Abstract
Technological advances in transmission electron microscopes and detectors have turned cryogenic electron microscopy (cryo-EM) into an essential tool for structural biology. A commonly used cryo-EM data analysis method, single particle analysis, averages hundreds of thousands of low-dose images of individual macromolecular complexes to determine a density map of the complex. The presence of symmetry in the complex is beneficial since each projection image can be assigned to multiple views of the complex. However, data processing that applies symmetry can average out asymmetric features and consequently data analysis methods are required to resolve asymmetric structural features. Scipion is a cryo-EM image processing framework that integrates functions from different image processing packages as plugins. To extend its functionality for handling symmetry mismatches, we present here a Scipion plugin termed LocalRec implementing the localized reconstruction method. When tested on an adenovirus data set, the plugin enables resolving the symmetry-mismatched trimeric fibre bound to the five-fold vertices of the capsid. Furthermore, it improves the structure determination of the icosahedral capsid by dealing with the defocus gradient across the particle. LocalRec is expected to be widely applicable in a range of cryo-EM investigations of flexible and symmetry mismatched complexes.
Highlights
Cryogenic electron microscopy is a powerful technique of structural biology
When combined with single particle analysis (SPA), it facilitates the study of biological macromolecules and their complexes by yielding three-dimensional (3D) reconstructions, which are 3D maps of the macromolecular electrostatic potential
We describe a plugin named LocalRec for the Scipion software framework
Summary
Cryogenic electron microscopy (cryo-EM) is a powerful technique of structural biology. If the resolution of the density map is sufficient, typically better than 4.0 Å, it is possible to derive an atomic model of the complex to gain insights into its function. Because cryo-EM images suffer from low signalto-noise ratio, in order to reach sufficient signal at the target resolution tens or hundreds of thousands of images of the target protein or complex (so-called ‘particles’) are required. The required number of particles is reduced by the presence of symmetry in the complex as each individual particle effectively contributes several symmetry-related views of the complex. When symmetry is utilized in calculating the density map, features that do not follow the assumed symmetry are incorrectly averaged. If the assumption of a certain symmetry is only an approximation, it may reduce the attainable resolution of the whole complex
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