Conformational Change of Nucleosome Arrays prior to Phase Separation.

Chromatin phase transition serves as a regulatory mechanism for eukaryotic transcription. Understanding this process requires the characterization of the nucleosome array structure in response to external stimuli prior to phase separation. However, the intrinsic flexibility and heterogeneity hinders the arrays' structure determination. Here we exploit advances in cryogenic electron tomography (cryo-ET) to determine the three-dimensional (3D) structure of each individual particle of mono-, di-, tri-, and tetranucleosome arrays. Statistical analysis reveals the ionic strength changes the angle between the DNA linker and nucleosome core particle (NCP), which regulate the overall morphology of nucleosome arrays. The finding that one-third of the arrays in the presence of H1 contain an NCP invaded by foreign DNA suggests an alternative function of H1 in constructing nucleosomal networks. The new insights into the nucleosome conformational changes prior to the intermolecular interaction stage extends our understanding of chromatin phase separation regulation.

Fully Mechanically Controlled Automated Electron Microscopic Tomography.

Knowledge of three-dimensional (3D) structures of each individual particles of asymmetric and flexible proteins is essential in understanding those proteins' functions; but their structures are difficult to determine. Electron tomography (ET) provides a tool for imaging a single and unique biological object from a series of tilted angles, but it is challenging to image a single protein for three-dimensional (3D) reconstruction due to the imperfect mechanical control capability of the specimen goniometer under both a medium to high magnification (approximately 50,000-160,000×) and an optimized beam coherence condition. Here, we report a fully mechanical control method for automating ET data acquisition without using beam tilt/shift processes. This method could reduce the accumulation of beam tilt/shift that used to compensate the error from the mechanical control, but downgraded the beam coherence. Our method was developed by minimizing the error of the target object center during the tilting process through a closed-loop proportional-integral (PI) control algorithm. The validations by both negative staining (NS) and cryo-electron microscopy (cryo-EM) suggest that this method has a comparable capability to other ET methods in tracking target proteins while maintaining optimized beam coherence conditions for imaging.