scholarly journals MicroED Structures from Micrometer Thick Protein Crystals

2017 ◽  
Author(s):  
Michael W. Martynowycz ◽  
Calina Glynn ◽  
Jennifer Miao ◽  
M. Jason de la Cruz ◽  
Johan Hattne ◽  
...  
Keyword(s):  
Theoretical Calculations ◽  
Protein Structures ◽  
Atomic Resolution ◽  
Proteinase K ◽  
Protein Crystals ◽  
Molecular Replacement ◽  
Varying Thickness ◽  
Order Of Magnitude ◽  
Human Prion Protein ◽  
Do So

AbstractTheoretical calculations suggest that crystals exceeding 100 nm thickness are excluded by dynamical scattering from successful structure determination using microcrystal electron diffraction (MicroED). These calculations are at odds with experimental results where MicroED structures have been determined from significantly thicker crystals. Here we systematically evaluate the influence of thickness on the accuracy of MicroED intensities and the ability to determine structures from protein crystals one micrometer thick. To do so, we compare ab initio structures of a human prion protein segment determined from thin crystals to those determined from crystals up to one micrometer thick. We also compare molecular replacement solutions from crystals of varying thickness for a larger globular protein, proteinase K. Our results indicate that structures can be reliably determined from crystals at least an order of magnitude thicker than previously suggested by simulation, opening the possibility for an even broader range of MicroED experiments.SummaryAtomic resolution protein structures can be determined by MicroED from crystals that surpass the theoretical maximum thickness limit by an order of magnitude.

Infrared spectra, rotational correlation functions, and intermolecular mean squared torques in compressed gaseous methane

1968 ◽  
Vol 46 (11) ◽  
pp. 1331-1340 ◽  
Author(s):  
R. L. Armstrong ◽  
S. M. Blumenfeld ◽  
C. G. Gray
Keyword(s):  
Correlation Functions ◽  
Theoretical Calculations ◽  
Dominant Contribution ◽  
Dispersion Interactions ◽  
Fundamental Vibration ◽  
Order Of Magnitude ◽  
The Mean ◽  
Vibration Rotation ◽  
Experimental Values ◽  
Rotational Correlation

Extensive measurements of the methane ν3 and ν4 fundamental vibration–rotation bands in CH4–He mixtures and the ν3 band in CH4–He, CH4–N2, and CD4–He mixtures have been carried out in infrared absorption at 295 °K to pressures of 3000 atm. Some profiles of the ν3 band in CH4–Ar mixtures and in pure CH4 have also been obtained. Rotational correlation functions, band moments, and intermolecular mean squared torques have been determined from the ν3 band profiles. Theoretical calculations of the mean squared torque due to anisotropic multipolar, induction and dispersion interactions have been carried out. The theoretical and experimental torques are in order-of-magnitude agreement for the CH4–N2 and CH4–CH4 systems; for CH4–He, CD4–He, and CH4–Ar the theoretical values are two to three orders of magnitude too small to account for the experimental values, indicating that in these cases the dominant contribution to the torques is given by the anisotropic overlap forces.

Protein phasing at non-atomic resolution by combining Patterson andVLDtechniques

2014 ◽  
Vol 70 (7) ◽  
pp. 1994-2006 ◽  
Author(s):  
Rocco Caliandro ◽  
Benedetta Carrozzini ◽  
Giovanni Luca Cascarano ◽  
Giuliana Comunale ◽  
Carmelo Giacovazzo ◽  
...  
Keyword(s):  
Model Building ◽  
Protein Structures ◽  
Experimental Information ◽  
Atomic Resolution ◽  
Data Sets ◽  
Density Maps ◽  
Combined Use ◽  
Final Electron ◽  
Automatic Model Building

Phasing proteins at non-atomic resolution is still a challenge for anyab initiomethod. A variety of algorithms [Patterson deconvolution, superposition techniques, a cross-correlation function (Cmap), theVLD(vive la difference) approach, the FF function, a nonlinear iterative peak-clipping algorithm (SNIP) for defining the background of a map and thefree lunchextrapolation method] have been combined to overcome the lack of experimental information at non-atomic resolution. The method has been applied to a large number of protein diffraction data sets with resolutions varying from atomic to 2.1 Å, with the condition that S or heavier atoms are present in the protein structure. The applications include the use ofARP/wARPto check the quality of the final electron-density maps in an objective way. The results show that resolution is still the maximum obstacle to protein phasing, but also suggest that the solution of protein structures at 2.1 Å resolution is a feasible, even if still an exceptional, task for the combined set of algorithms implemented in the phasing program. The approach described here is more efficient than the previously described procedures:e.g.the combined use of the algorithms mentioned above is frequently able to provide phases of sufficiently high quality to allow automatic model building. The method is implemented in the current version ofSIR2014.

scholarly journals Atomic resolution structure of serine protease proteinase K at ambient temperature

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Tetsuya Masuda ◽  
Mamoru Suzuki ◽  
Shigeyuki Inoue ◽  
Changyong Song ◽  
Takanori Nakane ◽  
...  
Keyword(s):  
Ambient Temperature ◽  
Serine Protease ◽  
Atomic Resolution ◽  
Proteinase K ◽  
Resolution Structure

scholarly journals Requirements for proteolysis during apoptosis.

1997 ◽  
Vol 17 (11) ◽  
pp. 6502-6507 ◽  
Author(s):  
D L Vaux ◽  
S Wilhelm ◽  
G Häcker
Keyword(s):  
Dna Cleavage ◽  
Morphological Changes ◽  
Cysteine Proteases ◽  
Dna Degradation ◽  
Effector Proteins ◽  
Proteinase K ◽  
Caspase Activation ◽  
Caspase Substrate ◽  
Do So

The key effector proteins of apoptosis are a family of cysteine proteases termed caspases. Following activation of caspases, biochemical events occur that lead to DNA degradation and the characteristic morphological changes associated with apoptosis. Here we show that cytoplasmic extracts activated in vitro by proteinase K were able to cleave the caspase substrate DEVD-7-amino-4-methylcoumarin, while neither proteinase K nor nonactivated extracts were able to do so alone. Caspase-like activity was inhibited by the specific caspase inhibitor DEVD-aldehyde and by the protease inhibitor iodoacetamide, but not by N-ethylmaleimide. When added to isolated nuclei, the activated extracts caused internucleosomal DNA degradation and morphological changes typical of apoptosis. As DNA cleavage and morphological changes could be inhibited by N-ethylmaleimide but not by iodoacetamide, we conclude that during apoptosis, caspase activation causes activation of another cytoplasmic enzyme that can be inhibited by N-ethylmaleimide. Activity of this enzyme is necessary for activation of endonucleases, DNA cleavage, and changes in nuclear morphology.

scholarly journals A novel microseeding method for the crystallization of membrane proteins in lipidic cubic phase

2016 ◽  
Vol 72 (4) ◽  
pp. 307-312 ◽  
Author(s):  
Stefan Andrew Kolek ◽  
Bastian Bräuning ◽  
Patrick Douglas Shaw Stewart
Keyword(s):  
Membrane Proteins ◽  
Soluble Protein ◽  
Protein Crystallization ◽  
Protein Structures ◽  
Integral Membrane Protein ◽  
Seed Stock ◽  
New Method ◽  
Cubic Phase ◽  
Seed Crystals ◽  
Order Of Magnitude

Random microseed matrix screening (rMMS), in which seed crystals are added to random crystallization screens, is an important breakthrough in soluble protein crystallization that increases the number of crystallization hits that are available for optimization. This greatly increases the number of soluble protein structures generated every year by typical structural biology laboratories. Inspired by this success, rMMS has been adapted to the crystallization of membrane proteins, making LCP seed stock by scaling up LCP crystallization conditions without changing the physical and chemical parameters that are critical for crystallization. Seed crystals are grown directly in LCP and, as with conventional rMMS, a seeding experiment is combined with an additive experiment. The new method was used with the bacterial integral membrane protein OmpF, and it was found that it increased the number of crystallization hits by almost an order of magnitude: without microseeding one new hit was found, whereas with LCP-rMMS eight new hits were found. It is anticipated that this new method will lead to better diffracting crystals of membrane proteins. A method of generating seed gradients, which allows the LCP seed stock to be diluted and the number of crystals in each LCP bolus to be reduced, if required for optimization, is also demonstrated.

scholarly journals Effects of DNA oxidation on the evolution of genomes

2017 ◽  
Author(s):  
Michael Sheinman ◽  
Rutger Hermsen
Keyword(s):  
Dna Replication ◽  
Mutation Rate ◽  
Dna Oxidation ◽  
Comparative Genomic ◽  
Computational Studies ◽  
Mutational Spectrum ◽  
Genomic Approach ◽  
Order Of Magnitude ◽  
Evolution Of Genomes ◽  
Do So

Oxidation of DNA increases its mutation rate, causing otherwise rare G → T transversions during DNA replication. Here we use a comparative genomic approach to assess the importance of DNA oxidation for the evolution of genomic sequences. To do so, we study the mutational spectrum of Gn-tracks on various timescales, ranging from one human generation to the divergence between primates, and compare it to the properties of guanines oxidation known from experimental and computational studies. Our results suggest that, in short Gntracks (n≤ 3), oxidation does not dominate the mutagenesis of guanines, except in cancerous tumors, especially in lungs. However, we consistently find that the G → T transversion rate is elevated by an order of magnitude in long Gntracks (n≳ 6). In such long Gn-tracks, G → T substitutions in fact dominate the mutational spectrum, suggesting that long Gntracks are oxidized more frequently and/or repaired less efficiently.

scholarly journals Atomic resolution structure of serine protease proteinase K at ambient temperature

2017 ◽  
Author(s):  
Tetsuya Masuda ◽  
Mamoru Suzuki ◽  
Shigeyuki Inoue ◽  
Changyong Song ◽  
Takanori Nakane ◽  
...  
Keyword(s):  
Ambient Temperature ◽  
Active Sites ◽  
Atomic Resolution ◽  
Proteinase K ◽  
Water Molecules ◽  
Specific Substrate ◽  
Hydrogen Atoms ◽  
X Ray Crystallography ◽  
Chain Conformations ◽  
Resolution Structure

AbstractAtomic resolution structures (beyond 1.20 Å) at ambient temperature, which is usually hampered by the radiation damage in synchrotron X-ray crystallography (SRX), will add to our understanding of the structure-function relationships of enzymes. Serial femtosecond crystallography (SFX) has attracted surging interest by providing a route to bypass such challenges. Yet the progress on atomic resolution analysis with SFX has been rather slow. In this report, we describe the 1.20 Å resolution structure of proteinase K using 13 keV photon energy. Hydrogen atoms, water molecules, and a number of alternative side-chain conformations have been resolved. The increase in the value of B-factor in SFX suggests that the residues and water molecules adjacent to active sites were flexible and exhibited dynamic motions at specific substrate-recognition sites.

scholarly journals Anti-symmetric Compton scattering in LiNiPO4: Towards a direct probe of the magneto-electric multipole moment

2021 ◽  
Vol 1 ◽  
pp. 132
Author(s):  
Sayantika Bhowal ◽  
Daniel O'Neill ◽  
Michael Fechner ◽  
Nicola A. Spaldin ◽  
Urs Staub ◽  
...  
Keyword(s):  
Compton Scattering ◽  
Density Functional ◽  
Theoretical Calculations ◽  
Theoretical Work ◽  
Multipole Moment ◽  
Compton Profile ◽  
Magnetoelectric Materials ◽  
Order Of Magnitude ◽  
The Difference ◽  
Electric Multipole Moment

Background: Magnetoelectric multipoles, which break both space-inversion and time-reversal symmetries, play an important role in the magnetoelectric response of a material. Motivated by uncovering the underlying fundamental physics of the magnetoelectric multipoles and the possible technological applications of magnetoelectric materials, understanding as well as detecting such magnetoelectric multipoles has become an active area of research in condensed matter physics. Here we employ the well-established Compton scattering effect as a possible probe for the magnetoelectric toroidal moments in LiNiPO4. Methods: We employ combined theoretical and experimental techniques to compute as well as detect the antisymmetric Compton profile in LiNiPO4. For the theoretical investigation we use density functional theory to compute the anti-symmetric part of the Compton profile for the magnetic and structural ground state of LiNiPO4. For the experimental verification, we measure the Compton signals for a single magnetoelectric domain sample of LiNiPO4, and then again for the same sample with its magnetoelectric domain reversed. We then take the difference between these two measured signals to extract the antisymmetric Compton profile in LiNiPO4. Results: Our theoretical calculations indicate an antisymmetric Compton profile in the direction of the ty toroidal moment in momentum space, with the computed antisymmetric profile around four orders of magnitude smaller than the total profile. The difference signal that we measure is consistent with the computed profile, but of the same order of magnitude as the statistical errors and systematic uncertainties of the experiment. Conclusions: While the weak difference signal in the measurements prevents an unambiguous determination of the antisymmetric Compton profile in LiNiPO4, our results motivate  further theoretical work to understand the factors that influence the size of the antisymmetric Compton profile, and to identify materials exhibiting larger effects.

Phasing in Crystallography

2013 ◽  
Author(s):  
Carmelo Giacovazzo
Keyword(s):  
Protein Structures ◽  
Data Bank ◽  
Molecular Replacement ◽  
Main Research ◽  
Elite Group ◽  
Isomorphous Replacement ◽  
Technological Advances ◽  
Data Resolution ◽  
Rational Classification

Modern crystallographic methods originate from the synergy of two main research streams, the small-molecule and the macro-molecular streams. The first stream was able to definitively solve the phase problem for molecules up to 200 atoms in the asymmetric unit. The achievements obtained by the macromolecular stream are also impressive. A huge number of protein structures have been deposited in the Protein Data Bank. The solution of them is no longer reserved to an elite group of scientists, but may be attained in a large number of laboratories around the world, even by young scientists. New probabilistic approaches have been tailored to deal with larger structures, errors in the experimental data, and modest data resolution. Traditional phasing techniques like ab initio, molecular replacement, isomorphous replacement, and anomalous dispersion techniques have been revisited. The new approaches have been implemented in robust phasing programs, which have been organized in automatic pipelines usable even by non-experts. Protein structures, which 50 years ago could take months or even years to solve, can now be solved in a matter of hours, partly also due to technological advances in computer science. This book describes all modern crystallographic phasing methods, and introduces a new rational classification of them. A didactic approach is used, with the techniques described simply and logically in the main text, and further mathematical details confined to the Appendices for motivated readers. Numerous figures and applicative details illustrate the text.

Effects of Confinement on Arborescent Graft Polystyrene Particle Shape

2000 ◽  
Vol 6 (S2) ◽  
pp. 1114-1115
Author(s):  
T.M. Chou ◽  
A. Aitouchen ◽  
M. Libera ◽  
R.M. Briber ◽  
M. Gauthier
Keyword(s):  
Molecular Weight ◽  
Carbon Film ◽  
Ccd Camera ◽  
Single Drop ◽  
Ambient Conditions ◽  
Polystyrene Particle ◽  
Graft Polymers ◽  
Order Of Magnitude ◽  
Controllable Size ◽  
Do So

Arborescent graft polymers are highly branched materials synthesized by grafting multiple generations of macromolecular precursors on a central core [1,2]. They can be synthesized with controllable size and molecular weight depending on the number of generations and the precursor molecular weight. They possess many of the unique structural [3], thermodynamic, and rheological [4] properties of dendrimers but do so on a length scale roughly one order of magnitude higher. This research describes the effect of confinement on the shape of arborescent graft polystyrene (PS).This work used 3rd generation arborescent graft PS synthesized with 5kg/mole linear polystyrene precursor (G3- 5K). The G3-5K PS sample was mixed to a dilution of approximately 0.005 wt% in toluene. A single drop of solution was placed on either a holey-carbon film or an ultrathin continuous carbon film. The solvent was evaporated under ambient conditions. A Philips CM20 FEG TEM/STEM equipped with a Gatan 666 PEELS and a 679 multiscan CCD camera was used to study the structure of these specimens.

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