scholarly journals SAD phasing of XFEL data depends critically on the error model

2019 ◽  
Vol 75 (11) ◽  
pp. 959-968 ◽  
Author(s):  
Aaron S. Brewster ◽  
Asmit Bhowmick ◽  
Robert Bolotovsky ◽  
Derek Mendez ◽  
Petrus H. Zwart ◽  
...  
Keyword(s):  
Protein Structure ◽  
Error Estimates ◽  
De Novo ◽  
Least Squares Method ◽  
Random Errors ◽  
X Ray ◽  
Counting Error ◽  
Structure Solution ◽  
Sad Phasing ◽  
Reflection Intensity

A nonlinear least-squares method for refining a parametric expression describing the estimated errors of reflection intensities in serial crystallographic (SX) data is presented. This approach, which is similar to that used in the rotation method of crystallographic data collection at synchrotrons, propagates error estimates from photon-counting statistics to the merged data. Here, it is demonstrated that the application of this approach to SX data provides better SAD phasing ability, enabling the autobuilding of a protein structure that had previously failed to be built. Estimating the error in the merged reflection intensities requires the understanding and propagation of all of the sources of error arising from the measurements. One type of error, which is well understood, is the counting error introduced when the detector counts X-ray photons. Thus, if other types of random errors (such as readout noise) as well as uncertainties in systematic corrections (such as from X-ray attenuation) are completely understood, they can be propagated along with the counting error, as appropriate. In practice, most software packages propagate as much error as they know how to model and then include error-adjustment terms that scale the error estimates until they explain the variance among the measurements. If this is performed carefully, then during SAD phasing likelihood-based approaches can make optimal use of these error estimates, increasing the chance of a successful structure solution. In serial crystallography, SAD phasing has remained challenging, with the few examples of de novo protein structure solution each requiring many thousands of diffraction patterns. Here, the effects of different methods of treating the error estimates are estimated and it is shown that using a parametric approach that includes terms proportional to the known experimental uncertainty, the reflection intensity and the squared reflection intensity to improve the error estimates can allow SAD phasing even from weak zinc anomalous signal.

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.

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 A defined structural unit enables de novo design of small-molecule–binding proteins

Science ◽  
2020 ◽  
Vol 369 (6508) ◽  
pp. 1227-1233 ◽  
Author(s):  
Nicholas F. Polizzi ◽  
William F. DeGrado
Keyword(s):  
Protein Structure ◽  
Amino Acid ◽  
Small Molecules ◽  
Structural Unit ◽  
De Novo ◽  
Computational Design ◽  
De Novo Design ◽  
X Ray ◽  
X Ray Crystallography ◽  
Chemical Groups

The de novo design of proteins that bind highly functionalized small molecules represents a great challenge. To enable computational design of binders, we developed a unit of protein structure—a van der Mer (vdM)—that maps the backbone of each amino acid to statistically preferred positions of interacting chemical groups. Using vdMs, we designed six de novo proteins to bind the drug apixaban; two bound with low and submicromolar affinity. X-ray crystallography and mutagenesis confirmed a structure with a precisely designed cavity that forms favorable interactions in the drug–protein complex. vdMs may enable design of functional proteins for applications in sensing, medicine, and catalysis.

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 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 Cadmium SAD phasing at CuKα wavelength

F1000Research ◽  
2019 ◽  
Vol 8 ◽  
pp. 84
Author(s):  
Igor E. Eliseev ◽  
Anna N. Yudenko ◽  
Valeria M. Ukrainskaya ◽  
Oleg B. Chakchir
Keyword(s):  
Experimental Model ◽  
De Novo ◽  
Common Method ◽  
Single Domain Antibody ◽  
Anomalous Diffraction ◽  
Cadmium Ions ◽  
X Ray ◽  
X Ray Crystallography ◽  
Domain Antibody ◽  
Sad Phasing

Single-wavelength anomalous diffraction (SAD) is the most common method for de novo elucidation of macromolecular structures by X-ray crystallography. It requires an anomalous scatterer in a crystal to calculate phases. A recent study by Panneerselvam et al. emphasized the utility of cadmium ions for SAD phasing at the standard synchrotron wavelength of 1 Å. Here we show that cadmium is also useful for phasing of crystals collected in-house with CuKα radiation. Using a crystal of single-domain antibody as an experimental model, we demonstrate how cadmium SAD can be conveniently employed to solve a CuKα dataset. We then discuss the factors which make this method generally applicable.

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.

scholarly journals Data collection with a tailored X-ray beam size at 2.69 Å wavelength (4.6 keV): sulfur SAD phasing of Cdc23Nterm

2016 ◽  
Vol 72 (3) ◽  
pp. 403-412 ◽  
Author(s):  
Michele Cianci ◽  
Matthew R. Groves ◽  
David Barford ◽  
Thomas R. Schneider
Keyword(s):  
Data Collection ◽  
Anaphase Promoting Complex ◽  
X Ray ◽  
Cell Parameters ◽  
Structure Solution ◽  
Sad Phasing ◽  
A Subunit ◽  
Anomalous Signal ◽  
Crystal Twinning ◽  
Methionine Residues

The capability to reach wavelengths of up to 3.1 Å at the newly established EMBL P13 beamline at PETRA III, the new third-generation synchrotron at DESY in Hamburg, provides the opportunity to explore very long wavelengths to harness the sulfur anomalous signal for phase determination. Data collection at λ = 2.69 Å (4.6 keV) allowed the crystal structure determination by sulfur SAD phasing of Cdc23Nterm, a subunit of the multimeric anaphase-promoting complex (APC/C). At this energy, Cdc23Ntermhas an expected Bijvoet ratio 〈|Fanom|〉/〈F〉 of 2.2%, with 282 residues, including six cysteines and five methionine residues, and two molecules in the asymmetric unit (65.4 kDa; 12 Cys and ten Met residues). Selectively illuminating two separate portions of the same crystal with an X-ray beam of 50 µm in diameter allowed crystal twinning to be overcome. The crystals diffracted to 3.1 Å resolution, with unit-cell parametersa=b= 61.2,c = 151.5 Å, and belonged to space groupP43. The refined structure to 3.1 Å resolution has anRfactor of 18.7% and anRfreeof 25.9%. This paper reports the structure solution, related methods and a discussion of the instrumentation.

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