Nature Communications · 2015 · 273 citations · 39 references
Life SciencesBiophysicsMolecular BiologyMolecular VirologyStructural BiologyStructural BiophysicsCryo-electron MicroscopyMacromolecular AssemblyRna Polymerase StructureViral AssemblyViral StructureVirus StructureCryo-electron Microscopy Model FittingCryo-electron Microscopy Of ComplexesCryo-electron Microscopy ReconstructionMacromolecular Complex Structure DeterminationSingle-particle ReconstructionSymmetry ReconstructionThreefold AxesViral Capsid Structure
Electron cryomicroscopy can achieve near‑atomic resolution for ordered complexes, but flexible subunits, differing symmetry, or sub‑stoichiometric presence limit attainable resolution. We present a general method for localized 3‑D reconstruction of such subunits. After determining particle orientations, local subunit regions are extracted and treated as single particles, and the method is demonstrated on three examples including a flexible assembly and complexes with partial occupancy or mismatched symmetry. The method enables accurate fitting of the monomeric RNA‑dependent RNA polymerase at the viral capsid’s threefold axis, revealing its exact orientation and interactions, and promises novel biological insights across challenging systems.
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.
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