A Workflow for Visualizing Protein Conformational Dynamics from Cryo-EM Maps Using Human Asparagine Synthetase

Area of Science:

• Structural Biology
• Biophysics
• Biochemistry

Background:

• Conformational heterogeneity in macromolecules presents a challenge for structural determination using cryo-electron microscopy (cryo-EM).
• Traditional discrete classification methods may not fully capture continuous or complex motions within protein structures.
• Understanding protein dynamics is crucial for elucidating biological function.

Purpose of the Study:

• To present a comprehensive workflow for analyzing conformational heterogeneity in cryo-EM data.
• To enable the visualization and refinement of atomic models representing different structural states.
• To provide a framework for linking protein dynamics to biochemical and functional hypotheses.

Main Methods:

• Utilizing 3D variability analysis (3DVA) in CryoSPARC to compute variability maps from particle images.
• Applying principal component analysis to identify modes of structural variation.
• Employing variability refinement in Phenix to generate ensembles of atomic models corresponding to different conformational frames.
• Demonstrating the workflow using human asparagine synthetase (ASNS).

Main Results:

• Generation of 3D variability maps highlighting principal modes of structural motion.
• Refinement of multi-model atomic structures representing discrete conformational states.
• Production of visualization files for comparative analysis and movie generation.
• Successful application to cryo-EM reconstructions in the 2.0-6.0 Å resolution range.

Conclusions:

• The presented workflow effectively analyzes conformational heterogeneity in cryo-EM data.
• It allows for the detailed study of continuous domain motions, hinge-bending, and local rearrangements.
• The approach facilitates direct comparison of conformational extremes and quantitative dynamic analysis (e.g., RMSF calculations).