Refinement of atomic models in high resolution EM reconstructions using Flex-EM and local assessment

Area of Science:

• Structural Biology
• Biophysics
• Computational Biology

Background:

• Advancements in 3D Electron Microscopy (EM) necessitate improved methods for atomic model fitting and refinement.
• Existing flexible fitting methods like Flex-EM/MODELLER have been validated for intermediate-to-low resolution density maps.

Purpose of the Study:

• To evaluate the suitability of Flex-EM/MODELLER for refining atomic models in high-resolution (2.5-4.5Å) 3D EM density maps.
• To demonstrate a hierarchical refinement protocol for enhanced structural accuracy.
• To assess the performance of the method using both simulated and experimental data.

Main Methods:

• Employed a hierarchical refinement protocol with progressively reduced atomic displacement steps.
• Utilized simulated and experimental 3D EM data, including a map of Apo-GroEL.
• Assessed local fit using the segment-based Manders' overlap coefficient (SMOC) score.
• Evaluated model quality using the Qmean score.

Main Results:

• Successfully refined atomic models in high-resolution 3D EM density maps (2.5-4.5Å).
• The hierarchical protocol demonstrated effectiveness in improving structural refinement.
• SMOC profiles aided in identifying poorly fitted regions within the models.
• Demonstrated the significant impact of initial model errors on the goodness-of-fit.

Conclusions:

• Flex-EM/MODELLER is suitable for high-resolution structure refinement in 3D EM.
• The hierarchical approach and assessment tools (SMOC, Qmean) enhance model accuracy and quality evaluation.
• The study highlights the importance of initial model quality and discusses Flex-EM's integration into CCP-EM.