title: Localized reconstruction of subunits from electron cryomicroscopy images of macromolecular complexes.
abstract: Electron cryomicroscopy can yield near-atomic resolution structures of highly ordered macromolecular complexes. Often however some subunits bind in a flexible manner, have different symmetry from the rest of the complex, or are present in sub-stoichiometric amounts, limiting the attainable resolution. Here we report a general method for the localized three-dimensional reconstruction of such subunits. After determining the particle orientations, local areas corresponding to the subunits can be extracted and treated as single particles. We demonstrate the method using three examples including a flexible assembly and complexes harbouring subunits with either partial occupancy or mismatched symmetry. Most notably, the method allows accurate fitting of the monomeric RNA-dependent RNA polymerase bound at the threefold axis of symmetry inside a viral capsid, revealing for the first time its exact orientation and interactions with the capsid proteins. Localized reconstruction is expected to provide novel biological insights in a range of challenging biological systems.
authors.affiliation: Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK.
authors.forename: Serban L
authors.initials: SL
authors.lastname: Ilca
authors.affiliation: Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK.
authors.forename: Abhay
authors.identifier: http://orcid.org/0000-0002-4480-5439
authors.initials: A
authors.lastname: Kotecha
authors.affiliation: Department of Biosciences, University of Helsinki, Viikinkaari 9, 00014 Helsinki, Finland.
authors.forename: Xiaoyu
authors.initials: X
authors.lastname: Sun
authors.affiliation: Department of Biosciences, University of Helsinki, Viikinkaari 9, 00014 Helsinki, Finland.
authors.forename: Minna M
authors.initials: MM
authors.lastname: Poranen
authors.affiliation: Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK.|Diamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE, UK.
authors.forename: David I
authors.initials: DI
authors.lastname: Stuart
authors.affiliation: Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK.
authors.forename: Juha T
authors.identifier: http://orcid.org/0000-0002-0348-7323
authors.initials: JT
authors.lastname: Huiskonen
mesh_terms: D036022:Capsid Proteins / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure*; D020285:Cryoelectron Microscopy / Q000379:methods*; D046911:Macromolecular Substances / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure*; D021122:Protein Subunits; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure*; D012401:Rotavirus
chemical_list: D036022:Capsid Proteins; D046911:Macromolecular Substances; D021122:Protein Subunits; D012324:RNA-Dependent RNA Polymerase