scholarly journals De novo determination of mosquitocidal Cry11Aa and Cry11Ba structures from naturally-occurring nanocrystals

2021 ◽  
Author(s):  
Guillaume Tetreau ◽  
Michael R. Sawaya ◽  
Elke De Zitter ◽  
Elena A. Andreeva ◽  
Anne-Sophie Banneville ◽  
...  
Keyword(s):  
De Novo ◽  
Mosquito Control ◽  
Crystal Packing ◽  
Molecular Replacement ◽  
Dispersion Method ◽  
X Ray ◽  
Naturally Occurring ◽  
Serial Femtosecond Crystallography

Cry11Aa and Cry11Ba are the two most potent toxins produced by mosquitocidal Bacillus thuringiensis subsp. israelensis and jegathesan, respectively. The toxins naturally crystallize within the host; however, the crystals are too small for structure determination at synchrotron sources. Therefore, we applied serial femtosecond crystallography at X-ray free electron lasers to in vivo-grown nanocrystals of these toxins. The structure of Cry11Aa was determined de novo using the single-wavelength anomalous dispersion method, which in turn enabled the determination of the Cry11Ba structure by molecular replacement. The two structures reveal a new pattern for in vivo crystallization of Cry toxins, whereby each of their three domains packs with a symmetrically identical domain, and a cleavable crystal packing motif is located within the protoxin rather than at the termini. The diversity of in vivo crystallization patterns suggests explanations for their varied levels of toxicity and rational approaches to improve these toxins for mosquito control.

The application of the molecular replacement method to the de novo determination of protein structure

1991 ◽  
Vol 24 (4) ◽  
pp. 399-424 ◽  
Author(s):  
Michael C. Lawrence
Keyword(s):  
Protein Structure ◽  
De Novo ◽  
Molecular Replacement ◽  
X Ray Diffraction ◽  
X Ray ◽  
Replacement Method ◽  
Molecular Replacement Method ◽  
Electron Density Map ◽  
Novel Protein

The determination of a novel protein structure by X-ray diffraction is seldom straightforward. Three hurdles present themselves (i) the protein must be purified in sufficient quantity to allow crystallization trials, (ii) crystals must be grown to adequate size and must diffract to a resolution that will allow atomic detail to be revealed, and (iii) phases must be determined for the diffracted X-ray beams in order that an initial electron-density map may be calculated.

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 Surface Engineering of Top7 to Facilitate Structure Determination

2022 ◽  
Vol 23 (2) ◽  
pp. 701
Author(s):  
Yuki Ito ◽  
Takuya Araki ◽  
Shota Shiga ◽  
Hiroyuki Konno ◽  
Koki Makabe
Keyword(s):  
Structure Determination ◽  
Surface Engineering ◽  
De Novo ◽  
Crystal Packing ◽  
Structure Model ◽  
X Ray ◽  
Hexahistidine Tag ◽  
Model Protein ◽  
Β Sheet ◽  
Packing Interactions

Top7 is a de novo designed protein whose amino acid sequence has no evolutional trace. Such a property makes Top7 a suitable scaffold for studying the pure nature of protein and protein engineering applications. To use Top7 as an engineering scaffold, we initially attempted structure determination and found that crystals of our construct, which lacked the terminal hexahistidine tag, showed weak diffraction in X-ray structure determination. Thus, we decided to introduce surface residue mutations to facilitate crystal structure determination. The resulting surface mutants, Top7sm1 and Top7sm2, crystallized easily and diffracted to the resolution around 1.7 Å. Despite the improved data, we could not finalize the structures due to high R values. Although we could not identify the origin of the high R values of the surface mutants, we found that all the structures shared common packing architecture with consecutive intermolecular β-sheet formation aligned in one direction. Thus, we mutated the intermolecular interface to disrupt the intermolecular β-sheet formation, expecting to form a new crystal packing. The resulting mutant, Top7sm2-I68R, formed new crystal packing interactions as intended and diffracted to the resolution of 1.4 Å. The surface mutations contributed to crystal packing and high resolution. We finalized the structure model with the R/Rfree values of 0.20/0.24. Top7sm2-I68R can be a useful model protein due to its convenient structure determination.

scholarly journals Protein–ligand complex structure from serial femtosecond crystallography using soaked thermolysin microcrystals and comparison with structures from synchrotron radiation

2017 ◽  
Vol 73 (8) ◽  
pp. 702-709 ◽  
Author(s):  
Hisashi Naitow ◽  
Yoshinori Matsuura ◽  
Kensuke Tono ◽  
Yasumasa Joti ◽  
Takashi Kameshima ◽  
...  
Keyword(s):  
Synchrotron Radiation ◽  
Room Temperature ◽  
Complex Structure ◽  
Binding Mode ◽  
Ligand Complex ◽  
X Ray ◽  
Cell Parameters ◽  
Serial Femtosecond Crystallography ◽  
Small Molecule Ligand

Serial femtosecond crystallography (SFX) with an X-ray free-electron laser is used for the structural determination of proteins from a large number of microcrystals at room temperature. To examine the feasibility of pharmaceutical applications of SFX, a ligand-soaking experiment using thermolysin microcrystals has been performed using SFX. The results were compared with those from a conventional experiment with synchrotron radiation (SR) at 100 K. A protein–ligand complex structure was successfully obtained from an SFX experiment using microcrystals soaked with a small-molecule ligand; both oil-based and water-based crystal carriers gave essentially the same results. In a comparison of the SFX and SR structures, clear differences were observed in the unit-cell parameters, in the alternate conformation of side chains, in the degree of water coordination and in the ligand-binding mode.

The determination of composition and thermal history of plagioclase by the X-ray powder method

1955 ◽  
Vol 30 (229) ◽  
pp. 648-656 ◽  
Author(s):  
J. Goodyear ◽  
W. J. Duffin
Keyword(s):  
Chemical Composition ◽  
Low Temperature ◽  
Similar Composition ◽  
X Ray ◽  
Naturally Occurring ◽  
Cell Dimensions ◽  
History Of ◽  
Unit Cell Dimensions ◽  
Ray Patterns

In a recent paper (hereafter referred to as GD) Goodyear and Dufiln (1954) described X-ray powder data for a number of synthetic and chemically analysed plagioclases of composition An0Abl00-Anl00Ab0. Important aspects of this work were a correlation of the X-ray patterns with chemical composition, and a distinction between the pattern of a naturally occurring material of low-temperature origin and that of a synthetic of similar composition. The investigation showed quite clearly that the unit-cell dimensions of a synthetic plagioelase depend but little on composition from An0Abl00 to An70Ab30, whilst they differ from those of the low-temperature modification greatly for albite, to a lessening degree as the composition approaches An70Ab30, and practically not at all in the range An70Ab30-Anl00Ab0.

scholarly journals From lows to highs: using low-resolution models to phase X-ray data

2013 ◽  
Vol 69 (11) ◽  
pp. 2257-2265 ◽  
Author(s):  
David I. Stuart ◽  
Nicola G. A. Abrescia
Keyword(s):  
Structural Biology ◽  
General Concept ◽  
Molecular Replacement ◽  
X Ray ◽  
X Ray Crystallography ◽  
X Ray Scattering ◽  
Phase Extension ◽  
Intact Virus ◽  
Structural Methods

The study of virus structures has contributed to methodological advances in structural biology that are generally applicable (molecular replacement and noncrystallographic symmetry are just two of the best known examples). Moreover, structural virology has been instrumental in forging the more general concept of exploiting phase information derived from multiple structural techniques. This hybridization of structural methods, primarily electron microscopy (EM) and X-ray crystallography, but also small-angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR) spectroscopy, is central to integrative structural biology. Here, the interplay of X-ray crystallography and EM is illustrated through the example of the structural determination of the marine lipid-containing bacteriophage PM2. Molecular replacement starting from an ∼13 Å cryo-EM reconstruction, followed by cycling density averaging, phase extension and solvent flattening, gave the X-ray structure of the intact virus at 7 Å resolution This in turn served as a bridge to phase, to 2.5 Å resolution, data from twinned crystals of the major coat protein (P2), ultimately yielding a quasi-atomic model of the particle, which provided significant insights into virus evolution and viral membrane biogenesis.

Sensitivity of in Vivo X-Ray Fluorescence Determination of Skeletal Lead Stores

1990 ◽  
Vol 45 (5) ◽  
pp. 268-272 ◽  
Author(s):  
Rosemary K. Sokas ◽  
Anatole Besarab ◽  
Melissa A. McDiarmid ◽  
Irving M. Shapiro ◽  
Peter Bloch
Keyword(s):  
X Ray ◽  
Fluorescence Determination

scholarly journals Serial crystallography using automated drop dispensing

2021 ◽  
Vol 28 (5) ◽  
Author(s):  
Zhen Su ◽  
Joshua Cantlon ◽  
Lacey Douthit ◽  
Max Wiedorn ◽  
Sébastien Boutet ◽  
...  
Keyword(s):  
Free Electron Laser ◽  
Protein Crystal ◽  
Cross Contamination ◽  
X Ray ◽  
On Demand ◽  
Drop On Demand ◽  
Serial Crystallography ◽  
Cycling System ◽  
Serial Femtosecond Crystallography

Automated, pulsed liquid-phase sample delivery has the potential to greatly improve the efficiency of both sample and photon use at pulsed X-ray facilities. In this work, an automated drop on demand (DOD) system that accelerates sample exchange for serial femtosecond crystallography (SFX) is demonstrated. Four different protein crystal slurries were tested, and this technique is further improved here with an automatic sample-cycling system whose effectiveness was verified by the indexing results. Here, high-throughput SFX screening is shown to be possible at free-electron laser facilities with very low risk of cross contamination and minimal downtime. The development of this technique will significantly reduce sample consumption and enable structure determination of proteins that are difficult to crystallize in large quantities. This work also lays the foundation for automating sample delivery.

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