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

DOI: 10.1016/j.ymeth.2016.03.007 DOI Help

Authors: Agnel Praveen Joseph (Birkbeck College) , Sony Malhotra (Science and Technology Facilities Council [STFC]) , Tom Burnley (Science and Technology Facilities Council [STFC]) , Chris Wood (Science and Technology Facilities Council [STFC]) , Daniel K. Clare (Diamond Light Source) , Martyn Winn (Science and Technology Facilities Council (STFC)) , Maya Topf (Birkbeck College)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Methods

State: Published (Approved)
Published: March 2016

Abstract: As the resolutions of Three Dimensional Electron Microscopic reconstructions of biological macromolecules are being improved, there is a need for better fitting and refinement methods at high resolutions and robust approaches for model assessment. Flex-EM/MODELLER has been used for flexible fitting of atomic models in intermediate-to-low resolution density maps of different biological systems. Here, we demonstrate the suitability of the method to successfully refine structures at higher resolutions (2.5-4.5 Å) using both simulated and experimental data, including a newly processed map of Apo-GroEL. A hierarchical refinement protocol was adopted where the rigid body definitions are relaxed and atom displacement steps are reduced progressively at successive stages of refinement. For the assessment of local fit, we used the SMOC (segment-based Manders’ overlap coefficient) score, while the model quality was checked using the Qmean score. Comparison of SMOC profiles at different stages of refinement helped in detecting regions that are poorly fitted. We also show how initial model errors can have significant impact on the goodness-of-fit. Finally, we discuss the implementation of Flex-EM in the CCP-EM software suite.

Journal Keywords: eBIC: Flexible fitting; Density fitting; Protein structure modelling; 3D-EM; Structure refinement; Model validation

Subject Areas: Biology and Bio-materials

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