The structure of the Pfp1 protease from the hyperthermophilic archaeonThermococcus thioreducensin two crystal forms

2017 ◽  
Vol 73 (9) ◽  
pp. 749-756 ◽  
Author(s):  
Steven B. Larson ◽  
Alexander McPherson
Keyword(s):  
Active Site ◽  
Disulfide Bonds ◽  
Room Temperature ◽  
Search Model ◽  
Molecular Replacement ◽  
Pyrococcus Horikoshii ◽  
Crystal Forms ◽  
X Ray ◽  
Laboratory Source ◽  
Intermolecular Disulfide Bonds

The Pfp1 protease, a cysteine protease of unknown specificity from the hyperthermophilic archaeonThermococcus thioreducens, was crystallized in two distinctive crystal forms: from concentrated citrate in one case and PEG in the other. X-ray data were collected from both crystal forms at room temperature to about 1.9 Å resolution using a laboratory source and detector, and the structures were solved by molecular replacement using the Pfp1 protease fromPyrococcus horikoshiias the search model. In theT. thioreducensprotease structures, Cys18 residues on adjacent molecules in the asymmetric units form intermolecular disulfide bonds, thereby yielding hexamers composed of three cross-linked, quasi-dyad-related dimers with crystallographically exact threefold axes and exhibiting almost exact 32 symmetry. The corresponding residue inP. horikoshiiPfp1 is Tyr18. An individual active site containing Cys100 and His101 also includes a Glu74 residue contributed by a quasi-twofold-related, non-cross-linked subunit. Two catalytic triads are therefore closely juxtaposed about the quasi-twofold axis at the interface of these subunits, and are relatively sequestered within the hexamer cavity. The cysteine in the active site is observed to be oxidized in both of the crystal forms that were studied.

Crystallization and preliminary X-ray diffraction studies of human catalase

1999 ◽  
Vol 55 (9) ◽  
pp. 1614-1615 ◽  
Author(s):  
R. A. P. Nagem ◽  
E. A. L. Martins ◽  
V. M. Gonçalves ◽  
R. Aparício ◽  
I. Polikarpov
Keyword(s):  
Search Model ◽  
Molecular Replacement ◽  
X Ray Diffraction ◽  
Beef Liver ◽  
X Ray ◽  
Diffusion Technique ◽  
Cell Dimensions ◽  
Synchrotron Radiation Diffraction ◽  
Replacement Solution ◽  
Unit Cell Dimensions

The enzyme catalase (H2O2–H2O2 oxidoreductase; E.C. 11.1.6) was purified from haemolysate of human placenta and crystallized using the vapour-diffusion technique. Synchrotron-radiation diffraction data have been collected to 1.76 Å resolution. The enzyme crystallized in the space group P212121, with unit-cell dimensions a = 83.6, b = 139.4, c = 227.5 Å. A molecular-replacement solution of the structure has been obtained using beef liver catalase (PDB code 4blc) as a search model.

scholarly journals X-ray crystal structure of a malonate-semialdehyde dehydrogenase fromPseudomonassp. strain AAC

2017 ◽  
Vol 73 (1) ◽  
pp. 24-28 ◽  
Author(s):  
Matthew Wilding ◽  
Colin Scott ◽  
Thomas S. Peat ◽  
Janet Newman
Keyword(s):  
Crystal Structure ◽  
Search Model ◽  
Molecular Replacement ◽  
X Rays ◽  
X Ray Diffraction ◽  
Acetyl Coa ◽  
Space Group ◽  
X Ray ◽  
Semialdehyde Dehydrogenase

The NAD-dependent malonate-semialdehyde dehydrogenase KES23460 fromPseudomonassp. strain AAC makes up half of a bicistronic operon responsible for β-alanine catabolism to produce acetyl-CoA. The KES23460 protein has been heterologously expressed, purified and used to generate crystals suitable for X-ray diffraction studies. The crystals belonged to space groupP212121and diffracted X-rays to beyond 3 Å resolution using the microfocus beamline of the Australian Synchrotron. The structure was solved using molecular replacement, with a monomer from PDB entry 4zz7 as the search model.

Recent insights into lytic polysaccharide monooxygenases (LPMOs)

2018 ◽  
Vol 46 (6) ◽  
pp. 1431-1447 ◽  
Author(s):  
Tobias Tandrup ◽  
Kristian E. H. Frandsen ◽  
Katja S. Johansen ◽  
Jean-Guy Berrin ◽  
Leila Lo Leggio
Keyword(s):  
Active Site ◽  
Room Temperature ◽  
Experimental Studies ◽  
Binding Mode ◽  
Structural Features ◽  
Oxygen Species ◽  
Animal Kingdom ◽  
X Ray ◽  
Lytic Polysaccharide Monooxygenases ◽  
Polysaccharide Monooxygenases

Lytic polysaccharide monooxygenases (LPMOs) are copper enzymes discovered within the last 10 years. By degrading recalcitrant substrates oxidatively, these enzymes are major contributors to the recycling of carbon in nature and are being used in the biorefinery industry. Recently, two new families of LPMOs have been defined and structurally characterized, AA14 and AA15, sharing many of previously found structural features. However, unlike most LPMOs to date, AA14 degrades xylan in the context of complex substrates, while AA15 is particularly interesting because they expand the presence of LPMOs from the predominantly microbial to the animal kingdom. The first two neutron crystallography structures have been determined, which, together with high-resolution room temperature X-ray structures, have putatively identified oxygen species at or near the active site of LPMOs. Many recent computational and experimental studies have also investigated the mechanism of action and substrate-binding mode of LPMOs. Perhaps, the most significant recent advance is the increasing structural and biochemical evidence, suggesting that LPMOs follow different mechanistic pathways with different substrates, co-substrates and reductants, by behaving as monooxygenases or peroxygenases with molecular oxygen or hydrogen peroxide as a co-substrate, respectively.

STRUCTURAL DETERMINATION OF THE Si/Cu(110) INTERFACE BY PHOTOELECTRON DIFFRACTION

1997 ◽  
Vol 04 (06) ◽  
pp. 1331-1335 ◽  
Author(s):  
C. ROJAS ◽  
J. A. MARTÍn-GAGO ◽  
E. ROMÁN ◽  
G. PAOLUCCI ◽  
B. BRENA ◽  
...  
Keyword(s):  
Room Temperature ◽  
Structural Parameters ◽  
Single Scattering ◽  
Structural Determination ◽  
X Ray ◽  
Photoelectron Diffraction ◽  
First Order ◽  
Proposed Model ◽  
Laboratory Source

Deposition of 0.5 Si monolayer (ML) on a Cu (110) surface at room temperature (RT) leads to the formation of a c(2×2) LEED pattern. In order to find out the surface atomic structure of this ordered phase, X-ray photoelectron diffraction (XPD) azimuthal scans at different photon energies and full hemispherical XPD patterns of the Si 2 p core level have been measured using both synchrotron radiation and a laboratory source. We present an atomic model for the surface structure based on the examination of forward scattering and first order interference XPD features. Refinement of the structural parameters was achieved by performing single scattering cluster (SSC) calculations. In the proposed model Si atoms replace Cu atoms at the surface along the [Formula: see text] atomic rows.

scholarly journals Ensemble-based enzyme design can recapitulate the effects of laboratory directed evolution in silico

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Aron Broom ◽  
Rojo V. Rakotoharisoa ◽  
Michael C. Thompson ◽  
Niayesh Zarifi ◽  
Erin Nguyen ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Protein Design ◽  
Active Site ◽  
Room Temperature ◽  
De Novo ◽  
Conformational Ensemble ◽  
Enzyme Design ◽  
X Ray ◽  
Conformational Ensembles ◽  
X Ray Crystallography

Abstract The creation of artificial enzymes is a key objective of computational protein design. Although de novo enzymes have been successfully designed, these exhibit low catalytic efficiencies, requiring directed evolution to improve activity. Here, we use room-temperature X-ray crystallography to study changes in the conformational ensemble during evolution of the designed Kemp eliminase HG3 (kcat/KM 146 M−1s−1). We observe that catalytic residues are increasingly rigidified, the active site becomes better pre-organized, and its entrance is widened. Based on these observations, we engineer HG4, an efficient biocatalyst (kcat/KM 103,000 M−1s−1) containing key first and second-shell mutations found during evolution. HG4 structures reveal that its active site is pre-organized and rigidified for efficient catalysis. Our results show how directed evolution circumvents challenges inherent to enzyme design by shifting conformational ensembles to favor catalytically-productive sub-states, and suggest improvements to the design methodology that incorporate ensemble modeling of crystallographic data.

scholarly journals Solving the RNA polymerase I structural puzzle

2014 ◽  
Vol 70 (10) ◽  
pp. 2570-2582 ◽  
Author(s):  
María Moreno-Morcillo ◽  
Nicholas M. I. Taylor ◽  
Tim Gruene ◽  
Pierre Legrand ◽  
Umar J. Rashid ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Ribosomal Rna ◽  
Active Site ◽  
Rna Polymerase I ◽  
Biological Information ◽  
Molecular Replacement ◽  
Cellular Functions ◽  
X Ray ◽  
Polymerase I

Knowing the structure of multi-subunit complexes is critical to understand basic cellular functions. However, when crystals of these complexes can be obtained they rarely diffract beyond 3 Å resolution, which complicates X-ray structure determination and refinement. The crystal structure of RNA polymerase I, an essential cellular machine that synthesizes the precursor of ribosomal RNA in the nucleolus of eukaryotic cells, has recently been solved. Here, the crucial steps that were undertaken to build the atomic model of this multi-subunit enzyme are reported, emphasizing how simple crystallographic experiments can be used to extract relevant biological information. In particular, this report discusses the combination of poor molecular replacement and experimental phases, the application of multi-crystal averaging and the use of anomalous scatterers as sequence markers to guide tracing and to locate the active site. The methods outlined here will likely serve as a reference for future structural determination of large complexes at low resolution.

scholarly journals Expression, purification, crystallization and preliminary X-ray analysis of glucose-1-phosphate uridylyltransferase (GalU) fromErwinia amylovora

2014 ◽  
Vol 70 (9) ◽  
pp. 1249-1251 ◽  
Author(s):  
Mirco Toccafondi ◽  
Michele Cianci ◽  
Stefano Benini
Keyword(s):  
Erwinia Amylovora ◽  
Search Model ◽  
Data Sets ◽  
Molecular Replacement ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
E Coli ◽  
Maximum Resolution ◽  
His Tag

Glucose-1-phosphate uridylyltransferase fromErwinia amylovoraCFPB1430 was expressed as a His-tag fusion protein inEscherichia coli. After tag removal, the purified protein was crystallized from 100 mMTris pH 8.5, 2 Mammonium sulfate, 5% ethylene glycol. Diffraction data sets were collected to a maximum resolution of 2.46 Å using synchrotron radiation. The crystals belonged to the hexagonal space groupP62, with unit-cell parametersa= 80.67,b= 80.67,c = 169.18. The structure was solved by molecular replacement using the structure of theE. colienzyme as a search model.

scholarly journals Structural plasticity of SARS-CoV-2 3CL Mpro active site cavity revealed by room temperature X-ray crystallography

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Daniel W. Kneller ◽  
Gwyndalyn Phillips ◽  
Hugh M. O’Neill ◽  
Robert Jedrzejczak ◽  
Lucy Stols ◽  
...  
Keyword(s):  
Active Site ◽  
Room Temperature ◽  
Structural Plasticity ◽  
X Ray ◽  
X Ray Crystallography ◽  
Active Site Cavity

Currierite, Na4Ca3MgAl4(AsO3OH)12·9H2O, a new acid arsenate with ferrinatrite-like heteropolyhedral chains from the Torrecillas mine, Iquique Province, Chile

2017 ◽  
Vol 81 (5) ◽  
pp. 1141-1149 ◽  
Author(s):  
Anthony R. Kampf ◽  
Stuart J. Mills ◽  
Barbara P. Nash ◽  
Maurizio Dini ◽  
Arturo A. Molina Donoso
Keyword(s):  
Room Temperature ◽  
Three Dimensional ◽  
Geometrical Isomer ◽  
New Mineral ◽  
X Ray Diffraction ◽  
Calculated Density ◽  
Secondary Alteration ◽  
Crystal Forms ◽  
X Ray ◽  
Mohs Hardness

AbstractThe new mineral currierite (IMA2016-030), Na4Ca3MgAl4(AsO3OH)12·9H2O, was found at the Torrecillas mine, Iquique Province, Chile, where it occurs as a secondary alteration phase in association with anhydrite, canutite, chudobaite, halite, lavendulan, magnesiokoritnigite, quartz, scorodite and torrecillasite. Currierite occurs as hexagonal prisms, needles and hair-like fibres up to ∼200 μm long, in sprays. The crystal forms are ﹛100﹜ and ﹛001﹜. Crystals are transparent, with vitreous to silky lustre and white streak. The Mohs hardness is ∼2, tenacity is brittle, but elastic in very thin fibres, and the fracture is irregular. Crystals exhibit at least one good cleavage parallel [001]. The measured density is 3.08(2) g cm -3 and the calculated density is 3.005 g cm -3. Optically, currierite is uniaxial (–) with ω= 1.614(1) and ε= 1.613(1) (measured in white light). The mineral is slowly soluble in dilute HCl at room temperature. The empirical formula, determined from electron-microprobe analyses, is (Na3.95A12.96Ca2.74Mg1.28Fe0.633+Cu0.13K0.08Co0.03Σ11.80 (AS11.685+Sb0.325+Σ12(O56.96Cl0.04)Σ57H30.81. Currierite is hexagonal, P622, with a = 12.2057(9), c = 9.2052(7) Å, V= 1187.7(2) Å3 and Z = 1. The eight strongest powder X-ray diffraction lines are [dobs Å(I)(hkl)]: 10.63(100)(100), 6.12(20)(110), 5.30(15)(200), 4.61(24)(002), 4.002(35)(210), 3.474(29)(202), 3.021(96)(212) and 1.5227(29)(440,334,612). The structure of currierite (R1 = 2.27% for 658 Fo > 4σF reflections) is based upon a heteropolyhedral chain along c in which AlO6 octahedra are triple-linked by sharing corners with AsO3OH tetrahedra. Chains are linked to one another by bonds to 8(4 + 4)-coordinated Na and 8-coordinated Ca forming a three-dimensional framework with large cavities that contain rotationally disordered Mg(H2O)6 octahedra. The chain in the structure of currierite is identical to that in kaatialaite and a geometrical isomer of that in ferrinatrite. The mineral is named in honour of Mr. Rock Henry Currier (1940–2015), American mineral dealer, collector, author and lecturer.

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