scholarly journals Low Multiplicity Sulfur SAD Phasing in the Home lab

2014 ◽  
Vol 70 (a1) ◽  
pp. C607-C607 ◽  
Author(s):  
Severine Freisz ◽  
Juergen Graf ◽  
Matthew Benning ◽  
Vernon Smith
Keyword(s):  
De Novo ◽  
Structural Information ◽  
Low Noise ◽  
Intensity Difference ◽  
Anomalous Scattering ◽  
High Quality ◽  
X Ray ◽  
Sad Phasing ◽  
Very High

Advances in crystallographic hardware and software are enabling structural biologists to investigate more challenging projects. Recent developments in hardware and software are greatly increasing the capabilities of in-house diffraction systems making it more routine to obtain de novo structural information in the home lab. Single-wavelength anomalous diffraction (SAD) techniques with Cu Ka or Ga Ka radiation are now widely used for structure solution even in cases involving weak anomalous scatterers, like sulfur. We have now introduced the D8 Venture solution for structural biology with the PHOTON 100 detector featuring the first CMOS active pixel sensor for X-ray crystallography. Our new microfocus source, the METALJET delivers beam intensity exceeding those of typical bending-magnet beamlines. The very high intensity, the small beam focus and the lower air scatter produced by Gallium Kα radiation help to greatly reduce the background scatter. This provides greater signal to noise essential to identify weak anomalous signal. Due to the very weak anomalous scattering of S, data multiplicities in the order of 40 are typically necessary to obtain phases by S-SAD. Collecting high-multiplicity data minimizes systematic experimental errors to measure with very high accuracy the minute intensity difference between Friedel Pairs (1.0 – 1.5 %) [1]. This requires software which optimizes the collection strategy, for example with respect to overall data collection time to minimize radiation damage. The combination of a brighter, more stable X-ray source with a high sensitivity low noise detector have greatly improved the quality of data collected in-house. The high quality allows successful SAD measurements far away from the absorption edge. Here we present a low multiplicity sulfur-SAD phasing experiment on a small Thaumatin crystal showing the high quality of the data collected on the D8 VENTURE with the METALJET.

scholarly journals Synchrotron microcrystal native-SAD phasing at a low energy

IUCrJ ◽  
2019 ◽  
Vol 6 (4) ◽  
pp. 532-542 ◽  
Author(s):  
Gongrui Guo ◽  
Ping Zhu ◽  
Martin R. Fuchs ◽  
Wuxian Shi ◽  
Babak Andi ◽  
...  
Keyword(s):  
Data Analysis ◽  
Data Collection ◽  
Diffraction Data ◽  
De Novo ◽  
Low Energy ◽  
Anomalous Scattering ◽  
Free Electron Lasers ◽  
X Ray ◽  
Structural Evaluation ◽  
Sad Phasing

De novo structural evaluation of native biomolecules from single-wavelength anomalous diffraction (SAD) is a challenge because of the weakness of the anomalous scattering. The anomalous scattering from relevant native elements – primarily sulfur in proteins and phosphorus in nucleic acids – increases as the X-ray energy decreases toward their K-edge transitions. Thus, measurements at a lowered X-ray energy are promising for making native SAD routine and robust. For microcrystals with sizes less than 10 µm, native-SAD phasing at synchrotron microdiffraction beamlines is even more challenging because of difficulties in sample manipulation, diffraction data collection and data analysis. Native-SAD analysis from microcrystals by using X-ray free-electron lasers has been demonstrated but has required use of thousands of thousands of microcrystals to achieve the necessary accuracy. Here it is shown that by exploitation of anomalous microdiffraction signals obtained at 5 keV, by the use of polyimide wellmounts, and by an iterative crystal and frame-rejection method, microcrystal native-SAD phasing is possible from as few as about 1 200 crystals. Our results show the utility of low-energy native-SAD phasing with microcrystals at synchrotron microdiffraction beamlines.

scholarly journals Protein structure determination by single-wavelength anomalous diffraction phasing of X-ray free-electron laser data

IUCrJ ◽  
2016 ◽  
Vol 3 (3) ◽  
pp. 180-191 ◽  
Author(s):  
Karol Nass ◽  
Anton Meinhart ◽  
Thomas R. M. Barends ◽  
Lutz Foucar ◽  
Alexander Gorel ◽  
...  
Keyword(s):  
Structure Determination ◽  
Free Electron ◽  
De Novo ◽  
Protein Structure Determination ◽  
Anomalous Scattering ◽  
X Ray ◽  
Sad Phasing ◽  
Anomalous Signal ◽  
Serial Femtosecond Crystallography

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.

Challenges of sulfur SAD phasing as a routine method in macromolecular crystallography

2011 ◽  
Vol 19 (1) ◽  
pp. 19-29 ◽  
Author(s):  
James Doutch ◽  
Michael A. Hough ◽  
S. Samar Hasnain ◽  
Richard W. Strange
Keyword(s):  
Model Building ◽  
De Novo ◽  
Protein Structures ◽  
Anomalous Scattering ◽  
X Ray ◽  
Long Wavelength ◽  
Structure Solution ◽  
Average Proportion ◽  
Sad Phasing

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.

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.

scholarly journals Short-pulse laser-driven x-ray radiography

2016 ◽  
Vol 4 ◽  
Author(s):  
E. Brambrink ◽  
S. Baton ◽  
M. Koenig ◽  
R. Yurchak ◽  
N. Bidaut ◽  
...  
Keyword(s):  
Pulse Laser ◽  
Short Pulse ◽  
Incident Angle ◽  
Laboratory Astrophysics ◽  
Signal To Noise ◽  
High Quality ◽  
X Ray ◽  
Short Pulse Laser ◽  
Short Pulse Lasers

We have developed a new radiography setup with a short-pulse laser-driven x-ray source. Using a radiography axis perpendicular to both long- and short-pulse lasers allowed optimizing the incident angle of the short-pulse laser on the x-ray source target. The setup has been tested with various x-ray source target materials and different laser wavelengths. Signal to noise ratios are presented as well as achieved spatial resolutions. The high quality of our technique is illustrated on a plasma flow radiograph obtained during a laboratory astrophysics experiment on POLARs.

scholarly journals Improving Accuracy in Studying the Interactions of Seismic Waves with Bottom Sediments

2021 ◽  
Vol 9 (2) ◽  
pp. 229
Author(s):  
Georgy Mitrofanov ◽  
Nikita Goreyavchev ◽  
Roman Kushnarev
Keyword(s):  
Bottom Sediments ◽  
Seismic Waves ◽  
Wide Frequency Range ◽  
Quality Of Data ◽  
High Quality ◽  
Relative Characteristic ◽  
Seabed Evolution ◽  
Marine Seismic ◽  
Very High

The emerging tasks of determining the features of bottom sediments, including the evolution of the seabed, require a significant improvement in the quality of data and methods for their processing. Marine seismic data has traditionally been perceived to be of high quality compared to land data. However, high quality is always a relative characteristic and is determined by the problem being solved. In a detailed study of complex processes, the interaction of waves with bottom sediments, as well as the processes of seabed evolution over short time intervals (not millions of years), we need very high accuracy of observations. If we also need significant volumes of research covering large areas, then a significant revision of questions about the quality of observations and methods of processing is required to improve the quality of data. The article provides an example of data obtained during high-precision marine surveys and containing a wide frequency range from hundreds of hertz to kilohertz. It is shown that these data, visually having a very high quality, have variations in wavelets at all analyzed frequencies. The corresponding variations reach tens of percent. The use of the method of factor decomposition in the spectral domain made it possible to significantly improve the quality of the data, reducing the variability of wavelets by several times.

scholarly journals Get Phases from Arsenic Anomalous Scattering: de novo SAD Phasing of Two Protein Structures Crystallized in Cacodylate Buffer

PLoS ONE ◽  
2011 ◽  
Vol 6 (9) ◽  
pp. e24227 ◽  
Author(s):  
Xiang Liu ◽  
Heng Zhang ◽  
Xiao-Jun Wang ◽  
Lan-Fen Li ◽  
Xiao-Dong Su
Keyword(s):  
De Novo ◽  
Protein Structures ◽  
Anomalous Scattering ◽  
Cacodylate Buffer ◽  
Sad Phasing

scholarly journals Facile, High Quality Sequencing of Bacterial Genomes from Small Amounts of DNA

2014 ◽  
Vol 2014 ◽  
pp. 1-8
Author(s):  
Momchilo Vuyisich ◽  
Ayesha Arefin ◽  
Karen Davenport ◽  
Shihai Feng ◽  
Cheryl Gleasner ◽  
...  
Keyword(s):  
Genomic Dna ◽  
De Novo ◽  
Gc Content ◽  
Library Preparation ◽  
Sequencing Data ◽  
Bacterial Genomes ◽  
Dna Amount ◽  
High Quality ◽  
Preparation Methods

Sequencing bacterial genomes has traditionally required large amounts of genomic DNA (~1 μg). There have been few studies to determine the effects of the input DNA amount or library preparation method on the quality of sequencing data. Several new commercially available library preparation methods enable shotgun sequencing from as little as 1 ng of input DNA. In this study, we evaluated the NEBNext Ultra library preparation reagents for sequencing bacterial genomes. We have evaluated the utility of NEBNext Ultra for resequencing andde novoassembly of four bacterial genomes and compared its performance with the TruSeq library preparation kit. The NEBNext Ultra reagents enable high quality resequencing andde novoassembly of a variety of bacterial genomes when using 100 ng of input genomic DNA. For the two most challenging genomes (Burkholderiaspp.), which have the highest GC content and are the longest, we also show that the quality of both resequencing andde novoassembly is not decreased when only 10 ng of input genomic DNA is used.

scholarly journals SAXS Merge: an automated statistical method to merge SAXS profiles using Gaussian processes

2013 ◽  
Vol 21 (1) ◽  
pp. 203-208 ◽  
Author(s):  
Yannick G. Spill ◽  
Seung Joong Kim ◽  
Dina Schneidman-Duhovny ◽  
Daniel Russel ◽  
Ben Webb ◽  
...  
Keyword(s):  
Statistical Method ◽  
High Throughput ◽  
Gaussian Processes ◽  
Small Angle ◽  
Structural Information ◽  
Linear Interpolation ◽  
High Quality ◽  
X Ray ◽  
X Ray Scattering ◽  
Specific Order

Small-angle X-ray scattering (SAXS) is an experimental technique that allows structural information on biomolecules in solution to be gathered. High-quality SAXS profiles have typically been obtained by manual merging of scattering profiles from different concentrations and exposure times. This procedure is very subjective and results vary from user to user. Up to now, no robust automatic procedure has been published to perform this step, preventing the application of SAXS to high-throughput projects. Here,SAXS Merge, a fully automated statistical method for merging SAXS profiles using Gaussian processes, is presented. This method requires only the buffer-subtracted SAXS profiles in a specific order. At the heart of its formulation is non-linear interpolation using Gaussian processes, which provides a statement of the problem that accounts for correlation in the data.

Why We Ought to Accept the Repugnant Conclusion

Utilitas ◽  
2002 ◽  
Vol 14 (3) ◽  
pp. 339-359 ◽  
Author(s):  
Torbjorn Tannsjo
Keyword(s):  
Quality Of Life ◽  
Well Being ◽  
Derek Parfit ◽  
Worth Living ◽  
Repugnant Conclusion ◽  
High Quality ◽  
Hedonistic Utilitarianism ◽  
Sentient Beings ◽  
Very High

Derek Parfit has famously pointed out that ‘total’ utilitarian views, such as classical hedonistic utilitarianism, lead to the conclusion that, to each population of quite happy persons there corresponds a more extensive population with people living lives just worth living, which is (on the whole) better. In particular, for any possible population of at least ten billion people, all with a very high quality of life, there must be some much larger imaginable population whose existence, if other things are equal, would be better, even though its members have lives that are barely worth living. This world is better if the sum total of well-being is great enough, and it is great enough if only enough sentient beings inhabit it. This conclusion has been considered by Parfit and others to be ‘repugnant’.

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