Localized reconstruction of subunits from electron cryomicroscopy images of macromolecular complexes

By S. Ilca, A. Kotecha, Xiaoyu Sun, M. Poranen, D. Stuart, J. Huiskonen. 2015

241 citations recorded by the source corpus.

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. Electron cryomicroscopy can allow the elucidation of macromolecular structures; however, mismatches in symmetry between different components limit the attainable resolution. Here, the authors set out a computational method for extracting and retaining information from such components.

Subfields: Molecular Biology, Structural Biology

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Citations in the corpus

Elicitation of broadly protective immunity to influenza by multivalent hemagglutinin nanoparticle vaccines (2020, methodology): “We subsequently implemented a previously reported localized reconstruction strategy 47 to determine a reconstruction of the H1 MI15 HA at 3.3 Å resolution using Relion.”
Characterisation of molecular motions in cryo-EM single-particle data by multi-body refinement in RELION (2018, methodology): “As this software is distributed as open-source, others can contribute their own modifications and improvements of the presented algorithms, as has happened for partial signal subtraction approaches in the past (Zhou et al., 2015; Ilca et al., 2015; Schoebel et al., 2017).”
Processing of Structurally Heterogeneous Cryo-EM Data in RELION. (2016, methodology): “…by Ilca and colleagues to subtract viral capsid densities in order to visualize an RNA polymerase bound inside double-stranded RNA bacteriophage φ6 (Ilca et al., 2015), while Zhou and colleagues used their own modified version of RELION to subtract projections of NSF rings to analyze structural…”