Utilizing guanine-coordinated Zn2+ ions to determine DNA crystal structures by single-wavelength anomalous diffraction

2019 ◽  
Vol 75 (1) ◽  
pp. 32-40 ◽  
Author(s):  
Caixia Hou ◽  
Oleg V. Tsodikov
Keyword(s):  
Nucleic Acids ◽  
Crystal Structures ◽  
Structural Changes ◽  
Dna Oligomers ◽  
Anomalous Diffraction ◽  
Double Stranded Dna ◽  
Experimental Phasing ◽  
Sad Phasing ◽  
Anomalous Signal

The experimental phase determination of crystal structures of nucleic acids and nucleic acid–ligand complexes would benefit from a facile method. Even for double-stranded DNA, software-generated models are generally insufficiently accurate to serve as molecular replacement search models, necessitating experimental phasing. Here, it is demonstrated that Zn2+ ions coordinated to the N7 atom of guanine bases generate sufficient anomalous signal for single-wavelength anomalous diffraction (SAD) phasing of DNA crystal structures. Using zinc SAD, three crystal structures of double-stranded DNA oligomers, 5′-AGGGATCCCT-3′, 5′-GGGATCCC-3′ and 5′-GAGGCCTC-3′, were determined. By determining the crystal structure of one of these oligomers, GAGGCCTC, in the presence of Mg2+ instead of Zn2+, it was demonstrated that Zn2+ is not structurally perturbing. These structures allowed the analysis of structural changes in the DNA on the binding of analogues of the natural product mithramycin to two of these oligomers, AGGGATCCCT and GAGGCCTC. Zinc SAD may become a routine approach for determining the crystal structures of nucleic acids and their complexes with small molecules.

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 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 Structure of the cyanobactin oxidase ThcOx fromCyanothecesp. PCC 7425, the first structure to be solved at Diamond Light Source beamline I23 by means of S-SAD

2016 ◽  
Vol 72 (11) ◽  
pp. 1174-1180 ◽  
Author(s):  
Andrew F. Bent ◽  
Greg Mann ◽  
Wael E. Houssen ◽  
Vitaliy Mykhaylyk ◽  
Ramona Duman ◽  
...  
Keyword(s):  
Light Source ◽  
Heavy Atom ◽  
Protein Crystal ◽  
Molecular Replacement ◽  
Anomalous Effect ◽  
Anomalous Diffraction ◽  
Native Sulfur ◽  
New Instrument ◽  
Sad Phasing

Determination of protein crystal structures requires that the phases are derived independently of the observed measurement of diffraction intensities. Many techniques have been developed to obtain phases, including heavy-atom substitution, molecular replacement and substitution during protein expression of the amino acid methionine with selenomethionine. Although the use of selenium-containing methionine has transformed the experimental determination of phases it is not always possible, either because the variant protein cannot be produced or does not crystallize. Phasing of structures by measuring the anomalous diffraction from S atoms could in theory be almost universal since almost all proteins contain methionine or cysteine. Indeed, many structures have been solved by the so-called native sulfur single-wavelength anomalous diffraction (S-SAD) phasing method. However, the anomalous effect is weak at the wavelengths where data are normally recorded (between 1 and 2 Å) and this limits the potential of this method to well diffracting crystals. Longer wavelengths increase the strength of the anomalous signal but at the cost of increasing air absorption and scatter, which degrade the precision of the anomalous measurement, consequently hindering phase determination. A new instrument, the long-wavelength beamline I23 at Diamond Light Source, was designed to work at significantly longer wavelengths compared with standard synchrotron beamlines in order to open up the native S-SAD method to projects of increasing complexity. Here, the first novel structure, that of the oxidase domain involved in the production of the natural product patellamide, solved on this beamline is reported using data collected to a resolution of 3.15 Å at a wavelength of 3.1 Å. The oxidase is an example of a protein that does not crystallize as the selenium variant and for which no suitable homology model for molecular replacement was available. Initial attempts collecting anomalous diffraction data for native sulfur phasing on a standard macromolecular crystallography beamline using a wavelength of 1.77 Å did not yield a structure. The new beamline thus has the potential to facilitate structure determination by native S-SAD phasing for what would previously have been regarded as very challenging cases with modestly diffracting crystals and low sulfur content.

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 Combining random microseed matrix screening and the magic triangle for the efficient structure solution of a potential lysin from bacteriophage P68

2019 ◽  
Vol 75 (7) ◽  
pp. 670-681
Author(s):  
Jia Quyen Truong ◽  
Santosh Panjikar ◽  
Linda Shearwin-Whyatt ◽  
John B. Bruning ◽  
Keith E. Shearwin
Keyword(s):  
Hen Egg White Lysozyme ◽  
High Quality ◽  
X Ray Crystallography ◽  
Egg White Lysozyme ◽  
Structure Solution ◽  
Experimental Phasing ◽  
Sad Phasing ◽  
Hen Egg White ◽  
Novel Structures

Two commonly encountered bottlenecks in the structure determination of a protein by X-ray crystallography are screening for conditions that give high-quality crystals and, in the case of novel structures, finding derivatization conditions for experimental phasing. In this study, the phasing molecule 5-amino-2,4,6-triiodoisophthalic acid (I3C) was added to a random microseed matrix screen to generate high-quality crystals derivatized with I3C in a single optimization experiment. I3C, often referred to as the magic triangle, contains an aromatic ring scaffold with three bound I atoms. This approach was applied to efficiently phase the structures of hen egg-white lysozyme and the N-terminal domain of the Orf11 protein from Staphylococcus phage P68 (Orf11 NTD) using SAD phasing. The structure of Orf11 NTD suggests that it may play a role as a virion-associated lysin or endolysin.

Structure determination of the nucleosome core particle by selenium SAD phasing

2019 ◽  
Vol 75 (10) ◽  
pp. 930-936
Author(s):  
Mika Saotome ◽  
Naoki Horikoshi ◽  
Kazuki Urano ◽  
Tomoya Kujirai ◽  
Hidetaka Yuzurihara ◽  
...  
Keyword(s):  
Molecular Replacement ◽  
Base Pairs ◽  
Simple Method ◽  
X Ray Crystallography ◽  
Nucleosome Core ◽  
Nucleosome Core Particles ◽  
Experimental Phasing ◽  
Eukaryotic Species ◽  
Sad Phasing

The eukaryotic genome is compacted inside the nucleus of the cell in the form called chromatin. The fundamental unit of chromatin is the nucleosome, which contains four types of histones (H3, H4, H2A and H2B) and approximately 150 base pairs of DNA wrapped around the histone complex. The structure of the nucleosome is highly conserved across several eukaryotic species, and molecular replacement has been the primary phasing method used to solve nucleosome structures by X-ray crystallography. However, there is currently no simple, widely applicable experimental phasing method for the nucleosome. In the present study, it is demonstrated that selenomethionine-incorporated histones H3, H2A and H2B can be reconstituted into nucleosomes and crystallized for structural determination. Unexpectedly, it was found that the nucleosome can be phased with a relatively small number of Se atoms. The structures of nucleosome core particles containing 12 and 16 Se atoms were solved by SAD phasing at 2.5 and 2.4 Å resolution, respectively. The present study demonstrates a simple method for determining nucleosome structures by experimental phasing, which may be particularly useful for noncanonical structures that cannot be solved by molecular replacement.

scholarly journals Rapid cadmium SAD phasing at the standard wavelength (1 Å)

2017 ◽  
Vol 73 (7) ◽  
pp. 581-590 ◽  
Author(s):  
Saravanan Panneerselvam ◽  
Esa-Pekka Kumpula ◽  
Inari Kursula ◽  
Anja Burkhardt ◽  
Alke Meents
Keyword(s):  
Crystal Growth ◽  
Data Set ◽  
Cadmium Ions ◽  
Experimental Phasing ◽  
Wide Range ◽  
Sad Phasing ◽  
Anomalous Signal ◽  
Single Data ◽  
Nucleotide Binding Proteins ◽  
Cation Complexes

Cadmium ions can be effectively used to promote crystal growth and for experimental phasing. Here, the use of cadmium ions as a suitable anomalous scatterer at the standard wavelength of 1 Å is demonstrated. The structures of three different proteins were determined using cadmium single-wavelength anomalous dispersion (SAD) phasing. Owing to the strong anomalous signal, the structure of lysozyme could be automatically phased and built using a very low anomalous multiplicity (1.1) and low-completeness (77%) data set. Additionally, it is shown that cadmium ions can easily substitute divalent ions in ATP–divalent cation complexes. This property could be generally applied for phasing experiments of a wide range of nucleotide-binding proteins. Improvements in crystal growth and quality, good anomalous signal at standard wavelengths (i.e.no need to change photon energy) and rapid phasing and refinement using a single data set are benefits that should allow cadmium ions to be widely used for experimental phasing.

scholarly journals Choosing your (Friedel) mates wisely: grouping data sets to improve anomalous signal

2019 ◽  
Vol 75 (2) ◽  
pp. 200-210 ◽  
Author(s):  
Nicolas Foos ◽  
Michele Cianci ◽  
Max H. Nanao
Keyword(s):  
Genetic Algorithm ◽  
Data Sets ◽  
Anomalous Diffraction ◽  
Sad Phasing ◽  
Anomalous Signal ◽  
The Relationship

Single-wavelength anomalous diffraction (SAD) phasing from multiple crystals can be especially challenging in samples with weak anomalous signals and/or strong non-isomorphism. Here, advantage is taken of the combinatorial diversity possible in such experiments to study the relationship between merging statistics and downstream metrics of phasing signals. It is furthermore shown that a genetic algorithm (GA) can be used to optimize the grouping of data sets to enhance weak anomalous signals based on these merging statistics.

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