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 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 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 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 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.

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 Lower resolution Laue neutron data yield correct nanocluster formulations

2014 ◽  
Vol 70 (a1) ◽  
pp. C187-C187
Author(s):  
Alison Edwards
Keyword(s):  
Neutron Diffraction ◽  
Single Crystal ◽  
Diffraction Pattern ◽  
Precise Determination ◽  
Data Sets ◽  
X Rays ◽  
Low Resolution ◽  
Neutron Data ◽  
X Ray ◽  
Product Formulation

"The renaissance in Laue studies - at neutron sources - provides us with access to single crystal neutron diffraction data for synthetic compounds without requiring synthesis of prohibitively large amounts of compound or improbably large crystals. Such neutron diffraction studies provide vital data where proof of the presence or absence of hydrogen in particular locations is required and which cannot validly be proved by X-ray studies. Since the commissioning of KOALA at OPAL in 2009[1] we have obtained numerous data sets which demonstrate the vital importance of measuring data even where the extent of the diffraction pattern is at relatively low resolution - especially when compared to that obtainable for the same compound with X-rays. In the Laue experiment performed with a fixed radius detector, data reduction is only feasible for crystals in the ""goldilocks"" zone – where the unit cell is relatively large for the detector, a correspondingly low resolution diffraction pattern in which adjacent spots are less affected by overlap will yield more data against which a structure can be refined than a pattern of higher resolution – one where neighbouring spots overlap rendering both unusable (in our current methodology). Analogous application of powder neutron diffraction in such determinations is also considered. Single crystal neutron diffraction studies of several important compounds (up to 5KDa see figure below)[2] in which precise determination of hydride content by neutron diffraction was pivotal to the final formulation will be presented. The neutron data sets typically possess 20% or fewer unique data at substantially "lower resolution" than the corresponding X-ray data sets. Careful refinement clearly reveals chemical detail which is typically unexplored in related X-ray diffraction studies reporting high profile chemistry despite the synthetic route being one which hydride ought to be considered/excluded in product formulation."

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 Data-collection strategy for challenging native SAD phasing

2016 ◽  
Vol 72 (3) ◽  
pp. 421-429 ◽  
Author(s):  
Vincent Olieric ◽  
Tobias Weinert ◽  
Aaron D. Finke ◽  
Carolin Anders ◽  
Dianfan Li ◽  
...  
Keyword(s):  
Data Collection ◽  
Data Sets ◽  
Yield Data ◽  
Guide Rna ◽  
Multiple Data ◽  
Data Collection Strategy ◽  
Sad Phasing ◽  
Anomalous Signal ◽  
Multiple Data Sets ◽  
Low X

Recent improvements in data-collection strategies have pushed the limits of native SAD (single-wavelength anomalous diffraction) phasing, a method that uses the weak anomalous signal of light elements naturally present in macromolecules. These involve the merging of multiple data sets from either multiple crystals or from a single crystal collected in multiple orientations at a low X-ray dose. Both approaches yield data of high multiplicity while minimizing radiation damage and systematic error, thus ensuring accurate measurements of the anomalous differences. Here, the combined use of these two strategies is described to solve cases of native SAD phasing that were particular challenges: the integral membrane diacylglycerol kinase (DgkA) with a low Bijvoet ratio of 1% and the large 200 kDa complex of the CRISPR-associated endonuclease (Cas9) bound to guide RNA and target DNA crystallized in the low-symmetry space groupC2. The optimal native SAD data-collection strategy based on systematic measurements performed on the 266 kDa multiprotein/multiligand tubulin complex is discussed.

Watching Photochemistry Happen: Recent Developments in Dynamic Single-Crystal X-Ray Diffraction Studies

2020 ◽  
Author(s):  
Lauren E. Hatcher ◽  
Mark R. Warren ◽  
Anuradha R. Pallipurath ◽  
Lucy K. Saunders ◽  
Jonathan M. Skelton
Keyword(s):  
Single Crystal ◽  
X Ray Diffraction ◽  
X Ray ◽  
Recent Developments
Sign in / Sign up

Export Citation Format

Share Document