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

2019 ◽  
Vol 75 (3) ◽  
pp. 262-271 ◽  
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.

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 Guidelines for de novo phasing using multiple small-wedge data collection

2021 ◽  
Vol 28 (5) ◽  
pp. 1284-1295 ◽  
Author(s):  
Seiki Baba ◽  
Hiroaki Matsuura ◽  
Takashi Kawamura ◽  
Naoki Sakai ◽  
Yuki Nakamura ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Data Collection ◽  
De Novo ◽  
Optimal Dose ◽  
Dose Dependence ◽  
Systematic Investigation ◽  
Quality Data ◽  
Anomalous Scattering ◽  
Automatic Data ◽  
Experimental Phasing

Intense micro-focus X-ray beamlines available at synchrotron facilities have achieved high-quality data collection even from the microcrystals of membrane proteins. The automatic data collection system developed at SPring-8, named ZOO, has contributed to many structure determinations of membrane proteins using small-wedge synchrotron crystallography (SWSX) datasets. The `small-wedge' (5–20°) datasets are collected from multiple crystals and then merged to obtain the final structure factors. To our knowledge, no systematic investigation on the dose dependence of data accuracy has so far been reported for SWSX, which is between `serial crystallography' and `rotation crystallography'. Thus, herein, we investigated the optimal dose conditions for experimental phasing with SWSX. Phase determination using anomalous scattering signals was found to be more difficult at higher doses. Furthermore, merging more homogeneous datasets grouped by hierarchical clustering with controlled doses mildly reduced the negative factors in data collection, such as `lack of signal' and `radiation damage'. In turn, as more datasets were merged, more probable phases could be obtained across a wider range of doses. Therefore, our findings show that it is essential to choose a lower dose than 10 MGy for de novo structure determination by SWSX. In particular, data collection using a dose of 5 MGy proved to be optimal in balancing the amount of signal available while reducing the amount of damage as much as possible.

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.

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 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 S-SAD phasing on SOLEIL Beamline PROXIMA 1

2014 ◽  
Vol 70 (a1) ◽  
pp. C609-C609
Author(s):  
Patrick Gourhant ◽  
Beatriz Guimaraes ◽  
Tatiana Isabet ◽  
Sebastian Klinke ◽  
Pierre Legrand ◽  
...  
Keyword(s):  
Model Building ◽  
De Novo ◽  
Signal To Noise Ratio ◽  
Beam Line ◽  
Synchrotron Source ◽  
Combining Data ◽  
Low Energies ◽  
Sad Phasing ◽  
Multiple Sample ◽  
Data Collections

"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."

scholarly journals Automatic loop centring with a high-precision goniometer head at the SLS macromolecular crystallography beamlines

2011 ◽  
Vol 18 (4) ◽  
pp. 595-600 ◽  
Author(s):  
Anuschka Pauluhn ◽  
Claude Pradervand ◽  
Daniel Rossetti ◽  
Marco Salathe ◽  
Clemens Schulze-Briese
Keyword(s):  
Data Collection ◽  
Light Source ◽  
High Precision ◽  
Initial Position ◽  
Macromolecular Crystallography ◽  
Centre Of Mass ◽  
Mass Detection ◽  
Swiss Light Source ◽  
Set Up ◽  
Goniometer Head

Automatic loop centring has been developed as part of the automation process in crystallographic data collection at the Swiss Light Source. The procedure described here consists of an optional set-up part, in which the background images are taken, and the actual centring part. The algorithm uses boundary and centre-of-mass detection at two different microscope image magnifications. Micromounts can be handled as well. Centring of the loops can be achieved in 15–26 s, depending on their initial position, and as fast as manual centring. The alignment of the sample is carried out by means of a new flexural-hinge-based compact goniometer head. The device features an electromagnet for robotic wet mounting of samples. The circle of confusion was measured to be smaller than 1 µm (r.m.s.); its bidirectional backlash is below 2 µm.

A solution-free crystal-mounting platform for native SAD

2020 ◽  
Vol 76 (10) ◽  
pp. 938-945
Author(s):  
Jian Yu ◽  
Akira Shinoda ◽  
Koji Kato ◽  
Isao Tanaka ◽  
Min Yao
Keyword(s):  
Data Collection ◽  
Nucleic Acids ◽  
Protein Structures ◽  
Anomalous Scattering ◽  
X Rays ◽  
Long Wavelength ◽  
Light Atoms ◽  
Sad Phasing ◽  
Anomalous Signal

The native SAD phasing method uses the anomalous scattering signals from the S atoms contained in most proteins, the P atoms in nucleic acids or other light atoms derived from the solution used for crystallization. These signals are very weak and careful data collection is required, which makes this method very difficult. One way to enhance the anomalous signal is to use long-wavelength X-rays; however, these wavelengths are more strongly absorbed by the materials in the pathway. Therefore, a crystal-mounting platform for native SAD data collection that removes solution around the crystals has been developed. This platform includes a novel solution-free mounting tool and an automatic robot, which extracts the surrounding solution, flash-cools the crystal and inserts the loop into a UniPuck cassette for use in the synchrotron. Eight protein structures (including two new structures) have been successfully solved by the native SAD method from crystals prepared using this platform.

scholarly journals Experimental phasing: best practice and pitfalls

2010 ◽  
Vol 66 (4) ◽  
pp. 458-469 ◽  
Author(s):  
Airlie J. McCoy ◽  
Randy J. Read
Keyword(s):  
Best Practice ◽  
Model Building ◽  
Protein Crystal ◽  
Protein Crystal Structure ◽  
Experimental Phasing ◽  
Log Likelihood ◽  
Map Generation ◽  
Sad Phasing ◽  
Supplementary Material ◽  
Crystal Twinning

Developments in protein crystal structure determination by experimental phasing are reviewed, emphasizing the theoretical continuum between experimental phasing, density modification, model building and refinement. Traditional notions of the composition of the substructure and the best coefficients for map generation are discussed. Pitfalls such as determining the enantiomorph, identifying centrosymmetry (or pseudo-symmetry) in the substructure and crystal twinning are discussed in detail. An appendix introduces combined real–imaginary log-likelihood gradient map coefficients for SAD phasing and their use for substructure completion as implemented in the softwarePhaser. Supplementary material includes animated probabilistic Harker diagrams showing how maximum-likelihood-based phasing methods can be used to refine parameters in the case of SIR and MIR; it is hoped that these will be useful for those teaching best practice in experimental phasing methods.

scholarly journals Native SAD Structure Solution from Merohedrally Twinned Data

2014 ◽  
Vol 70 (a1) ◽  
pp. C614-C614
Author(s):  
Tobias Weinert ◽  
Sandro Waltersperger ◽  
Vincent Olieric ◽  
Federica Basilico ◽  
Valentina Cecatiello ◽  
...  
Keyword(s):  
Light Source ◽  
Model Building ◽  
Experimental Setup ◽  
Difficult Case ◽  
Space Group ◽  
Human Centromere ◽  
Centromere Protein ◽  
Structure Solution ◽  
Swiss Light Source ◽  
Automatic Model Building

Up until now, comparatively few structures were solved by native SAD. Recent advances in multi crystal averaging [1] have shown that native SAD can be applied to an increasing number of cases. Though theoretically possible [2], successful structure solutions from twinned data have not been reported yet. Here, we report the structure solution of the human Centromere protein M from a merohedrally twinned crystal with a twinning fraction of 0.45 in the space group P3. The data were collected at the bending magnet beamline X06DA at the Swiss Light Source, which is equipped with the in-house developed multi-axis goniometer PRIGo and the PILATUS 2M detector. A highly redundant 2.2 Å dataset was collected in a number of different crystal orientations. A substructure solution could only be obtained after 50000 SHELXD [3] tries. Automatic model building after phasing and density modification resulted in a model with the majority of residues built correctly. We will present this particularly difficult case together with other more routine cases, all solved with the same experimental setup and at the beamline X06DA.

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