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

IUCrJ ◽  
2016 ◽  
Vol 3 (3) ◽  
pp. 180-191 ◽  
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.

scholarly journals Membrane protein structure determination by SAD, SIR, or SIRAS phasing in serial femtosecond crystallography using an iododetergent

2016 ◽  
Vol 113 (46) ◽  
pp. 13039-13044 ◽  
Author(s):  
Takanori Nakane ◽  
Shinya Hanashima ◽  
Mamoru Suzuki ◽  
Haruka Saiki ◽  
Taichi Hayashi ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Structure Determination ◽  
Free Electron ◽  
Heavy Atom ◽  
Anomalous Scattering ◽  
X Ray ◽  
Isomorphous Replacement ◽  
Experimental Phasing ◽  
Serial Femtosecond Crystallography

The 3D structure determination of biological macromolecules by X-ray crystallography suffers from a phase problem: to perform Fourier transformation to calculate real space density maps, both intensities and phases of structure factors are necessary; however, measured diffraction patterns give only intensities. Although serial femtosecond crystallography (SFX) using X-ray free electron lasers (XFELs) has been steadily developed since 2009, experimental phasing still remains challenging. Here, using 7.0-keV (1.771 Å) X-ray pulses from the SPring-8 Angstrom Compact Free Electron Laser (SACLA), iodine single-wavelength anomalous diffraction (SAD), single isomorphous replacement (SIR), and single isomorphous replacement with anomalous scattering (SIRAS) phasing were performed in an SFX regime for a model membrane protein bacteriorhodopsin (bR). The crystals grown in bicelles were derivatized with an iodine-labeled detergent heavy-atom additive 13a (HAD13a), which contains the magic triangle, I3C head group with three iodine atoms. The alkyl tail was essential for binding of the detergent to the surface of bR. Strong anomalous and isomorphous difference signals from HAD13a enabled successful phasing using reflections up to 2.1-Å resolution from only 3,000 and 4,000 indexed images from native and derivative crystals, respectively. When more images were merged, structure solution was possible with data truncated at 3.3-Å resolution, which is the lowest resolution among the reported cases of SFX phasing. Moreover, preliminary SFX experiment showed that HAD13a successfully derivatized the G protein-coupled A2a adenosine receptor crystallized in lipidic cubic phases. These results pave the way for de novo structure determination of membrane proteins, which often diffract poorly, even with the brightest XFEL beams.

scholarly journals Experimental phase determination with selenomethionine or mercury-derivatization in serial femtosecond crystallography

IUCrJ ◽  
2017 ◽  
Vol 4 (5) ◽  
pp. 639-647 ◽  
Author(s):  
Keitaro Yamashita ◽  
Naoyuki Kuwabara ◽  
Takanori Nakane ◽  
Tomohiro Murai ◽  
Eiichi Mizohata ◽  
...  
Keyword(s):  
Structure Determination ◽  
Free Electron ◽  
De Novo ◽  
Heavy Atom ◽  
Protein Structure Determination ◽  
High Energy ◽  
X Rays ◽  
Replacement Method ◽  
Serial Femtosecond Crystallography

Serial femtosecond crystallography (SFX) using X-ray free-electron lasers (XFELs) holds enormous potential for the structure determination of proteins for which it is difficult to produce large and high-quality crystals. SFX has been applied to various systems, but rarely to proteins that have previously unknown structures. Consequently, the majority of previously obtained SFX structures have been solved by the molecular replacement method. To facilitate protein structure determination by SFX, it is essential to establish phasing methods that work efficiently for SFX. Here, selenomethionine derivatization and mercury soaking have been investigated for SFX experiments using the high-energy XFEL at the SPring-8 Angstrom Compact Free-Electron Laser (SACLA), Hyogo, Japan. Three successful cases are reported of single-wavelength anomalous diffraction (SAD) phasing using X-rays of less than 1 Å wavelength with reasonable numbers of diffraction patterns (13 000, 60 000 and 11 000). It is demonstrated that the combination of high-energy X-rays from an XFEL and commonly used heavy-atom incorporation techniques will enable routinede novostructural determination of biomacromolecules.

scholarly journals Native sulfur/chlorine SAD phasing for serial femtosecond crystallography

2015 ◽  
Vol 71 (12) ◽  
pp. 2519-2525 ◽  
Author(s):  
Takanori Nakane ◽  
Changyong Song ◽  
Mamoru Suzuki ◽  
Eriko Nango ◽  
Jun Kobayashi ◽  
...  
Keyword(s):  
Radiation Damage ◽  
Crystal Structures ◽  
Structure Determination ◽  
Anomalous Diffraction ◽  
Native Sulfur ◽  
Wide Range ◽  
Sad Phasing ◽  
Anomalous Signal ◽  
Serial Femtosecond Crystallography

Serial femtosecond crystallography (SFX) allows structures to be determined with minimal radiation damage. However, phasing native crystals in SFX is not very common. Here, the structure determination of native lysozyme from single-wavelength anomalous diffraction (SAD) by utilizing the anomalous signal of sulfur and chlorine at a wavelength of 1.77 Å is successfully demonstrated. This sulfur SAD method can be applied to a wide range of proteins, which will improve the determination of native crystal structures.

scholarly journals Native phasing of x-ray free-electron laser data for a G protein–coupled receptor

2016 ◽  
Vol 2 (9) ◽  
pp. e1600292 ◽  
Author(s):  
Alexander Batyuk ◽  
Lorenzo Galli ◽  
Andrii Ishchenko ◽  
Gye Won Han ◽  
Cornelius Gati ◽  
...  
Keyword(s):  
Free Electron ◽  
Room Temperature ◽  
De Novo ◽  
Ultrashort Pulses ◽  
Temperature Structure ◽  
X Ray ◽  
Anomalous Signal ◽  
G Protein Coupled ◽  
Serial Femtosecond Crystallography ◽  
General Method

Serial femtosecond crystallography (SFX) takes advantage of extremely bright and ultrashort pulses produced by x-ray free-electron lasers (XFELs), allowing for the collection of high-resolution diffraction intensities from micrometer-sized crystals at room temperature with minimal radiation damage, using the principle of “diffraction-before-destruction.” However, de novo structure factor phase determination using XFELs has been difficult so far. We demonstrate the ability to solve the crystallographic phase problem for SFX data collected with an XFEL using the anomalous signal from native sulfur atoms, leading to a bias-free room temperature structure of the human A2A adenosine receptor at 1.9 Å resolution. The advancement was made possible by recent improvements in SFX data analysis and the design of injectors and delivery media for streaming hydrated microcrystals. This general method should accelerate structural studies of novel difficult-to-crystallize macromolecules and their complexes.

scholarly journals Pink-beam serial femtosecond crystallography for accurate structure-factor determination at an X-ray free-electron laser

IUCrJ ◽  
2021 ◽  
Vol 8 (6) ◽  
Author(s):  
Karol Nass ◽  
Camila Bacellar ◽  
Claudio Cirelli ◽  
Florian Dworkowski ◽  
Yaroslav Gevorkov ◽  
...  
Keyword(s):  
Data Quality ◽  
Structure Determination ◽  
Structure Factor ◽  
Free Electron ◽  
Free Electron Laser ◽  
De Novo ◽  
Similar Data ◽  
X Ray ◽  
Redundant Data ◽  
Serial Femtosecond Crystallography

Serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs) enables essentially radiation-damage-free macromolecular structure determination using microcrystals that are too small for synchrotron studies. However, SFX experiments often require large amounts of sample in order to collect highly redundant data where some of the many stochastic errors can be averaged out to determine accurate structure-factor amplitudes. In this work, the capability of the Swiss X-ray free-electron laser (SwissFEL) was used to generate large-bandwidth X-ray pulses [Δλ/λ = 2.2% full width at half-maximum (FWHM)], which were applied in SFX with the aim of improving the partiality of Bragg spots and thus decreasing sample consumption while maintaining the data quality. Sensitive data-quality indicators such as anomalous signal from native thaumatin micro-crystals and de novo phasing results were used to quantify the benefits of using pink X-ray pulses to obtain accurate structure-factor amplitudes. Compared with data measured using the same setup but using X-ray pulses with typical quasi-monochromatic XFEL bandwidth (Δλ/λ = 0.17% FWHM), up to fourfold reduction in the number of indexed diffraction patterns required to obtain similar data quality was achieved. This novel approach, pink-beam SFX, facilitates the yet underutilized de novo structure determination of challenging proteins at XFELs, thereby opening the door to more scientific breakthroughs.

scholarly journals Advances in long-wavelength native phasing at X-ray free-electron lasers

IUCrJ ◽  
2020 ◽  
Vol 7 (6) ◽  
pp. 965-975 ◽  
Author(s):  
Karol Nass ◽  
Robert Cheng ◽  
Laura Vera ◽  
Aldo Mozzanica ◽  
Sophie Redford ◽  
...  
Keyword(s):  
Data Quality ◽  
Structure Determination ◽  
Free Electron ◽  
De Novo ◽  
Geometry Optimization ◽  
Protein Structure Determination ◽  
Time Resolved ◽  
X Ray ◽  
Long Wavelength ◽  
Order Of Magnitude

Long-wavelength pulses from the Swiss X-ray free-electron laser (XFEL) have been used for de novo protein structure determination by native single-wavelength anomalous diffraction (native-SAD) phasing of serial femtosecond crystallography (SFX) data. In this work, sensitive anomalous data-quality indicators and model proteins were used to quantify improvements in native-SAD at XFELs such as utilization of longer wavelengths, careful experimental geometry optimization, and better post-refinement and partiality correction. Compared with studies using shorter wavelengths at other XFELs and older software versions, up to one order of magnitude reduction in the required number of indexed images for native-SAD was achieved, hence lowering sample consumption and beam-time requirements significantly. Improved data quality and higher anomalous signal facilitate so-far underutilized de novo structure determination of challenging proteins at XFELs. Improvements presented in this work can be used in other types of SFX experiments that require accurate measurements of weak signals, for example time-resolved studies.

scholarly journals Long-wavelength native-SAD phasing: opportunities and challenges

IUCrJ ◽  
2019 ◽  
Vol 6 (3) ◽  
pp. 373-386 ◽  
Author(s):  
Shibom Basu ◽  
Vincent Olieric ◽  
Filip Leonarski ◽  
Naohiro Matsugaki ◽  
Yoshiaki Kawano ◽  
...  
Keyword(s):  
Anomalous Scattering ◽  
X Rays ◽  
X Ray ◽  
Long Wavelength ◽  
Experimental Phasing ◽  
Sad Phasing ◽  
Anomalous Signal ◽  
Scattering Factor ◽  
X Ray Absorption

Native single-wavelength anomalous dispersion (SAD) is an attractive experimental phasing technique as it exploits weak anomalous signals from intrinsic light scatterers (Z < 20). The anomalous signal of sulfur in particular, is enhanced at long wavelengths, however the absorption of diffracted X-rays owing to the crystal, the sample support and air affects the recorded intensities. Thereby, the optimal measurable anomalous signals primarily depend on the counterplay of the absorption and the anomalous scattering factor at a given X-ray wavelength. Here, the benefit of using a wavelength of 2.7 over 1.9 Å is demonstrated for native-SAD phasing on a 266 kDa multiprotein-ligand tubulin complex (T2R-TTL) and is applied in the structure determination of an 86 kDa helicase Sen1 protein at beamline BL-1A of the KEK Photon Factory, Japan. Furthermore, X-ray absorption at long wavelengths was controlled by shaping a lysozyme crystal into spheres of defined thicknesses using a deep-UV laser, and a systematic comparison between wavelengths of 2.7 and 3.3 Å is reported for native SAD. The potential of laser-shaping technology and other challenges for an optimized native-SAD experiment at wavelengths >3 Å are discussed.

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.

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