

Jian-Hong Liu

Keyword(s):

Binuclear Complexes ◽

Two Dimensional ◽

X Ray Diffraction ◽

Orthorhombic Space Group ◽

Reaction Conditions ◽

Triclinic Space Group ◽

Space Group ◽

X Ray ◽

Cell Parameters ◽

Dimensional Network

The complexes Mn(Cl3CCOO)2(4,4′-bpy) (1) and [Cu2(ClCH2COO)(2,2′-bpy)2(OH)(H2O)]-(NO3)2(2) (bpy = bipyridine) were generated under mild reaction conditions and characterized by IR spectra, thermogravimetric analysis (TGA), X-ray powder diffraction (XRD), and single crystal X-ray diffraction. Compound 1 exhibits a two-dimensional network with octahedrally coordinated Mn(II) atoms linked by 4,4′-bpy ligands and Cl3COO− ligands. Compound 2 features a supramolecular structure of binuclear complexes, with edge-sharing five-coordinated square-pyramidal units bridged by the ClCH2COO− ligand, an OH− group and a water molecule. Complex 1 crystallizes in the orthorhombic space group Pbcn with cell parameters: a = 16.5390(17), b = 11.6396(17), c = 9.9181(12) Å, V = 1909.3(4) Å3, Z = 4, wR2 = 0.1576. Complex 2 crystallizes in the triclinic space group P1̅ with cell parameters: a = 7.6190(15), b = 11.151(2), c = 16.640(3) Å , α = 73.13(3), β = 80.89(3), γ = 74.51(3)°, V = 1298.73(4) Å3, Z = 2, wR2 = 0.1265.

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

Acta Crystallographica Section F Structural Biology and Crystallization Communications ◽

10.1107/s1744309113004077 ◽

2013 ◽

Vol 69 (4) ◽

pp. 421-424 ◽

Cited By ~ 2

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.

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Synthese und Struktur saurer Ubergangsmetallsulfate - Ti(H5O2)(SO4)2(H2O)2 und Zr(H3O)2(SO4)3 / Synthesis and Structure of Acid Transition Metal Sulfates - Ti(H5O2)(SO4)2(H2O)2 und Zr(H3O)2(SO4)3

Zeitschrift für Naturforschung B ◽

10.1515/znb-1996-0105 ◽

1996 ◽

Vol 51 (1) ◽

pp. 19-24 ◽

Cited By ~ 10

Author(s):

S. Trojanov ◽

A. Stiewe ◽

E. Kemnitz

Keyword(s):

Transition Metal ◽

Hydrogen Bonds ◽

Ternary System ◽

Orthorhombic Space Group ◽

Triclinic Space Group ◽

Space Group ◽

Cell Parameters ◽

Titanium Compound

Abstract From the ternary system Mn(SO4)n/2 / SO3 / H2O (Mn+ = Zr4+, Ti3+) two new acid metal sulfates, Ti(H5O2)(SO4)2(H2O)2 and Zr(H3O) 2(SO4)3, have been synthesized and structurally characterized. Ti(H5O2)(SO4)2(H2O)2 crystallizes in the orthorhombic space group Pnma with cell parameters a = 9.858(5), b = 18.32(2), c = 5.437(7)Å (Z = 4); Zr(H3O)2(SO4)3 crystallizes in the triclinic space group P1 with cell parameters a = 5.092(2), b = 8.533(3), c = 12.388(6)A, α = 92.95(3)°, β = 90.89(3)°, γ = 94.35(3)° (Z= 2). In the titanium compound there are slightly distorted TiO6 octahedra with Ti-O distances between 1.99 and 2.09 Å which are sharing corners with SO4 tetrahedra. Through hydrogen bonds layers are formed perpendicular to the b-axis. These layers are connected by H3O+ ions only. The structure of Zr(H3O)2(SO4)3 consists of three different SO4 tetrahedra and distorted ZrO7 polyhedra which are connected via common corners and edges to form columns parallel to the a-axis. The Zr-O distances vary from 2.13 to 2.18Å for equatorial positions, significantly longer than the axial Zr-O distances with 2.05Å.

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Purification, crystallization and initial crystallographic analysis of the α-catenin homologue HMP-1 fromCaenorhabditis elegans

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x16001862 ◽

2016 ◽

Vol 72 (3) ◽

pp. 234-239 ◽

Cited By ~ 2

Author(s):

Hyunook Kang ◽

Injin Bang ◽

William I. Weis ◽

Hee-Jung Choi

Keyword(s):

Crystallographic Analysis ◽

Unit Cell ◽

Molecular Replacement ◽

Binding Region ◽

Unit Cell Parameters ◽

Mechanical Tension ◽

Space Group ◽

Cell Parameters ◽

Terminal Domain ◽

Mammalian Homologue

Adherens junctions transmit mechanical force between cells. In these junctions, β-catenin binds to cadherins and to the N-terminal domain of α-catenin, which in turn binds to actin filamentsviaits C-terminal domain. The middle (M) domain of α-catenin plays an important role in responding to mechanical tension. The nematodeCaenorhabditis eleganscontains α- and β-catenin homologues called HMP-1 and HMP-2, respectively, but HMP-1 behaves differently from its mammalian homologue. Thus, structural and biochemical studies of HMP-1 have been initiated to understand the mechanism of HMP-1 and the evolution of α-catenin. The N-terminal domain of HMP-1 in complex with the minimal HMP-1-binding region of HMP-2 was purified and crystallized. These crystals diffracted to 1.6 Å resolution and belonged to space groupP3121, with unit-cell parametersa=b= 57.1,c= 155.4 Å. The M domain of HMP-1 was also purified and crystallized. The M-domain crystals diffracted to 2.4 Å resolution and belonged to space groupP212121, with unit-cell parametersa = 72.8,b= 81.5,c = 151.4 Å. Diffraction data were collected and processed from each crystal, and the structures were solved by molecular replacement.

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