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

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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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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A solution-free crystal-mounting platform for native SAD

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798320011584 ◽

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.

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Low Multiplicity Sulfur SAD Phasing in the Home lab

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314093929 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C607-C607 ◽

Cited By ~ 1

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.

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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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Synchrotron microcrystal native-SAD phasing at a low energy

IUCrJ ◽

10.1107/s2052252519004536 ◽

2019 ◽

Vol 6 (4) ◽

pp. 532-542 ◽

Cited By ~ 5

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.

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

IUCrJ ◽

10.1107/s2052252516002980 ◽

2016 ◽

Vol 3 (3) ◽

pp. 180-191 ◽

Cited By ~ 54

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.

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Native SAD is maturing

IUCrJ ◽

10.1107/s2052252515008337 ◽

2015 ◽

Vol 2 (4) ◽

pp. 431-440 ◽

Cited By ~ 36

Author(s):

John P. Rose ◽

Bi-Cheng Wang ◽

Manfred S. Weiss

Keyword(s):

De Novo ◽

Anomalous Scattering ◽

Accurate Data ◽

X Ray Diffraction ◽

The Past ◽

Methods Development ◽

Use Of Data ◽

First Choice ◽

Sodium Magnesium ◽

Sad Phasing

Native SAD phasing uses the anomalous scattering signal of light atoms in the crystalline, native samples of macromolecules collected from single-wavelength X-ray diffraction experiments. These atoms include sodium, magnesium, phosphorus, sulfur, chlorine, potassium and calcium. Native SAD phasing is challenging and is critically dependent on the collection of accurate data. Over the past five years, advances in diffraction hardware, crystallographic software, data-collection methods and strategies, and the use of data statistics have been witnessed which allow `highly accurate data' to be routinely collected. Today, native SAD sits on the verge of becoming a `first-choice' method for bothde novoand molecular-replacement structure determination. This article will focus on advances that have caught the attention of the community over the past five years. It will also highlight bothde novonative SAD structures and recent structures that were key to methods development.

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De novo design of a reversible phosphorylation-dependent switch for membrane targeting

Nature Communications ◽

10.1038/s41467-021-21622-5 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Leon Harrington ◽

Jordan M. Fletcher ◽

Tamara Heermann ◽

Derek N. Woolfson ◽

Petra Schwille

Keyword(s):

Protein Interactions ◽

Lipid Membrane ◽

De Novo ◽

Protein Localization ◽

Protein Structures ◽

Spatiotemporal Pattern ◽

Membrane Targeting ◽

Protein Protein Interactions ◽

Reversible Phosphorylation ◽

Potential Applications

AbstractModules that switch protein-protein interactions on and off are essential to develop synthetic biology; for example, to construct orthogonal signaling pathways, to control artificial protein structures dynamically, and for protein localization in cells or protocells. In nature, the E. coli MinCDE system couples nucleotide-dependent switching of MinD dimerization to membrane targeting to trigger spatiotemporal pattern formation. Here we present a de novo peptide-based molecular switch that toggles reversibly between monomer and dimer in response to phosphorylation and dephosphorylation. In combination with other modules, we construct fusion proteins that couple switching to lipid-membrane targeting by: (i) tethering a ‘cargo’ molecule reversibly to a permanent membrane ‘anchor’; and (ii) creating a ‘membrane-avidity switch’ that mimics the MinD system but operates by reversible phosphorylation. These minimal, de novo molecular switches have potential applications for introducing dynamic processes into designed and engineered proteins to augment functions in living cells and add functionality to protocells.

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S-SAD phasing on SOLEIL Beamline PROXIMA 1

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314093905 ◽

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."

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