scholarly journals Softer but stronger? Sulfur SAD with low energy X-rays of 2.7 Å

2014 ◽  
Vol 70 (a1) ◽  
pp. C604-C604
Author(s):  
Dorothee Liebschner ◽  
Naohiro Matsugaki ◽  
Miki Senda ◽  
Yusuke Yamada ◽  
Toshiya Senda
Keyword(s):  
Absorption Edge ◽  
Protein Crystal ◽  
X Rays ◽  
Long Wavelength ◽  
Heavy Atoms ◽  
Statistical Validity ◽  
Experimental Phasing ◽  
Recent Developments ◽  
Long Time ◽  
Anomalous Signal

Single wavelength anomalous diffraction (SAD) is a powerful experimental phasing technique used in macromolecular crystallography (MX). SAD is based on the absorption of X-rays by heavy atoms, which can be either incorporated into the protein (crystal) or naturally present in the structure, such as sulfur or metal ions. In particular, sulfur seems to be an attractive candidate for phasing, because most proteins contain a considerable number of S atoms. However, the K-absorption edge of sulfur is around 5.1 Å wavelength (2.4 keV), which is far from the optimal wavelength of most MX-beamlines at synchrotrons. Therefore, phasing experiments have to be performed further away from the absorption edge, which results in weaker anomalous signal. This explains why S-SAD was not commonly used for a long time, although its feasibility was illustrated by the ground-breaking study by Hendrickson and Teeter [1]. Recent developments in instrumentation, software and methodology made it possible to measure intensities more accurately, and, as a consequence, S-SAD has lately obtained more and more attention [2]. The beamline BL-1A at Photon factory (KEK, Japan) is designed to take full advantage of a long wavelength X-ray beam at around 3 Å to further enhance anomalous signals. We performed S-SAD experiments at BL-1A using two different wavelengths (1.9 Å and 2.7 Å) and compared their phasing capabilities. This methodological study was performed with ferredoxin reductase crystals of various sizes. In order to guarantee statistical validity and to exclude the influence of a particular sample, we repeated the comparison with several crystals. The novelty in the approach consists in using very long wavelengths (2.7 Å), not fully exploited in the literature so far. According to our study, the 2.7 Å wavelength shows - despite strong absorption effects of the diffracted X-rays - more successful phasing results than at 1.9 Å.

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.

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 In-vacuum long-wavelength macromolecular crystallography

2016 ◽  
Vol 72 (3) ◽  
pp. 430-439 ◽  
Author(s):  
Armin Wagner ◽  
Ramona Duman ◽  
Keith Henderson ◽  
Vitaliy Mykhaylyk
Keyword(s):  
Protein Structure ◽  
Diffraction Data ◽  
X Rays ◽  
Macromolecular Crystallography ◽  
Native Protein ◽  
Long Wavelength ◽  
Structure Solution ◽  
Dna Crystals ◽  
Anomalous Signal ◽  
Theoretical Considerations

Structure solution based on the weak anomalous signal from native (protein and DNA) crystals is increasingly being attempted as part of synchrotron experiments. Maximizing the measurable anomalous signal by collecting diffraction data at longer wavelengths presents a series of technical challenges caused by the increased absorption of X-rays and larger diffraction angles. A new beamline at Diamond Light Source has been built specifically for collecting data at wavelengths beyond the capability of other synchrotron macromolecular crystallography beamlines. Here, the theoretical considerations in support of the long-wavelength beamline are outlined and the in-vacuum design of the endstation is discussed, as well as other hardware features aimed at enhancing the accuracy of the diffraction data. The first commissioning results, representing the first in-vacuum protein structure solution, demonstrate the promising potential of the beamline.

scholarly journals The role of wavelength and source in the search for sulfur-atom positions evaluated in two case studies: lysozyme at room temperature and cryo apocrustacyanin A1

2004 ◽  
Vol 37 (4) ◽  
pp. 555-564 ◽  
Author(s):  
Michele Cianci ◽  
John R. Helliwell ◽  
David Moorcroft ◽  
Andrzej Olczak ◽  
James Raftery ◽  
...  
Keyword(s):  
Synchrotron Radiation ◽  
Data Collection ◽  
Room Temperature ◽  
Protein Crystal ◽  
Medium Size ◽  
Data Sets ◽  
X Rays ◽  
Anomalous Signal ◽  
Selection Of

Synchrotron radiation beamlines offer automated data collection with faster and larger detectors, a choice of wavelength(s), intense beams and fine collimation. An increasing output of protein crystal structures sustains an interest in streamlining data collection protocols. Thus, more and more investigators are looking into the use of the anomalous signal from sulfur to obtain initial phase information for medium-size proteins. This type of experiment ideally requires the use of synchrotron radiation, softer X-rays and cryocooling of the sample. Here the results are reported of an investigation into locating the weak,i.e.sulfur, anomalous scatterers in lysozyme using rotating anode or synchrotron radiation data recorded at room temperature. It was indeed possible to locate the sulfur atoms from a lysozyme crystal at room temperature. Accurate selection of images during scaling was needed where radiation damage effects were detected. Most interestingly, comparisons are provided of high-redundancy data sets recorded with synchrotron radiation at λ = 2.0 and 1.488 Å, and with CuKα and MoKα radiation. Apocrustacyanin A1 was also investigated; from the results of a very high redundancy data collection using softer synchrotron X-rays and a cryo-cooled crystal, it was possible to find the sulfur atoms.

On the influence of crystal size and wavelength on native SAD phasing

2016 ◽  
Vol 72 (6) ◽  
pp. 728-741 ◽  
Author(s):  
Dorothee Liebschner ◽  
Yusuke Yamada ◽  
Naohiro Matsugaki ◽  
Miki Senda ◽  
Toshiya Senda
Keyword(s):  
Crystal Size ◽  
Surrounding Medium ◽  
Small Samples ◽  
High Symmetry ◽  
X Rays ◽  
Small Unit ◽  
Heavy Atoms ◽  
Unit Cells ◽  
Sad Phasing ◽  
Anomalous Signal

Native SAD is an emerging phasing technique that uses the anomalous signal of native heavy atoms to obtain crystallographic phases. The method does not require specific sample preparation to add anomalous scatterers, as the light atoms contained in the native sample are used as marker atoms. The most abundant anomalous scatterer used for native SAD, which is present in almost all proteins, is sulfur. However, the absorption edge of sulfur is at low energy (2.472 keV = 5.016 Å), which makes it challenging to carry out native SAD phasing experiments as most synchrotron beamlines are optimized for shorter wavelength ranges where the anomalous signal of sulfur is weak; for longer wavelengths, which produce larger anomalous differences, the absorption of X-rays by the sample, solvent, loop and surrounding medium (e.g.air) increases tremendously. Therefore, a compromise has to be found between measuring strong anomalous signal and minimizing absorption. It was thus hypothesized that shorter wavelengths should be used for large crystals and longer wavelengths for small crystals, but no thorough experimental analyses have been reported to date. To study the influence of crystal size and wavelength, native SAD experiments were carried out at different wavelengths (1.9 and 2.7 Å with a helium cone; 3.0 and 3.3 Å with a helium chamber) using lysozyme and ferredoxin reductase crystals of various sizes. For the tested crystals, the results suggest that larger sample sizes do not have a detrimental effect on native SAD data and that long wavelengths give a clear advantage with small samples compared with short wavelengths. The resolution dependency of substructure determination was analyzed and showed that high-symmetry crystals with small unit cells require higher resolution for the successful placement of heavy atoms.

Recent Developments in Txrf of Light Elements

1995 ◽  
Vol 39 ◽  
pp. 771-779 ◽  
Author(s):  
Christina Streli ◽  
V. Bauer ◽  
P. Wobrauschek
Keyword(s):  
Absorption Edge ◽  
Detection Efficiency ◽  
Fluorescence Analysis ◽  
Total Reflection ◽  
Low Energy ◽  
X Rays ◽  
Light Elements ◽  
Energy Dispersive Analysis ◽  
X Ray ◽  
Recent Developments

Total Reflection X-ray Fluorescence Analysis (TXRF) has been proved to be well suited for the energy dispersive analysis of light elements, as B, C, N, O, F, Na, Mg,.,. using a special spectrometer. It is equipped with a Ge(HP) detector offering a sufficient detection efficiency from 180 eV upwards. The obtainable detection limits especially of the light elements are mainly influenced by the excitation source, which should provide a large number of photons with an energy near the K-absorption edge of these elements (from 200 eV upwards). Commercially available X-ray tubes do not offer characteristic X-rays in that range. In former experiments a windowless X-ray tube was built to prevent the low energy X-rays from being attenuated in the Be window. Experiments have been performed using Cu as anode material.

Getting the best out of long-wavelength X-rays:de novochlorine/sulfur SAD phasing of a structural protein from ATV

2010 ◽  
Vol 66 (3) ◽  
pp. 304-308 ◽  
Author(s):  
Adeline Goulet ◽  
Gisle Vestergaard ◽  
Catarina Felisberto-Rodrigues ◽  
Valérie Campanacci ◽  
Roger A. Garrett ◽  
...  
Keyword(s):  
Heavy Atom ◽  
Chloride Ion ◽  
Chloride Ions ◽  
Protein Crystal ◽  
Structural Protein ◽  
Long Wavelength ◽  
Sad Phasing ◽  
Anomalous Signal ◽  
Efficient Alternative ◽  
Minimal Data

The structure of a 14 kDa structural protein fromAcidianustwo-tailed virus (ATV) was solved by single-wavelength anomalous diffraction (SAD) phasing using X-ray data collected at 2.0 Å wavelength. Although the anomalous signal from methionine sulfurs was expected to suffice to solve the structure, one chloride ion turned out to be essential to achieve phasing. The minimal data requirements and the relative contributions of the Cl and S atoms to phasing are discussed. This work supports the feasibility of a systematic approach for the solution of protein crystal structures by SAD based on intrinsic protein light atoms along with associated chloride ions from the solvent. In such cases, data collection at long wavelengths may be a time-efficient alternative to selenomethionine substitution and heavy-atom derivatization.

X-Ray absorption edge elemental imaging of B. thuringienis

1989 ◽  
Vol 47 ◽  
pp. 212-213
Author(s):  
R. L. Stears
Keyword(s):  
Absorption Edge ◽  
Elemental Distribution ◽  
X Rays ◽  
Differential Absorption ◽  
Synchrotron Source ◽  
High Brightness ◽  
X Ray ◽  
Elemental Imaging ◽  
Cellular Integrity ◽  
X Ray Absorption

Because of the nature of the bacterial endospore, little work has been done on analyzing their elemental distribution and composition in the intact, living, hydrated state. The majority of the qualitative analysis entailed intensive disruption and processing of the endospores, which effects their cellular integrity and composition.Absorption edge imaging permits elemental analysis of hydrated, unstained specimens at high resolution. By taking advantage of differential absorption of x-ray photons in regions of varying elemental composition, and using a high brightness, tuneable synchrotron source to obtain monochromatic x-rays, contact x-ray micrographs can be made of unfixed, intact endospores that reveal sites of elemental localization. This study presents new data demonstrating the application of x-ray absorption edge imaging to produce elemental information about nitrogen (N) and calcium (Ca) localization using Bacillus thuringiensis as the test specimen.

Focusing of Long-Wavelength X-Rays by Means of Spherical and Planar Microchannel Plates

JETP Letters ◽  
2020 ◽  
Vol 112 (3) ◽  
pp. 138-144
Author(s):  
M. I. Mazuritskiy ◽  
A. M. Lerer
Keyword(s):  
X Rays ◽  
Long Wavelength ◽  
Microchannel Plates

Recent Developments In Monochromatic Microprobe X-Ray Fluorescence (MMXRF)

1998 ◽  
Vol 4 (S2) ◽  
pp. 378-379
Author(s):  
Z. W. Chen ◽  
D. B. Wittry
Keyword(s):  
Materials Science ◽  
High Vacuum ◽  
Three Dimensional ◽  
High Sensitivity ◽  
X Rays ◽  
Fluorescence Excitation ◽  
X Ray ◽  
Quantitative Microanalysis ◽  
Recent Developments ◽  
Fifth Order

A monochromatic x-ray microprobe based on a laboratory source has recently been developed in our laboratory and used for fluorescence excitation. This technique provides high sensitivity (ppm to ppb), nondestructive, quantitative microanalysis with minimum sample preparation and does not require a high vacuum specimen chamber. It is expected that this technique (MMXRF) will have important applications in materials science, geological sciences and biological science.Three-dimensional focusing of x-rays can be obtained by using diffraction from doubly curved crystals. In our MMXRF setup, a small x-ray source was produced by the bombardment of a selected target with a focused electron beam and a toroidal mica diffractor with Johann pointfocusing geometry was used to focus characteristic x-rays from the source. In the previous work ∼ 108 photons/s were obtained in a Cu Kα probe of 75 μm × 43 μm in the specimen plane using the fifth order reflection of the (002) planes of mica.

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