S-SAD phasing on SOLEIL Beamline PROXIMA 1

"PROXIMA 1, a beamline for macro-molecular crystallography at the 3rd generation synchrotron source SOLEIL, is equipped with a multi-circle goniometer (alpha 50 degrees) as well as a PILATUS 6M detector. These features, along with the extended energy range of the beam line towards the low energies (down to 5.5 keV) and the possibility to adapt the source size to the sample in order to optimize signal to noise ratio, have made the beam line very attractive for S-SAD phasing with more than seven examples of successful de novo phasing achieved over the last two years. The use of low energies has also proved a significant aid in assisting with MODEL building. The technical capabilities of the beam line for low energy data collections will be presented, along with a number of examples of the successful use of low wavelengths on the beam line. The importance of combining data from multiple sample orientations in order to achieve ""true multiplicity"" will be highlighted, as well as the importance of combining data from multiple crystals in order to achieve high multiplicity."

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

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s205327331409398x ◽

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.

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Making routine native SAD a reality: lessons from beamline X06DA at the Swiss Light Source

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798319003103 ◽

2019 ◽

Vol 75 (3) ◽

pp. 262-271 ◽

Cited By ~ 5

Author(s):

Shibom Basu ◽

Aaron Finke ◽

Laura Vera ◽

Meitian Wang ◽

Vincent Olieric

Keyword(s):

Data Collection ◽

Light Source ◽

Low Dose ◽

Model Building ◽

De Novo ◽

Anomalous Scattering ◽

Orientation Data ◽

Swiss Light Source ◽

Experimental Phasing ◽

Sad Phasing

Native single-wavelength anomalous dispersion (SAD) is the most attractive de novo phasing method in macromolecular crystallography, as it directly utilizes intrinsic anomalous scattering from native crystals. However, the success of such an experiment depends on accurate measurements of the reflection intensities and therefore on careful data-collection protocols. Here, the low-dose, multiple-orientation data-collection protocol for native SAD phasing developed at beamline X06DA (PXIII) at the Swiss Light Source is reviewed, and its usage over the last four years on conventional crystals (>50 µm) is reported. Being experimentally very simple and fast, this method has gained popularity and has delivered 45 de novo structures to date (13 of which have been published). Native SAD is currently the primary choice for experimental phasing among X06DA users. The method can address challenging cases: here, native SAD phasing performed on a streptavidin–biotin crystal with P21 symmetry and a low Bijvoet ratio of 0.6% is highlighted. The use of intrinsic anomalous signals as sequence markers for model building and the assignment of ions is also briefly described.

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Phasing with calcium at home

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x19004151 ◽

2019 ◽

Vol 75 (5) ◽

pp. 377-384 ◽

Cited By ~ 1

Author(s):

Shuaiqi Guo ◽

Robert Campbell ◽

Peter L. Davies ◽

John S. Allingham

Keyword(s):

Model Building ◽

De Novo ◽

Protein Structure Determination ◽

Adhesion Protein ◽

Calcium Dependent ◽

Protein Model ◽

First Choice ◽

Sad Phasing ◽

Metal Cofactors ◽

Automated Model Building

With better tools for data processing and with synchrotron beamlines that are capable of collecting data at longer wavelengths, sulfur-based native single-wavelength anomalous dispersion (SAD) phasing has become the `first-choice' method for de novo protein structure determination. However, for many proteins native SAD phasing can be simplified by taking advantage of their interactions with natural metal cofactors that are stronger anomalous scatterers than sulfur. This is demonstrated here for four unique domains of a 1.5 MDa calcium-dependent adhesion protein using the anomalous diffraction of the chelated calcium ions. In all cases, low anomalous multiplicity X-ray data were collected on a home-source diffractometer equipped with a chromium rotating anode (λ = 2.2909 Å). In all but one case, calcium SAD phasing alone was sufficient to allow automated model building and refinement of the protein model after the calcium substructure had been determined. Given that Ca atoms will be present in a significant percentage of proteins that remain uncharacterized, many aspects of the data-collection and processing methods described here could be broadly applied for routine de novo structure elucidation.

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

Journal of Synchrotron Radiation ◽

10.1107/s0909049511049004 ◽

2011 ◽

Vol 19 (1) ◽

pp. 19-29 ◽

Cited By ~ 7

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.

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An Unbiased Predictive Model to Detect DNA Methylation Propensity of CpG Islands in the Human Genome

Current Bioinformatics ◽

10.2174/1574893615999200724145835 ◽

2020 ◽

Vol 15 ◽

Author(s):

Dicle Yalcin ◽

Hasan H. Otu

Keyword(s):

Model Building ◽

De Novo ◽

Cpg Islands ◽

Treatment Strategies ◽

Area Under The Curve ◽

Global Methylation ◽

Sequence Features ◽

A Genome ◽

Combined Features ◽

Epigenetic Repression

Background: Epigenetic repression mechanisms play an important role in gene regulation, specifically in cancer development. In many cases, a CpG island’s (CGI) susceptibility or resistance to methylation are shown to be contributed by local DNA sequence features. Objective: To develop unbiased machine learning models–individually and combined for different biological features–that predict the methylation propensity of a CGI. Methods: We developed our model consisting of CGI sequence features on a dataset of 75 sequences (28 prone, 47 resistant) representing a genome-wide methylation structure. We tested our model on two independent datasets that are chromosome (132 sequences) and disease (70 sequences) specific. Results: We provided improvements in prediction accuracy over previous models. Our results indicate that combined features better predict the methylation propensity of a CGI (area under the curve (AUC) ~0.81). Our global methylation classifier performs well on independent datasets reaching an AUC of ~0.82 for the complete model and an AUC of ~0.88 for the model using select sequences that better represent their classes in the training set. We report certain de novo motifs and transcription factor binding site (TFBS) motifs that are consistently better in separating prone and resistant CGIs. Conclusion: Predictive models for the methylation propensity of CGIs lead to a better understanding of disease mechanisms and can be used to classify genes based on their tendency to contain methylation prone CGIs, which may lead to preventative treatment strategies. MATLAB and Python™ scripts used for model building, prediction, and downstream analyses are available at https://github.com/dicleyalcin/methylProp_predictor.

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

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314093863 ◽

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.

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

PLoS ONE ◽

10.1371/journal.pone.0024227 ◽

2011 ◽

Vol 6 (9) ◽

pp. e24227 ◽

Cited By ~ 20

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

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Handling techniques for channel spectra in synchrotron-based Fourier transform spectra

Canadian Journal of Physics ◽

10.1139/cjp-2013-0050 ◽

2013 ◽

Vol 91 (11) ◽

pp. 910-923 ◽

Cited By ~ 3

Author(s):

Amr Ibrahim ◽

Adriana Predoi-Cross ◽

Chad Povey

Keyword(s):

Signal To Noise Ratio ◽

Test Group ◽

Least Square ◽

Spectral Lines ◽

Spectral Parameters ◽

Synchrotron Source ◽

Group Of Seven ◽

Spectral Fitting ◽

Least Square Fit ◽

Fourier Transform Spectra

Recently, the high radiance of synchrotron sources has been used to enhance FTIR spectrometer performance. However, excessive channel spectra when synchrotron sources are used degrade the quality of retrieved spectral parameters. We have investigated seven different techniques for handling channel spectra. These techniques were used to reduce channel spectra for a test group of seven samples of CO2 mixed with air recorded using the synchrotron source at the Canadian Light Source. The increases in signal to noise ratio (SNR) of spectra handled with each technique were calculated. SNR results showed that transmission spectra, produced using synthetic background spectra with simulated channel spectra, achieved the highest SNR improvement. However, when the spectra groups were fitted using a nonlinear least square fit algorithm, the technique using channel spectra fitting produced the smallest fitting residual. Moreover, the retrieved intensities and air broadening coefficients of 21 spectral lines showed that the spectral fitting technique produced the most accurate values as compared to the HITRAN 2008 database (Rothman et al. J. Quant. Spectrosc. Radiat. Transfer, 110, 533 (2009)). Although the spectral fitting technique was accurate in retrieving spectral line parameters, applying the technique at wider spectral ranges reduced this accuracy.

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Multi-crystal native SAD analysis at 6 keV

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s1399004714013376 ◽

2014 ◽

Vol 70 (10) ◽

pp. 2544-2557 ◽

Cited By ~ 24

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.

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