title: Refinement of atomic models in high resolution EM reconstructions using Flex-EM and local assessment.
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 (2.5-4.5Å) 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.
authors.affiliation: Institute of Structural and Molecular Biology, Department of Biological Sciences, Birkbeck College, University of London, Malet Street, London WC1E 7HX, United Kingdom.
authors.forename: Agnel Praveen
authors.initials: AP
authors.lastname: Joseph
authors.affiliation: Scientific Computing Department, Science and Technology Facilities Council, Research Complex at Harwell, Didcot OX11 0FA, United Kingdom.
authors.forename: Sony
authors.initials: S
authors.lastname: Malhotra
authors.affiliation: Scientific Computing Department, Science and Technology Facilities Council, Research Complex at Harwell, Didcot OX11 0FA, United Kingdom.
authors.forename: Tom
authors.initials: T
authors.lastname: Burnley
authors.affiliation: Scientific Computing Department, Science and Technology Facilities Council, Research Complex at Harwell, Didcot OX11 0FA, United Kingdom.
authors.forename: Chris
authors.initials: C
authors.lastname: Wood
authors.affiliation: Electron Bio-Imaging Centre (eBIC), Diamond Light Source, Harwell Science & Innovation Campus, OX11 0DE, United Kingdom.
authors.forename: Daniel K
authors.initials: DK
authors.lastname: Clare
authors.affiliation: Scientific Computing Department, Science and Technology Facilities Council, Research Complex at Harwell, Didcot OX11 0FA, United Kingdom. Electronic address: martyn.winn@stfc.ac.uk.
authors.forename: Martyn
authors.initials: M
authors.lastname: Winn
authors.affiliation: Institute of Structural and Molecular Biology, Department of Biological Sciences, Birkbeck College, University of London, Malet Street, London WC1E 7HX, United Kingdom. Electronic address: m.topf@mail.cryst.bbk.ac.uk.
authors.forename: Maya
authors.initials: M
authors.lastname: Topf
mesh_terms: D000263:Adenylate Kinase / Q000737:chemistry / Q000648:ultrastructure; D018834:Chaperonin 60 / Q000737:chemistry / Q000648:ultrastructure; D020285:Cryoelectron Microscopy; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000648:ultrastructure; D039642:Eukaryotic Initiation Factors / Q000737:chemistry / Q000648:ultrastructure; D021621:Imaging, Three-Dimensional; D008958:Models, Molecular; D054683:Ribosome Subunits, Large, Eukaryotic / Q000737:chemistry / Q000648:ultrastructure; D012984:Software
chemical_list: D018834:Chaperonin 60; D029968:Escherichia coli Proteins; D039642:Eukaryotic Initiation Factors; D000263:Adenylate Kinase
keywords: 3D-EM; Density fitting; Flexible fitting; Model validation; Protein structure modelling; Structure refinement