HEMNMA-3D: Cryo Electron Tomography Method Based on Normal Mode Analysis to Study Continuous Conformational...

Area of Science:

• Structural Biology
• Biophysics
• Computational Biology

Background:

• Cryo-electron tomography (cryo-ET) enables *in situ* structural determination of biomolecules.
• Analyzing cryo-ET data for macromolecular complex dynamics is challenging due to cellular crowding and conformational heterogeneity.
• Existing methods struggle to resolve continuous conformational changes within complexes.

Purpose of the Study:

• To introduce HEMNMA-3D, the first method for analyzing cryo-electron subtomograms for continuous conformational changes in complexes.
• To provide a computational tool for visualizing and interpreting the dynamics of macromolecular complexes *in situ*.
• To enable deeper insights into the functional states of biomolecular machinery within their native cellular environment.

Main Methods:

• HEMNMA-3D employs iterative 3D-to-3D alignments (elastic and rigid-body) of a flexible reference structure.
• Elastic matching integrates Normal Mode Analysis (NMA) with experimental subtomogram data.
• Rigid-body alignment corrects for the 'missing wedge' artifact inherent in cryo-ET data acquisition.

Main Results:

• Conformational parameters derived from alignments are visualized in a low-dimensional 'space of conformations'.
• 3D averages of similar conformations and movies of reference displacement reveal complex dynamics.
• Validation using synthetic datasets and application to *in situ* nucleosome conformational variability demonstrate method efficacy.

Conclusions:

• HEMNMA-3D effectively analyzes cryo-ET data to reveal continuous conformational dynamics of macromolecular complexes.
• The method provides a visually interpretable insight into molecular motion within the cellular context.
• HEMNMA-3D, available as open-source software, significantly advances the potential of cryo-ET for studying biological systems.