scholarly journals Improving the accuracy for Photosystem II and other XFEL structures

2014 ◽  
Vol 70 (a1) ◽  
pp. C571-C571
Author(s):  
Nicholas Sauter ◽  
Aaron Brewster ◽  
Johan Hattne ◽  
Muhamed Amin ◽  
Jan Kern ◽  
...  

Femtosecond-scale XFEL pulses can produce diffraction free from radiation damage, under functional physiological conditions where reaction dynamics can be studied for systems such as photosystem II. However, it has been extremely difficult to derive accurate structure factors from the data since every shot is a still exposure from a distinct specimen. Accuracy can be improved by software methods implemented in the program cctbx.xfel, including optimal indexing and retention of data from multiple lattices, and separate determination of the resolution cutoff for individual lattices. Various techniques can produce well-conforming descriptions of the Bragg spot shape and crystal mosaicity, enabled in part by sub-pixel characterization of the detector geometry. By carefully discriminating between image pixels known to contain diffraction signal and the surrounding pixels containing only background noise, and by extending postrefinement techniques that lead to a better crystal orientation, we derive accurate structure factors with substantially fewer crystal specimen exposures. It is hoped that these developments will make it easier to measure small structure factor differences, such as those from anomalous scattering that will enable the de novo determination of macromolecular structure.

1999 ◽  
Vol 55 (4) ◽  
pp. 719-728 ◽  
Author(s):  
K. Burger ◽  
W. Prandl

Anomalous scattering of X-rays at a synchrotron source can be used for the ab initio structure determination of unknown crystal structures using only powder diffraction data. For noncentrosymmetric crystals, the phases of structure factors can only be determined with a remaining ambiguity, when one chemical element is used as resonant scatterer. A corresponding additional constraint function has been built into an enhanced version of the program MEED, so that now all types of information gained from an anomalous-scattering powder diffraction experiment can be used in a maximum-entropy calculation of the electron-density distribution: phased reflections, unphased reflections, intensities of groups of overlapping reflections, and now also reflections with a remaining ambiguity in the phase. This is important for practical use, since a lot of information is already lost in the powder diagram compared with single-crystal datasets and it is essential to use all remaining information. The new constraint is demonstrated with the structure of Cu5Zn8.


Author(s):  
P. Verwer ◽  
R. B. G. Ravelli ◽  
H. Krabbendam ◽  
J. Kroon

AbstractThe method of single-wavelength anomalous scattering (SAS) allows the determination of structure-factor phases apart from a two-fold phase ambiguity. A statistical procedure is described, based on the joint probability distribution of three structure-factors, in which


2019 ◽  
Vol 632 ◽  
pp. A128 ◽  
Author(s):  
Iris Breda ◽  
Polychronis Papaderos ◽  
Jean Michel Gomes ◽  
Stergios Amarantidis

Context. The Sérsic law (SL) offers a versatile, widely used functional form for the structural characterization of galaxies near and far. Whereas fitting this three-parameter function to galaxies with a genuine SL luminosity distribution (e.g., several local early-type galaxies–ETGs) yields a robust determination of the Sérsic exponent η and effective surface brightness μeff, this is not necessarily the case for galaxies whose surface brightness profiles (SBPs) appreciably deviate, either in their centers or over an extended radius interval, from the SL (e.g., ETGs with a “depleted” core and nucleated dwarf ellipticals, or most late-type galaxies-LTGs). In this general case of “imperfect” SL profiles, the best-fitting solution may significantly depend on the radius (or surface brightness) interval fit, the photometric passbands considered and the specifics of the fitting procedure (photometric uncertainties of SBP data points or image pixels, and corrections for point spread function (PSF) convolution effects). Such uncertainties may then affect, in a non-easily predictable manner, automated structural studies of large heterogeneous galaxy samples and introduce a scatter, if not a bias, in galaxy scaling relations and their evolution across redshift (z). Aims. Our goal is to devise a fitting concept that permits a robust determination of the equivalent SL model for the general case of galaxies with imperfect SL profiles. Methods. The distinctive feature of the concept proposed here (iFIT) is that the fit is not constrained through standard χ2 minimization between an observed SBP and the SL model of it, but instead through the search for the best match between the observationally determined and theoretically expected radial variation of the mean surface brightness and light growth curve. This approach ensures quick convergence to a unique solution for both perfect and imperfect Sérsic profiles, even shallow and resolution-degraded SBPs. iFIT allows for correction of PSF convolution effects, offering the user the option of choosing between a Moffat, Gaussian, or user-supplied PSF. iFIT, which is a standalone FORTRAN code, can be applied to any SBP that is provided in ASCII format and it has the capability of convenient graphical storage of its output. The iFIT distribution package is supplemented with an auxiliary SBP derivation tool in python. Results. iFIT has been extensively tested on synthetic data with a Sérsic index 0.3 ≤ η ≤ 4.2 and an effective radius 1 ≤ Reff  (″)≤20. Applied to non PSF-convolved data, iFIT can infer the Sérsic exponent η with an absolute error of ≤ 0.2 even for shallow SBPs. As for PSF-degraded data, iFIT can recover the input SL model parameters with a satisfactorily accuracy almost over the entire considered parameter space as long as FWHM(PSF) ≤ Reff. This study also includes examples of applications of iFIT to ETGs and local low-mass starburst galaxies. These tests confirm that iFIT shows little sensitivity on PSF corrections and SBP limiting surface brightness, and that subtraction of the best-fitting SL model in two different bands generally yields a good match to the observed radial color profile. Conclusions. It is pointed out that the publicly available iFIT offers an efficient tool for the non-supervised structural characterization of large galaxy samples, as those expected to become available with Euclid and LSST.


IUCrJ ◽  
2016 ◽  
Vol 3 (3) ◽  
pp. 180-191 ◽  
Author(s):  
Karol Nass ◽  
Anton Meinhart ◽  
Thomas R. M. Barends ◽  
Lutz Foucar ◽  
Alexander Gorel ◽  
...  

Serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs) offers unprecedented possibilities for macromolecular structure determination of systems that are prone to radiation damage. However, phasing XFEL datade novois complicated by the inherent inaccuracy of SFX data, and only a few successful examples, mostly based on exceedingly strong anomalous or isomorphous difference signals, have been reported. Here, it is shown that SFX data from thaumatin microcrystals can be successfully phased using only the weak anomalous scattering from the endogenous S atoms. Moreover, a step-by-step investigation is presented of the particular problems of SAD phasing of SFX data, analysing data from a derivative with a strong anomalous signal as well as the weak signal from endogenous S atoms.


2011 ◽  
Vol 19 (1) ◽  
pp. 19-29 ◽  
Author(s):  
James Doutch ◽  
Michael A. Hough ◽  
S. Samar Hasnain ◽  
Richard W. Strange

The sulfur SAD phasing method allows the determination of protein structuresde novowithout reference to derivatives such as Se-methionine. The feasibility for routine automated sulfur SAD phasing using a number of current protein crystallography beamlines at several synchrotrons was examined using crystals of trimericAchromobacter cycloclastesnitrite reductase (AcNiR), which contains a near average proportion of sulfur-containing residues and two Cu atoms per subunit. Experiments using X-ray wavelengths in the range 1.9–2.4 Å show that we are not yet at the level where sulfur SAD is routinely successful forautomatedstructure solution and model building using existing beamlines and current software tools. On the other hand, experiments using the shortest X-ray wavelengths available on existing beamlines could be routinely exploited to solve and produce unbiased structural models using the similarly weak anomalous scattering signals from the intrinsic metal atoms in proteins. The comparison of long-wavelength phasing (the Bijvoet ratio for nine S atoms and two Cu atoms is ∼1.25% at ∼2 Å) and copper phasing (the Bijvoet ratio for two Cu atoms is 0.81% at ∼0.75 Å) forAcNiR suggests that lower data multiplicity than is currently required for success should in general be possible for sulfur phasing if appropriate improvements to beamlines and data collection strategies can be implemented.


2018 ◽  
Vol 74 (2) ◽  
pp. 106-116 ◽  
Author(s):  
Isabel Usón ◽  
George M. Sheldrick

For the purpose of this article, experimental phasing is understood to mean the determination of macromolecular structures by exploiting small intensity differences of Friedel opposites and possibly of reflections measured at different wavelengths or for heavy-atom derivatives, without the use of specific structural models. TheSHELXprograms provide a robust and efficient route for routine structure solution by the SAD, MAD and related methods, but involve a number of simplifying assumptions that may limit their applicability in borderline cases. The substructure atoms (i.e.those with significant anomalous scattering) are first located by direct methods, and the experimental data are then used to estimate phase shifts that are added to the substructure phases to obtain starting phases for the native reflections. These are then improved by density modification and, if the resolution of the data and the type of structure permit, polyalanine tracing. A number of extensions to the tracing algorithm are discussed; these are designed to improve its performance at low resolution. Given native data to 2.5 Å resolution or better, a correlation coefficient greater than 25% between the structure factors calculated from such a trace and the native data is usually a good indication that the structure has been solved.


1999 ◽  
Vol 354 (1384) ◽  
pp. 701-710 ◽  
Author(s):  
Brian G. Spratt ◽  
Martin C. J. Maiden

Asexual bacterial populations inevitably consist of an assemblage of distinct clonal lineages. However, bacterial populations are not entirely asexual since recombinational exchanges occur, mobilizing small genome segments among lineages and species. The relative contribution of recombination, as opposed to de novo mutation, in the generation of new bacterial genotypes varies among bacterial populations and, as this contribution increases, the clonality of a given population decreases. In consequence, a spectrum of possible population structures exists, with few bacterial species occupying the extremes of highly clonal and completely non–clonal, most containing both clonal and non–clonal elements. The analysis of collections of bacterial isolates, which accurately represent the natural population, by nucleotide sequence determination of multiple housekeeping loci provides data that can be used both to investigate the population structure of bacterial pathogens and for the molecular characterization of bacterial isolates. Understanding the population structure of a given pathogen is important since it impacts on the questions that can be addressed by, and the methods and samples required for, effective molecular epidemiological studies.


2014 ◽  
Vol 70 (6) ◽  
pp. 1506-1516 ◽  
Author(s):  
Meike Stelter ◽  
Rafael Molina ◽  
Sandra Jeudy ◽  
Richard Kahn ◽  
Chantal Abergel ◽  
...  

A set of seven caged gadolinium complexes were used as vectors for introducing the chelated Gd3+ion into protein crystals in order to provide strong anomalous scattering forde novophasing. The complexes contained multidentate ligand molecules with different functional groups to provide a panel of possible interactions with the protein. An exhaustive crystallographic analysis showed them to be nondisruptive to the diffraction quality of the prepared derivative crystals, and as many as 50% of the derivatives allowed the determination of accurate phases, leading to high-quality experimental electron-density maps. At least two successful derivatives were identified for all tested proteins. Structure refinement showed that the complexes bind to the protein surface or solvent-accessible cavities, involving hydrogen bonds, electrostatic and CH–π interactions, explaining their versatile binding modes. Their high phasing power, complementary binding modes and ease of use make them highly suitable as a heavy-atom screen for high-throughputde novostructure determination, in combination with the SAD method. They can also provide a reliable tool for the development of new methods such as serial femtosecond crystallography.


Author(s):  
Kathleen M. Marr ◽  
Mary K. Lyon

Photosystem II (PSII) is different from all other reaction centers in that it splits water to evolve oxygen and hydrogen ions. This unique ability to evolve oxygen is partly due to three oxygen evolving polypeptides (OEPs) associated with the PSII complex. Freeze etching on grana derived insideout membranes revealed that the OEPs contribute to the observed tetrameric nature of the PSIl particle; when the OEPs are removed, a distinct dimer emerges. Thus, the surface of the PSII complex changes dramatically upon removal of these polypeptides. The atomic force microscope (AFM) is ideal for examining surface topography. The instrument provides a topographical view of individual PSII complexes, giving relatively high resolution three-dimensional information without image averaging techniques. In addition, the use of a fluid cell allows a biologically active sample to be maintained under fully hydrated and physiologically buffered conditions. The OEPs associated with PSII may be sequentially removed, thereby changing the surface of the complex by one polypeptide at a time.


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