Nucleosomes are elementary units of chromatin compaction, which play crucial role in genome functioning. X-ray crystallographic studies of the nucleosome core consistently revealed a compact struct...
Formation of compact dinucleosomes (CODIs) occurs after collision between adjacent nucleosomes at active regulatory DNA regions. Although CODIs are likely dynamic structures, their structural heterogeneity and dynamics were not systematically addressed. Here, single-particle Förster resonance energy transfer (spFRET) and electron microscopy were employed to study the structure and dynamics of CODIs. spFRET microscopy in solution and in gel revealed considerable uncoiling of nucleosomal DNA from the histone octamer in a fraction of CODIs, suggesting that at least one of the nucleosomes is destabilized in the presence of the adjacent closely positioned nucleosome. Accordingly, electron microscopy analysis suggests that up to 30 bp of nucleosomal DNA are involved in transient uncoiling/recoiling on the octamer. The more open and dynamic nucleosome structure in CODIs cannot be stabilized by histone chaperone Spt6. The data suggest that proper internucleosomal spacing is an important determinant of chromatin stability and support the possibility that CODIs could be intermediates of chromatin disruption.
Background: Tick-borne encephalitis virus (TBEV) is a dangerous human pathogen which envelope structure is already known from cryoEM study. TBEV mature viral particle size (~50 nm in diameter) makes it suitable for single-particle imaging (SPI) on X-ray free-electron laser (XFEL). XFEL SPI studies are at the early stages of development; thus, a well-described and conformationally homogeneous sample is required to develop approaches for experimental setup and data analysis. Here we present the image analysis results of data collected in October 2019 during the European XFEL experiment #2316. Methods: The detector was placed at 1.62 m from the injector; photon energy was around 6 keV, pulse energy 4 mJ, beam diameter ~ 500 nm. All runs were processed to detect hits with threshold filter (5th percentile of lit pixels) and further filtered to omit low-intensity images and images that lack detector modules. Filtered hits were background and geometry corrected with SPImage library and custom python scripts. Then hits were azimuthally integrated using PyFAI library. Scattering profiles were further clustered using the affinity propagation algorithm with cosine similarity metric in log space. Extracted classes were used to build averaged images. All hit profiles were fitted with model scattering to estimate the diameter of the particle. Simulated diffraction patterns were prepared using Condor from the cryoEM electron density map (EMDB ID 3752). Results: During the analysis after the filtering, only 276 clean and bright hits were collected per 135 min of injection (from 27287 hits detected via lit pixels threshold). Thus the hit rate was around ~ 2 hits/min, which is expected to rise in the future. The majority of hits correspond to the 40-50 nm particles (Fig. 1a), which is expected for TBEV. However, the exact size may vary due to solvent evaporation, ion condensation, and possible variability in the sample. Conclusion: The averaged images and their scattering profiles correlate with the simulated scattering patterns, though not ideally (Fig. 1 bc). Such discrepancy is expected due to the absence of electron density in the center of modeled viral structures.
Background: Protein structure determination using X-ray free-electron laser (XFEL) includes analysis and merging a large number of snapshot diffraction patterns.Convolutional neural networks are widely used to solve numerous computer vision problems, e.g.image classification, and can be used for diffraction pattern analysis.But the task of protein structure determination with the use of CNNs only is not yet solved.Methods: We collected a number of predominantly alpha-helical protein structures from PDB and analyzed their geometry.Relatively straight helices were left unchanged while curved ones were split into helices of smaller length.Finally, 88 two-helical protein structures were selected with the length of helices from 5 to 38 residues (7 to 57Å).For every structure radii, lengths and relative position and orientation of helices were calculated.Diffraction patterns were calculated by means of straight modeling.Every structure was approximated as a pair of cylinders of given length and radius and then its diffraction image was calculated with the explicit formula:, where I(R) is intensity generated on the point of detector with radius-vector R, V is the volume of structure, A 0 is an amplitude of w-ray wave, k 0 is a vector of initial wave, k is a vector of scattered wave.The obtained collection of diffraction patterns was used to train and test the convolutional neural network (CNN).A number of convolutional layers is used to extract features from input images.Then, a dense layer is used to solve a multi-class classification problem.In order to obtain learnable parameters, we have to solve the minimization problem of the cross-entropy loss function.Results: Preliminary length and radius of helices with given sequence could be obtained from molecular modeling.Taking this into account, our model demonstrates a possibility to classify helix pairs into up to 50 disjoint classes. Conclusion:CNNs could be successively used for the purpose of classification of two-helical idealized protein structures.This could be used for preliminary analysis of protein conformation.Our further efforts will be directed towards enlarging the number of classes and expanding our approach to more complex structures.
Histone N-terminal tails and their post-translational modifications affect various biological processes, often in a context-specific manner; the underlying mechanisms are poorly studied. Here, the role of individual N-terminal tails of histones H2A/H2B during transcription through chromatin was analyzed in vitro. spFRET data suggest that the tail of histone H2B (but not of histone H2A) affects nucleosome stability. Accordingly, deletion of the H2B tail (amino acids 1-31, but not 1-26) causes a partial relief of the nucleosomal barrier to transcribing RNA polymerase II (Pol II), likely facilitating uncoiling of DNA from the histone octamer during transcription. Taken together, the data suggest that residues 27-31 of histone H2B stabilize DNA-histone interactions at the DNA region localized ~25 bp in the nucleosome and thus interfere with Pol II progression through the region localized 11-15 bp in the nucleosome. This function of histone H2B requires the presence of the histone H2A N-tail that mediates formation of nucleosome-nucleosome dimers; however, nucleosome dimerization per se plays only a minimal role during transcription. Histone chaperone FACT facilitates transcription through all analyzed nucleosome variants, suggesting that H2A/H2B tails minimally interact with FACT during transcription; therefore, an alternative FACT-interacting domain(s) is likely involved in this process.
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Transcription through chromatin by different RNA polymerases produces different biological outcomes and is accompanied by either nucleosome survival at the original location (Pol II-type mechanism) or backward nucleosome translocation along DNA (Pol III-type mechanism). It has been proposed that differences in the structure of the key intermediates formed during transcription dictate the fate of the nucleosomes. To evaluate this possibility, structure of the key intermediate formed during transcription by Pol III-type mechanism was studied by DNase I footprinting and molecular modeling. The Pol III-type mechanism is characterized by less efficient formation of the key intermediate required for nucleosome survival (Ø-loop, Pol II-type mechanism), most likely due to steric interference between the RNA polymerase and DNA in the Ø-loop. The data suggest that the lower efficiency of Ø-loop formation induces formation of a lower nucleosomal barrier and nucleosome translocation during transcription by Pol III-type mechanism.
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