Crystallization and Preliminary X-Ray Analysis of Rotavirus Protein VP6

ABSTRACT As a first step to gain insight into the structure of the rotavirus virion at atomic resolution, we report here the expression, purification, and crystallization of recombinant rotavirus protein VP6. This protein has the property of polymerizing in the form of tubular structures in solution which have hindered crystallization thus far. Using a combination of electron microscopy and small-angle X-ray scattering, we found that addition of Ca2+ at concentrations higher than 100 mM results in depolymerization of the tubes, leading to an essentially monodisperse solution of trimeric VP6 even at high protein concentrations (higher than 10 mg/ml), thereby enabling us to search for crystallization conditions. We have thus obtained crystals of VP6 which diffract to better than 2.4 Å resolution and belong to the cubic space group P4132 with a cell dimension a of 160 Å. The crystals contain a trimer of VP6 lying along the diagonal of the cubic unit cell, resulting in one VP6 monomer per asymmetric unit and a solvent content of roughly 70%.

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Crystallization of nepenthesin I using a low-pH crystallization screen

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x15022323 ◽

2016 ◽

Vol 72 (1) ◽

pp. 24-28 ◽

Cited By ~ 3

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.

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Crystallization and preliminary X-ray analysis of a ribokinase fromVibrio choleraeO395

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x14014411 ◽

2014 ◽

Vol 70 (8) ◽

pp. 1098-1102 ◽

Cited By ~ 2

Author(s):

Rakhi Paul ◽

Madhumita Dandopath Patra ◽

Ramanuj Banerjee ◽

Udayaditya Sen

Keyword(s):

Escherichia Coli ◽

Polyethylene Glycol ◽

Catalytic Mechanism ◽

Empty Space ◽

Adenosine Diphosphate ◽

Asymmetric Unit ◽

Solvent Content ◽

X Ray ◽

Polyethylene Glycol 6000 ◽

Insight Into

Ribokinase (RK) is one of the principal enzymes in carbohydrate metabolism, catalyzing the reaction of D-ribose and adenosine triphosphate to produce ribose-5-phosphate and adenosine diphosphate (ADP). To provide further insight into the catalytic mechanism, therbsKgene fromVibrio choleraeO395 encoding ribokinase was cloned and the protein was overexpressed inEscherichia coliBL21 (DE3) and purified using Ni2+–NTA affinity chromatography. Crystals ofV. choleraeRK (Vc-RK) and of its complex with ribose and ADP were grown in the presence of polyethylene glycol 6000 and diffracted to 3.4 and 1.75 Å resolution, respectively. Analysis of the diffraction data showed that both crystals possess symmetry consistent with space groupP1. In the Vc-RK crystals, 16 molecules in the asymmetric unit were arranged in a spiral fashion, leaving a large empty space inside the crystal, which is consistent with its high Matthews coefficient (3.9 Å3 Da−1) and solvent content (68%). In the Vc-RK co-crystals four molecules were located in the asymmetric unit with a Matthews coefficient of 2.4 Å3 Da−1, corresponding to a solvent content of 50%.

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Copper–oxygen Dynamics in Tyrosinase

10.26434/chemrxiv.9255251 ◽

2019 ◽

Author(s):

Nobutaka Fujieda ◽

Sachiko Yanagisawa ◽

Minoru Kubo ◽

Genji Kurisu ◽

Shinobu Itoh

Keyword(s):

Catalytic Mechanism ◽

Substrate Binding ◽

Active Form ◽

Copper Ion ◽

Atomic Resolution ◽

Oxygen Species ◽

X Ray ◽

Oxygen Dynamics ◽

Insight Into ◽

Copper Centre

To unveil the activation of dioxygen on the copper centre (Cu2O2core) of tyrosinase, we performed X-ray crystallograpy with active-form tyrosinase at near atomic resolution. This study provided a novel insight into the catalytic mechanism of the tyrosinase, including the rearrangement of copper-oxygen species as well as the intramolecular migration of copper ion induced by substrate-binding.

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

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x17015102 ◽

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.

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Novel insight into structural magnetism by polarized synchrotron X-ray scattering

Advances in Physics X ◽

10.1080/23746149.2016.1150202 ◽

2016 ◽

Vol 1 (1) ◽

pp. 128-145 ◽

Cited By ~ 3

Author(s):

Hiroyuki Ohsumi ◽

Taka-hisa Arima

Keyword(s):

X Ray ◽

X Ray Scattering ◽

Insight Into ◽

Ray Scattering

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Initial Studies on The Heteroepitaxial Growth of Thin Films of (AI/In)N on Ain-Seeded (00.1) Sapphire by Single-Target Reactive Magnetron Sputtering

MRS Proceedings ◽

10.1557/proc-263-95 ◽

1992 ◽

Vol 263 ◽

Author(s):

T. J. Kistenmacher ◽

S. A. Ecelberger ◽

W. A. Bryden

Keyword(s):

Thin Films ◽

Chemical Composition ◽

Magnetron Sputtering ◽

Electrical Transport ◽

Growth Parameters ◽

Heteroepitaxial Growth ◽

X Ray ◽

X Ray Scattering ◽

Single Target ◽

A Cell

ABSTRACTThin films of (AI/In)N alloys have been deposited on AIN-nucleated (00.1) sapphire by reactive (pure N2 gas) magnetron sputtering and characterized by X-ray scattering, stylus profilometry, optical spectroscopy, and electrical transport measurements. Initial efforts have concentrated on producing films with compositions near Al0.31In0.69N (bandgap tailored to GaN). The alloy sputtering targets were disks fabricated by cold pressing appropriate molar mixtures of beads of 99.99% purity Al and In. The resulting thin films are composed of heteroepitaxial grains {(00.1)InNll(00.1)sapphire; (10.0)InNll(11.0)Sapphire} and their chemical composition has been deduced from the variation in the a cell constant (as measured by the X-ray precession method) to yield equilibrium film compositions near Al0.04In0.96N and Al0.25In0.75N, respectively. Preliminary results are presented on the dependence of the quality of heteroepitaxial growth and electrical and optical properties of. these AlxIn1−xN alloy films on various growth parameters: such as chemical composition; film thickness; morphology; and substrate temperature.

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Fiber Formation during Melt Blowing

International Nonwovens Journal ◽

10.1177/1558925003os-1200209 ◽

2003 ◽

Vol os-12 (2) ◽

pp. 1558925003os-12 ◽

Cited By ~ 12

Author(s):

Randall R. Bresee ◽

Wen-Chien Ko

Keyword(s):

Fiber Diameter ◽

Fiber Formation ◽

Experimental Measurements ◽

Wide Angle ◽

X Ray Diffraction ◽

Air Velocity ◽

Melt Blowing ◽

X Ray ◽

X Ray Scattering ◽

Insight Into

Experimental measurements are presented to provide phenomenological insight into the commercial melt blowing process. In particular, we discuss the following experimental measurements obtained at various die-collector locations: fiber diameter, fiber velocity, air velocity, fiber acceleration, fiber entanglement, fiber temperature, birefringence, wide-angle x-ray diffraction and small-angle x-ray scattering. Our discussion focuses on how these measurements provide insight into fiber formation during melt blowing.

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Real-time insight into nanostructure evolution during the rapid formation of ultra-thin gold layers on polymers

Nanoscale Horizons ◽

10.1039/d0nh00538j ◽

2021 ◽

Author(s):

Matthias Schwartzkopf ◽

Sven-Jannik Wöhnert ◽

Vivian Waclawek ◽

Niko Carstens ◽

André Rothkirch ◽

...

Keyword(s):

Polymer Matrix ◽

Hybrid Material ◽

Time Resolved ◽

X Ray ◽

Polymer Hybrid ◽

X Ray Scattering ◽

Rapid Formation ◽

Metal Polymer ◽

Insight Into

At the nascence of a metal–polymer hybrid material primarily vertical Au dimers and free adatoms diffuse on and into the polymer matrix revealed in situ by sub-millisecond time-resolved surface-sensitive X-ray scattering (GISAXS).

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

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x15000606 ◽

2015 ◽

Vol 71 (3) ◽

pp. 261-265 ◽

Cited By ~ 1

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%.

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