scholarly journals Native-SAD Phasing at Synchrotrons: A Low-dose and High-redundancy Strategy

2014 ◽  
Vol 70 (a1) ◽  
pp. C600-C600
Author(s):  
Qun Liu ◽  
Wayne Hendrickson
Keyword(s):  
De Novo ◽  
Protein Complexes ◽  
Heavy Atom ◽  
Real Life ◽  
Biological Macromolecules ◽  
X Rays ◽  
Light Elements ◽  
X Ray ◽  
Sad Phasing ◽  
Synchrotron Beamlines

Native biological macromolecules contain intrinsic light elements such as sulfur in proteins and phosphorus in nucleic acids. Native-SAD phasing utilizes the anomalous signals from light elements for de novo structure determination: first the substructure of anomalous scatterers is determined; phase evaluation for the entire structure then follows. Synchrotron beamlines are expected to be ideal instruments for native-SAD phasing due to their brilliant and energy-tunable x-rays. However, anomalous signals from light elements are typically very weak at x-ray energies accessible to most synchrotron beamlines. Efforts have been made to promote the utility of synchrotrons for routine native-SAD phasing with no requirement for heavy-atom incorporation. Our strategy is to limit the x-ray dose per crystal and to enhance the signal-to-noise ratio by increasing data redundancy through use of multiple crystals. We have devised a robust procedure and applied it for routine native-SAD analyses on real-life membrane proteins, protein-protein complexes, and recalcitrant proteins. Here we use these real-life case studies to illustrate our procedures in sample preparation, x-ray energy selection, data collection, data analysis and phasing.

scholarly journals Multi-crystal native SAD analysis at 6 keV

2014 ◽  
Vol 70 (10) ◽  
pp. 2544-2557 ◽  
Author(s):  
Qun Liu ◽  
Youzhong Guo ◽  
Yanqi Chang ◽  
Zheng Cai ◽  
Zahra Assur ◽  
...  
Keyword(s):  
De Novo ◽  
Real Life ◽  
Kinase Domain ◽  
X Rays ◽  
Protein Kinase Domain ◽  
Anomalous Diffraction ◽  
X Ray ◽  
Tyrosine Protein Kinase ◽  
Sad Phasing ◽  
Feasibility Test

Anomalous diffraction signals from typical native macromolecules are very weak, frustrating their use inde novostructure determination. Here, native SAD procedures are described to enhance signal to noise in anomalous diffraction by using multiple crystals in combination with synchrotron X-rays at 6 keV. Increased anomalous signals were obtained at 6 keV compared with 7 keV X-ray energy, which was used for previous native SAD analyses. A feasibility test of multi-crystal-based native SAD phasing was performed at 3.2 Å resolution for a known tyrosine protein kinase domain, and real-life applications were made to two novel membrane proteins at about 3.0 Å resolution. The three applications collectively serve to validate the robust feasibility of native SAD phasing at lower energy.

scholarly journals Advancing Native-SAD Phasing at Synchrotron with 13 Real-life Case Studies

2014 ◽  
Vol 70 (a1) ◽  
pp. C601-C601
Author(s):  
Meitian Wang
Keyword(s):  
Data Collection ◽  
Single Crystal ◽  
Measurement Errors ◽  
Model Building ◽  
De Novo ◽  
Real Life ◽  
Data Sets ◽  
X Ray ◽  
Recent Developments ◽  
Sad Phasing

The key step in elucidating de novo 3D X-ray structures relies on the incorporation of heavy elements into proteins or crystals. Selenomethionine incorporation or heavy metal derivatization are however not always possible and require additional efforts. Exploiting anomalous signals from intrinsically present elements like S, P, and Ca2+ from proteins and nucleic acids, as well as Cl-, SO42-, and PO42- from crystallization solutions, is therefore an appealing alternative. Such a method has been shown to be valid by collecting data from several crystals and combining them(1). Recent developments at macromolecular crystallography beamlines are however pushing the limits of what could be obtained out of a single crystal. Here we introduce a novel data collection routine for native-SAD phasing, which distributes tolerable X-ray life-doses to very high multiplicity X-ray diffraction data sets measured at 6 keV energy and at different crystal orientations on a single crystal. This allows the extraction of weak anomalous signals reliably by reducing both systematic and random measurement errors. The data collection method has been applied successfully to thirteen real-life examples including membrane proteins, a protein/DNA complex, and a large protein complex. In addition to de novo structure determination, we advocate such a data collection protocol for molecular replacement solvable structures where unbiased phase information is crucial in objective map interpretation and model building, especially for medium and low-resolution cases.

scholarly journals Macromolecular crystallography at synchrotron radiation sources: current status and future developments

2010 ◽  
Vol 466 (2124) ◽  
pp. 3421-3452 ◽  
Author(s):  
E. M. H. Duke ◽  
L. N. Johnson
Keyword(s):  
Synchrotron Radiation ◽  
Drug Targets ◽  
Protein Complexes ◽  
Current Status ◽  
Anomalous Scattering ◽  
Biological Macromolecules ◽  
X Rays ◽  
Time Resolved ◽  
X Ray ◽  
Atomic Structures

X-ray diffraction with synchrotron radiation (SR) has revealed the atomic structures of numerous biological macromolecules including proteins and protein complexes, nucleic acids and their protein complexes, viruses, membrane proteins and drug targets. The bright SR X-ray beam with its small divergence has made the study of weakly diffracting crystals of large biological molecules possible. The ability to tune the wavelength of the SR beam to the absorption edge of certain elements has allowed anomalous scattering to be exploited for phase determination. We review the developments at synchrotron sources and beamlines from the early days to the present time, and discuss the significance of the results in providing a deeper understanding of the biological function, the design of new therapeutic molecules and time-resolved studies of dynamic events using pump–probe techniques. Radiation damage, a problem with bright X-ray sources, has been partially alleviated by collecting data at low temperature (100 K) but work is ongoing. In the most recent development, free electron laser sources can offer a peak brightness of hard X-rays approximately 10 8 times brighter than that achieved at SR sources. We describe briefly how early experiments at FLASH and Linear Coherent Light Source have shown exciting possibilities for the future.

scholarly journals Optimization in S-SAD phasing - difference between solved and unsolved structure

2014 ◽  
Vol 70 (a1) ◽  
pp. C613-C613
Author(s):  
Jan Stránský ◽  
Tomáš Kovaľ ◽  
Lars Østergaard ◽  
Jarmila Dušková ◽  
Tereza Skálová ◽  
...  
Keyword(s):  
Data Processing ◽  
De Novo ◽  
Protein Structures ◽  
X Ray Diffraction ◽  
X Ray ◽  
Structure Solution ◽  
Sad Phasing ◽  
Optimal Values ◽  
Grant Agency ◽  
Intensity Reading

Development of X-ray diffraction technologies have made de novo phasing of protein structures by single-wavelength anomalous dispersion by sulphur (S-SAD) more common. As anomalous differences in the sulphur atomic factors are in the order of errors of measurement, careful intensity reading and data processing are crucial. S-SAD was used for de novo phasing of a small 12 kDa protein with 4 sulphur atoms per molecule at 2.3 Å, where the data did not enable a straightforward structure solution. Data processing was performed using XDS [1] and scaling using XSCALE. The sulphur substructure was determined by SHELXD [2] and phases were obtained from SHELXE [2]. Both algorithms strongly depend on input parameters and default values did not lead to the correct phases. Therefore a systematic search of optimal values of several parameters was used to find a solution. This method helped to confirm sulphur substructure and to differentiate the handedness of the solutions. Moreover, a script for comfortable conversion of SHELX outputs to MTZ format was developed, using programmes included in the CCP4 package [3]. The previously unsolvable protein structure was successfully resolved with the described procedure. This work was supported by the Grant Agency of the Czech Technical University in Prague, (SGS13/219/OHK4/3T/14), the Czech Science Foundation (P302/11/0855), project BIOCEV CZ.1.05/1.1.00/02.0109 from the ERDF.

Three-dimensional crystallization of membrane proteins

1988 ◽  
Vol 21 (4) ◽  
pp. 429-477 ◽  
Author(s):  
W. Kühlbrandt
Keyword(s):  
High Resolution ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Complexes ◽  
Three Dimensional ◽  
Biological Macromolecules ◽  
X Ray ◽  
X Ray Crystallography ◽  
Membrane Protein Complexes ◽  
Essential Prerequisite

As recently as 10 years ago, the prospect of solving the structure of any membrane protein by X-ray crystallography seemed remote. Since then, the threedimensional (3-D) structures of two membrane protein complexes, the bacterial photosynthetic reaction centres of Rhodopseudomonas viridis (Deisenhofer et al. 1984, 1985) and of Rhodobacter sphaeroides (Allen et al. 1986, 1987 a, 6; Chang et al. 1986) have been determined at high resolution. This astonishing progress would not have been possible without the pioneering work of Michel and Garavito who first succeeded in growing 3-D crystals of the membrane proteins bacteriorhodopsin (Michel & Oesterhelt, 1980) and matrix porin (Garavito & Rosenbusch, 1980). X-ray crystallography is still the only routine method for determining the 3-D structures of biological macromolecules at high resolution and well-ordered 3-D crystals of sufficient size are the essential prerequisite.

HIGH-RESOLUTION PIXE USING BRAGG’S SPECTROMETER

1991 ◽  
Vol 01 (03) ◽  
pp. 251-258 ◽  
Author(s):  
M. TERASAWA
Keyword(s):  
High Resolution ◽  
Heavy Ions ◽  
X Rays ◽  
Light Elements ◽  
Element Analysis ◽  
X Ray ◽  
Peak Intensity ◽  
Moderate Energy ◽  
Practical Analysis ◽  
Wavelength Selectivity

K, L, and M X-rays in the wavelengths between 6Å and 130Å generated by the bombardment of 200 keV protons and other heavy ions were measured by means of a wavelength dispersive Bragg’s spectrometer. The X-ray peak intensity was fairly high in general, while the background was very low. The technique was favorably applied to a practical analysis of several light elements (Be, B, C, N, O, and F). Use of moderate-energy heavy ions considering the wavelength selectivity in X-ray generation was effective for the element analysis. The high-resolution spectrometry in the analytical application of ion-induced X-ray generation was found to be useful for the study of fine electronic structure, e.g. satellite and hypersatellite X-ray study, and of the chemical state of materials.

Measurements of Soft and Ultrasoft X-Rays with Total Reflection Monochromator

1986 ◽  
Vol 30 ◽  
pp. 213-223
Author(s):  
Tomoya Arai
Keyword(s):  
Gas Flow ◽  
Bragg Reflection ◽  
Optical Resolution ◽  
Fluorescence Analysis ◽  
Total Reflection ◽  
X Rays ◽  
Light Elements ◽  
Lower Intensity ◽  
Measured Intensity ◽  
X Ray

The development of X-ray spectrographic analysis of light elements, which are O, C and B, has bee n performed for many applications using an end-window type X-ray tube with Rh-target and thin Be-window, wavelength dispersing devices, which are synthetic multilayers or total reflection mirror (with a specific filter) and a gas flow proportional counter with a thin film window. In Fig. 1 factors related to the intensity measurements in X-ray fluorescence analysis are shown. The excitation efficiency in the soft and ultrasoft X-ray region is very low because of the lower intensity of primary X-rays and low fluorescence yield of light elements. Instead of the wavelength dispersive method of Bragg reflection, having high resolution and low reflectivity, monochromatization combining total reflection by a selected mirror and an appropriate filter offered an alternate approach in order to increase measured intensity with reasonable optical resolution. Synthetic multilayers which have higher resolution and lower intensity compared with the performance of the mirror method have become popular for the detection of soft and ultrasoft X-ray region.

On the Primary X-Ray Analyzer

1965 ◽  
Vol 9 ◽  
pp. 508-514 ◽  
Author(s):  
Shizuo Kimoto ◽  
Masayuki Sato ◽  
Hitoshi Kamada ◽  
Takuzi Ui
Keyword(s):  
Electron Beams ◽  
Spectrochemical Analysis ◽  
Specimen Surface ◽  
X Rays ◽  
Light Elements ◽  
X Ray ◽  
Very High

AbstractThe primary X-ray analyzer is used for nondestructive spectrochemical analysis of solid specimens. Accelerated electron beams bombard the specimen surface directly and generate primary X-rays which are measured in a vacuum spectrometer. The method of primary X-ray spectroscopy is superior to the fluorescence X-ray spectroscopy because (1) detectable sensitivity for such light elements as magnesium and aluminum is very high, and (2) the correction of the measured value for self-absorption of X-rays by the specimen itself is low. The performance of the instrument and applications are reported.

Small-angle X-ray and neutron scattering as a tool for structural systems biology

2010 ◽  
Vol 391 (7) ◽  
Author(s):  
Dmitri I. Svergun
Keyword(s):  
Systems Biology ◽  
Neutron Scattering ◽  
Conformational Changes ◽  
Small Angle ◽  
Structural Systems ◽  
Biological Macromolecules ◽  
X Rays ◽  
Hierarchical Systems ◽  
X Ray ◽  
Structural Systems Biology

Abstract Small-angle scattering (SAS) of X-rays and neutrons reveals low-resolution structures of biological macromolecules in solution. With the recent experimental and methodological advances, SAS became a unique tool for characterising biological systems. The method covers an extremely broad range of molecule sizes (from a few kDa to hundreds of MDa) and experimental conditions (temperature, pH, salinity, ligand addition, etc.), which is of primary importance for a systemic approach in structural biology. The method provides unique information about the overall structure and conformational changes of native individual proteins, functional complexes, flexible macromolecules and hierarchical systems. New developments in small-angle X-ray and neutron scattering studies of biological macromolecules in solution are briefly reviewed, with a special emphasis on technical and methodological approaches useful for structural systems biology. Possibilities of synergistic use of the method with other techniques are considered.

scholarly journals Macromolecular crystallography using neutrons

2014 ◽  
Vol 36 (3) ◽  
pp. 40-42
Author(s):  
Matthew Blakeley
Keyword(s):  
Nucleic Acids ◽  
Biological Systems ◽  
Biological Macromolecules ◽  
X Rays ◽  
Macromolecular Crystallography ◽  
X Ray Diffraction ◽  
Vast Array ◽  
X Ray ◽  
Macromolecular Assemblies

When you think about macromolecular crystallography, the technique that most often comes to mind is X-ray diffraction and it's no wonder. Over 88000 structures of biological macromolecules – from proteins and nucleic acids to viruses and macromolecular assemblies – have been determined using X-rays, and these have contributed significantly to our understanding of a vast array of biological systems and processes.

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