Four-dimensional microED of conformational dynamics in protein microcrystals on the femto-to-microsecond timescales

Whether time-modulated pulsed-electron imaging in ultrafast electron microscopy (UEM) can mitigate the electron radiation damage that occurs to samples, is still controversial. The effectiveness of such mitigation effect and relevant potential application in cryo-EM remain to be explored. Herein, we built an ultrafast cryo-EM (cryo-UEM) device based on an ultrafast laser system. Using such equipment and the saturated aliphatic hydrocarbon compounds (C44H90), the fading curves of diffraction intensity and corresponding critical electron doses (Ne) of the samples were carefully measured under different imaging modes, temperatures, imaging dose rates and pulsed repetition rates. Our experimental results show that, the fading curves and Ne values of the C44H90 crystals are uncorrelated with the imaging electron dose rates and do not show dependence on the dose-rate effect. As the temperature decreased, the Ne values of the sample increased, indicating the cryoprotective effect on radiation damage to the samples. Surprisingly, at a constant temperature, the fading curves and Ne values of the sample in multi-electrons-packet and near-single-electron-packet pulsed modes are all approximately the same as those in conventional continuous electron-beam mode, even when the results are obtained at different pulsed repetition rates. These results show that the time-modulated pulsed electron beam does not seem to mitigate the electron radiation damage that occurs on samples. Our findings offer new insights and experimental basis for the radiation damage behavior of samples under electron beams, and provide guidance and inspiration for elucidating the fundamental principles of radiation damage.

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Utilizing Molecular Dynamics Simulations, Machine Learning, Cryo-EM, and NMR Spectroscopy to Predict and Validate Protein Dynamics

Son,

Kim,

Park

et al. 2024

IJMS

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Protein dynamics play a crucial role in biological function, encompassing motions ranging from atomic vibrations to large-scale conformational changes. Recent advancements in experimental techniques, computational methods, and artificial intelligence have revolutionized our understanding of protein dynamics. Nuclear magnetic resonance spectroscopy provides atomic-resolution insights, while molecular dynamics simulations offer detailed trajectories of protein motions. Computational methods applied to X-ray crystallography and cryo-electron microscopy (cryo-EM) have enabled the exploration of protein dynamics, capturing conformational ensembles that were previously unattainable. The integration of machine learning, exemplified by AlphaFold2, has accelerated structure prediction and dynamics analysis. These approaches have revealed the importance of protein dynamics in allosteric regulation, enzyme catalysis, and intrinsically disordered proteins. The shift towards ensemble representations of protein structures and the application of single-molecule techniques have further enhanced our ability to capture the dynamic nature of proteins. Understanding protein dynamics is essential for elucidating biological mechanisms, designing drugs, and developing novel biocatalysts, marking a significant paradigm shift in structural biology and drug discovery.

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Four-dimensional microED of conformational dynamics in protein microcrystals on the femto-to-microsecond timescales

Cited by 3 publications

References 101 publications

Scaling up cryo-EM for biology and chemistry: The journey from niche technology to mainstream method

Scaling up cryo-EM for biology and chemistry: The journey from niche technology to mainstream method

Radiation damage behavior of soft matter in ultrafast cryo-electron microscopy (cryo-UEM)

Utilizing Molecular Dynamics Simulations, Machine Learning, Cryo-EM, and NMR Spectroscopy to Predict and Validate Protein Dynamics

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