Use of CrKα radiation to enhance the signal from anomalous scatterers including sulfur

2000 ◽  
Vol 33 (3) ◽  
pp. 876-881 ◽  
Author(s):  
Witek Kwiatkowski ◽  
Joseph P. Noel ◽  
Senyon Choe
Keyword(s):  
Disulfide Bonds ◽  
Diffraction Method ◽  
Protein Molecule ◽  
Test Case ◽  
Data Sets ◽  
X Ray ◽  
Multi Wavelength ◽  
Anomalous Signal ◽  
Difference Fourier ◽  
Hiv 1

The anomalous signals from scatterers such as sulfur (S) and arsenic (As) were compared in diffraction data sets collected from an X-ray source with three different targets, Au, Cu and Cr, on a multi-target rotating anode. HIV-1 integrase crystals served as the test case for this study. The crystalline specimen of HIV-1 integrase contains in each protein molecule two As atoms, each covalently bound to a cysteine S atom, and two additional S atoms derived from methionine. It was found that the CrKα radiation gave the clearest peaks in anomalous difference Fourier maps, although the signal-to-noise ratios of the anomalous signal for the CuKα and CrKα data were similar but better than that for AuLα. This result was in spite of the fourfold higher flux from the Cu anodeversusthe Cr anode. For all three X-ray wavelengths, anomalous difference Fourier maps calculated with bias-removed phases derived from the known atomic model revealed clear peaks at the two As sites. However, only in the map calculated using the CrKα data were both peaks of the expected ellipsoidal shape, enveloping the As atom and the adjacent S atom. None of the S sites was apparent in difference maps calculated using the AuLα data. The ability to enhance the S-derived anomalous signal using CrKα radiation has particularly useful applications in the structure determination of proteins, for example in resolving ambiguities in the chain tracing of a protein with numerous disulfide bonds and in assigning amino acid identities. Additionally, anomalous difference Patterson maps calculated from the CrKα data were sufficiently clear to identify the As-related peaks. These results form the groundwork for in-house phase determination with the multi-wavelength anomalous diffraction method.

Combining cross-crystal averaging and MRSAD to phase a 4354-amino-acid structure

2016 ◽  
Vol 72 (2) ◽  
pp. 182-191
Author(s):  
Jason Nicholas Busby ◽  
J. Shaun Lott ◽  
Santosh Panjikar
Keyword(s):  
Radiation Damage ◽  
Model Building ◽  
Data Sets ◽  
Low Resolution ◽  
Data Set ◽  
C Protein ◽  
Large Size ◽  
Anomalous Signal ◽  
Difference Fourier ◽  
Combined Data

The B and C proteins from the ABC toxin complex ofYersinia entomophagaform a large heterodimer that cleaves and encapsulates the C-terminal toxin domain of the C protein. Determining the structure of the complex formed by B and the N-terminal region of C was challenging owing to its large size, the non-isomorphism of different crystals and their sensitivity to radiation damage. A native data set was collected to 2.5 Å resolution and a non-isomorphous Ta6Br12-derivative data set was collected that showed strong anomalous signal at low resolution. The tantalum-cluster sites could be found, but the anomalous signal did not extend to a high enough resolution to allow model building. Selenomethionine (SeMet)-derivatized protein crystals were produced, but the high number (60) of SeMet sites and the sensitivity of the crystals to radiation damage made phasing using the SAD or MAD methods difficult. Multiple SeMet data sets were combined to provide 30-fold multiplicity, and the low-resolution phase information from the Ta6Br12data set was transferred to this combined data set by cross-crystal averaging. This allowed the Se atoms to be located in an anomalous difference Fourier map; they were then used inAuto-Rickshawfor multiple rounds of autobuilding and MRSAD.

scholarly journals SpaB, an atypically adhesive basal pilin from the lactobacillar SpaCBA pilus: crystallization and X-ray diffraction analysis

2019 ◽  
Vol 75 (12) ◽  
pp. 731-737 ◽  
Author(s):  
Abhin Kumar Megta ◽  
Airi Palva ◽  
Ingemar von Ossowski ◽  
Vengadesan Krishnan
Keyword(s):  
Lactobacillus Rhamnosus ◽  
Crystal Form ◽  
Data Sets ◽  
Structural Factors ◽  
Lysine Methylation ◽  
X Ray Diffraction ◽  
X Ray ◽  
Cell Parameters ◽  
Dual Functional ◽  
Anomalous Signal

The SpaB pilin is recognized as the basal subunit of the sortase-dependent SpaCBA pilus, which is known to be produced by the Gram-positive Lactobacillus rhamnosus GG, a gut-adapted commensal advocated to have health benefits. Despite seeming to function as an archetypal basal pilin by serving as the terminal subunit in pilus assembly, SpaB also assumes an atypical role as a mucoadhesive protein. To shed light on the structural factors that contribute to this dual functional behaviour, a recombinant form of the L. rhamnosus GG SpaB pilin was produced and purified for crystallization and X-ray diffraction experiments. The crystallization of SpaB remained particularly challenging until the implementation of a three-pronged crystallization approach involving C-terminal tail truncation, surface lysine methylation and magnesium additives. Ultimately, hexagonal crystals of SpaB were produced and were able to diffract to a resolution of 2.4 Å. This crystal form belonged to space group P6522 or P6122, with unit-cell parameters a = b = 51.53, c = 408.22 Å, α = β = 90.0, γ = 120.0°. Obtaining an interpretable electron-density map via single-wavelength anomalous diffraction (SAD) using iodide-derivative data sets did not succeed owing to the weak anomalous signal. As an alternative, attempts to provide phases by molecular replacement using the iodide-SAD data from SpaB and a collection of distant homology models (<28% sequence identity) are in progress.

A new algorithm for the reconstruction of protein molecular envelopes from X-ray solution scattering data

2019 ◽  
Vol 52 (5) ◽  
pp. 937-944
Author(s):  
John Badger
Keyword(s):  
Protein Structures ◽  
Three Dimensional ◽  
Protein Molecule ◽  
Scattering Data ◽  
Data Sets ◽  
Low Resolution ◽  
Jones Potential ◽  
X Ray ◽  
Pair Distance Distribution Function ◽  
Protein Shape

At sufficiently low resolution, the scattering density within the volume occupied by a well folded protein molecule appears relatively flat. By enforcing this condition, three-dimensional protein molecular envelopes may be reconstructed using information obtained from X-ray solution scattering profiles. A practical approach for solving the low-resolution structures of protein molecules from solution scattering data involves modelling the protein shape using a set of volume-filling points (`beads') and transforming the scattering data to a more convenient target, the pair distance distribution function, P(r). Using algorithms described here, the beads interact via a modified Lennard–Jones potential and their positions are adjusted and confined until they fit the expected protein volume and agreement with P(r) is obtained. This methodology allows the protein volume to be modelled by an arbitrary, user-defined number of beads, enabling the rapid reconstruction of protein structures of widely varying sizes. Tests carried out with a variety of synthetic and experimental data sets show that this approach gives efficient and reliable determinations of protein molecular envelopes.

RuII–hydride–tropcomplexes: X-ray single-crystal determination and quantum-chemical calculations

2013 ◽  
Vol 69 (12) ◽  
pp. 1421-1426 ◽  
Author(s):  
Gustavo Santiso-Quinones ◽  
Rafael Rodriguez-Lugo ◽  
Vittorio Sacchetti ◽  
Hansjörg Grützmacher
Keyword(s):  
Quantum Chemical Calculations ◽  
Quantum Chemical ◽  
Theoretical Calculations ◽  
Data Sets ◽  
Space Group ◽  
X Ray ◽  
H Nmr ◽  
Axial Ligands ◽  
Catalytically Active ◽  
Difference Fourier

As part of our search for catalytically active RuII–hydride complexes, we have synthesized and crystallographically characterized three different ruthenium species, namely dihydrido[(SR)-(10,11-η)-N-(pyridin-2-ylmethyl-κN)-5H-dibenzo[a,d]cyclohepten-5-amine](triphenylphosphane-κP)ruthenium(II) tetrahydrofuran monosolvate, [RuH2(C21H18N2)(C18H15P)]·C4H8O or (SR)-[RuII(H)2{N-(pyridin-2-ylmethyl)tropNH}(PPh3)]·THF, (1), chlorido{(1SR,2RS)-N,N′-bis[(10,11-η)-5H-dibenzo[a,d]cyclohepten-5-amine]ethane-1,2-diamine-κ2N,N′}hydridoruthenium(II) dimethoxyethane hemisolvate, [RuClH(C32H28N2)]·0.5C4H10O2or (1SR,2RS)-[RuII(H)(Cl){tropNH(CH2)2HNtrop}]·DME, (2), and chlorido{(1SR,2RS)-N,N′-bis[(10,11-η)-5H-dibenzo[a,d]cyclohepten-5-amine]propane-1,3-diamine-κ2N,N′}hydridoruthenium(II), [RuClH(C33H30N2)] or (1SR,2RS)-[RuII(H)(Cl){tropNH(CH2)3HNtrop}], (3), wheretropis 5H-dibenzo[a,d]cycloheptene. In all three complexes, the RuIIcenter resides in an octahedral coordination environment. For (1)–(3), the hydride atoms were located in a difference Fourier map and were refined freely. In solution, the1H NMR spectra of all species show the presence of the hydride resonance. Comparison with quantum-chemical calculations reveals that the crystallographic data sets are plausible. In every case, the prediction is in very good agreement with the observed X-ray data. Not only the observed geometry is predicted well but also the Ru—H(hydride) bond lengths are reproduced remarkably well. Complexes (1) and (2) crystallized in the triclinicP\overline{1} space group, while (3) crystallized in the tetragonal space groupI41/a. For (3), there is disorder of the axial ligands producing two isomers (in a 98.7:1.3 ratio). Details of the synthesis, characterization, X-ray analysis, and theoretical calculations for complexes (1)–(3) are presented.

scholarly journals GRS1915+105: a comparison of the plateau state to the canonical hard state

2010 ◽  
Vol 6 (S275) ◽  
pp. 294-298 ◽  
Author(s):  
Pieter van Oers ◽  
Sera Markoff
Keyword(s):  
Black Hole ◽  
Near Infrared ◽  
Model Parameters ◽  
Data Sets ◽  
X Ray ◽  
Black Hole Binaries ◽  
Hard State ◽  
Multi Wavelength ◽  
Plateau State ◽  
The Relationship

AbstractGRS 1915+105 is a very peculiar black hole binary that exhibits accretion-related states that are not observed in any other stellar-mass black hole system. One of these states, however – referred to as the plateau state – may be related to the canonical hard state of black hole X-ray binaries. Both the plateau and hard state are associated with steady, relatively lower X-ray emission and flat/inverted radio emission, that is sometimes resolved into compact, self-absorbed jets. To investigate the relationship between the plateau and the hard state, we fit two multi-wavelength observations using a steady-state outflow-dominated model, developed for hard state black hole binaries. The data sets consist of quasi-simultaneous observations in radio, near-infrared and X-ray bands. Interestingly, we find both significant differences between the two plateau states, as well as between the best-fit model parameters and those representative of the hard state. We discuss our interpretation of these results, and the possible implications for GRS 1915+105's relationship to canonical black hole candidates.

Against the odds?De novostructure determination of a pilin with two cysteine residues by sulfur SAD

2015 ◽  
Vol 71 (5) ◽  
pp. 1095-1101 ◽  
Author(s):  
Manuela Gorgel ◽  
Andreas Bøggild ◽  
Jakob Jensen Ulstrup ◽  
Manfred S. Weiss ◽  
Uwe Müller ◽  
...  
Keyword(s):  
Protein Structure ◽  
Sodium Ion ◽  
Data Sets ◽  
Threshold Values ◽  
X Ray Diffraction ◽  
X Ray ◽  
Single Well ◽  
Anomalous Signal ◽  
Pilin Protein

Exploiting the anomalous signal of the intrinsic S atoms to phase a protein structure is advantageous, as ideally only a single well diffracting native crystal is required. However, sulfur is a weak anomalous scatterer at the typical wavelengths used for X-ray diffraction experiments, and therefore sulfur SAD data sets need to be recorded with a high multiplicity. In this study, the structure of a small pilin protein was determined by sulfur SAD despite several obstacles such as a low anomalous signal (a theoretical Bijvoet ratio of 0.9% at a wavelength of 1.8 Å), radiation damage-induced reduction of the cysteines and a multiplicity of only 5.5. The anomalous signal was improved by merging three data sets from different volumes of a single crystal, yielding a multiplicity of 17.5, and a sodium ion was added to the substructure of anomalous scatterers. In general, all data sets were balanced around the threshold values for a successful phasing strategy. In addition, a collection of statistics on structures from the PDB that were solved by sulfur SAD are presented and compared with the data. Looking at the quality indicatorRanom/Rp.i.m., an inconsistency in the documentation of the anomalousRfactor is noted and reported.

scholarly journals Crystallization and preliminary X-ray crystallographic studies of dipeptidyl aminopeptidase BII fromPseudoxanthomonas mexicanaWO24

2014 ◽  
Vol 70 (2) ◽  
pp. 221-224 ◽  
Author(s):  
Yasumitsu Sakamoto ◽  
Yoshiyuki Suzuki ◽  
Ippei Iizuka ◽  
Chika Tateoka ◽  
Saori Roppongi ◽  
...  
Keyword(s):  
Diffraction Method ◽  
Crystal Form ◽  
Diffusion Method ◽  
X Ray Diffraction ◽  
Hanging Drop ◽  
Orthorhombic Crystal ◽  
X Ray ◽  
Cell Parameters ◽  
Multi Wavelength ◽  
Dipeptidyl Aminopeptidase

Dipeptidyl aminopeptidase BII fromPseudoxanthomonas mexicanaWO24 (DAP BII) is able to cleave a variety of dipeptides from the amino-terminus of substrate peptides. For crystallographic studies, DAP BII was overproduced inEscherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data to 2.3 Å resolution were collected using an orthorhombic crystal form belonging to space groupP212121, with unit-cell parametersa= 76.55,b= 130.86,c= 170.87 Å. Structural analysis by the multi-wavelength anomalous diffraction method is in progress.

scholarly journals Making a difference in multi-data-set crystallography: simple and deterministic data-scaling/selection methods

2020 ◽  
Vol 76 (7) ◽  
pp. 636-652 ◽  
Author(s):  
Greta M. Assmann ◽  
Meitian Wang ◽  
Kay Diederichs
Keyword(s):  
Simulated Data ◽  
Test Case ◽  
Data Sets ◽  
Selection Methods ◽  
Data Set ◽  
Partial Data ◽  
Structure Solution ◽  
Making A Difference ◽  
Data Scaling ◽  
Anomalous Signal

Phasing by single-wavelength anomalous diffraction (SAD) from multiple crystallographic data sets can be particularly demanding because of the weak anomalous signal and possible non-isomorphism. The identification and exclusion of non-isomorphous data sets by suitable indicators is therefore indispensable. Here, simple and robust data-selection methods are described. A multi-dimensional scaling procedure is first used to identify data sets with large non-isomorphism relative to clusters of other data sets. Within each cluster that it identifies, further selection is based on the weighted ΔCC1/2, a quantity representing the influence of a set of reflections on the overall CC1/2 of the merged data. The anomalous signal is further improved by optimizing the scaling protocol. The success of iterating the selection and scaling steps was verified by substructure determination and subsequent structure solution. Three serial synchrotron crystallography (SSX) SAD test cases with hundreds of partial data sets and one test case with 62 complete data sets were analyzed. Structure solution was dramatically simplified with this procedure, and enabled solution of the structures after a few selection/scaling iterations. To explore the limits, the procedure was tested with much fewer data than originally required and could still solve the structure in several cases. In addition, an SSX data challenge, minimizing the number of (simulated) data sets necessary to solve the structure, was significantly underbid.

scholarly journals Comparison of helical scan and standard rotation methods in single-crystal X-ray data collection strategies

2017 ◽  
Vol 24 (1) ◽  
pp. 42-52 ◽  
Author(s):  
Ivan Polsinelli ◽  
Martin Savko ◽  
Cecile Rouanet-Mehouas ◽  
Lidia Ciccone ◽  
Susanna Nencetti ◽  
...  
Keyword(s):  
Radiation Damage ◽  
Disulfide Bonds ◽  
X Ray ◽  
Rotation Method ◽  
Rotation Methods ◽  
Anomalous Signal ◽  
Effects Of Radiation ◽  
Helical Scan ◽  
Collection Strategies ◽  
Meaningful Data

X-ray radiation in macromolecular crystallography can chemically alter the biological material and deteriorate the integrity of the crystal lattice with concomitant loss of resolution. Typical alterations include decarboxylation of glutamic and aspartic residues, breaking of disulfide bonds and the reduction of metal centres. Helical scans add a small translation to the crystal in the rotation method, so that for every image the crystal is shifted to expose a fresh part. On beamline PROXIMA 2A at Synchrotron SOLEIL, this procedure has been tested with various parameters in an attempt to understand how to mitigate the effects of radiation damage. Here, the strategies used and the crystallographic metrics for various scenarios are reported. Among these, the loss of bromine from bromophenyl moieties appears to be a useful monitor of radiation damage as the carbon–bromine bond is very sensitive to X-ray irradiation. Two cases are focused on where helical scans are shown to be superior in obtaining meaningful data compared with conventional methods. In one case the initial resolution of the crystal is extended over time, and in the second case the anomalous signal is preserved to provide greater effective multiplicity and easier phasing.

The Application of Resonant Scattering Techniques toAb InitioStructure Solution from Powder Data using SrSO4as a Test Case

1998 ◽  
Vol 31 (5) ◽  
pp. 789-797 ◽  
Author(s):  
K. Burger ◽  
D. Cox ◽  
R. Papoular ◽  
W. Prandl
Keyword(s):  
Complete Solution ◽  
Resonant Scattering ◽  
Test Case ◽  
Anomalous Scattering ◽  
Data Sets ◽  
Fourier Methods ◽  
X Ray ◽  
Structure Solution ◽  
Overlapping Peaks ◽  
Complex Powder

The application of resonant (anomalous) scattering techniques toab initiostructure solution from powder data is demonstrated for the compound SrSO4. This material is orthorhombic, in space groupPnma(a = 8.359,b = 5.351,c = 6.869 Å,Z = 4), with 11 variable atomic positional parameters, and gives a fairly complex powder pattern with many overlapping peaks. Accurate high-resolution synchrotron X-ray powder diffraction data were obtained at three energies: 14, 225 and 5133 eV below the SrKedge. From combinations of two of the three data-sets, partial Patterson densities were derived and used to phase the 71 uniquely resolved reflections, allowing a complete solution of the rest of the structure. Maximum entropy methods were found to be far superior to standard Fourier methods in the reconstruction of the electron-density distribution. The techniques of data collection, analysis and phase determination required for resonant scattering experiments of this type are described in detail.

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