Abstract
Helical reconstruction from electron cryomicrographs has become a routine technique for macromolecular structure determination of helical assemblies since the first days of Fourier-based three-dimensional image reconstruction. In the past decade, the single-particle technique has had an important impact on the advancement of helical reconstruction. Here, we present the software package SPRING that combines Fourier based symmetry analysis and real-space helical processing into a single workflow. One of the most time-consuming steps in helical reconstruction is the determination of the initial symmetry parameters. First, we propose a class-based helical reconstruction approach that enables the simultaneous exploration and evaluation of many symmetry combinations at low resolution. Second, multiple symmetry solutions can be further assessed and refined by single-particle based helical reconstruction using the correlation of simulated and experimental power spectra. Finally, the 3D structure can be determined to high resolution. In order to validate the procedure, we use the reference specimen Tobacco Mosaic Virus (TMV). After refinement of the helical symmetry, a total of 50,000 asymmetric units from two micrographs are sufficient to reconstruct a subnanometer 3D structure of TMV at 6.4Å resolution. Furthermore, we introduce the individual programs of the software and discuss enhancements of the helical reconstruction workflow. Thanks to its user-friendly interface and documentation, SPRING can be utilized by the novice as well as the expert user. In addition to the study of well-ordered helical structures, the development of a streamlined workflow for single-particle based helical reconstruction opens new possibilities to analyze specimens that are heterogeneous in symmetries.
Highlights
Structure determination of large macromolecular assemblies embedded in vitreous ice using electron microscopy (EM) is becoming increasingly popular as evidenced by the steady increase in the number of structure depositions into the EM databank (EMDB) (Lawson et al, 2011)
The most widely used approach is the implementation of the iterative helical real-space reconstruction (IHRSR) based on the SPIDER package (Frank et al, 1996) and additional tools for helical symmetry determination and imposition (Egelman, 2000)
We demonstrate the full functionality of the package by processing a subset of previously published micrographs of Tobacco Mosaic Virus (TMV) (Sachse et al, 2007)
Summary
Structure determination of large macromolecular assemblies embedded in vitreous ice using electron microscopy (EM) is becoming increasingly popular as evidenced by the steady increase in the number of structure depositions into the EM databank (EMDB) (Lawson et al, 2011). The first 3D reconstructions were computed from electron micrographs of helical assemblies (De Rosier and Klug, 1968) These assemblies have the advantage that a single helix already represents many views of the asymmetric unit whose structure needs to be determined. Helical assemblies make up $10% of the determined structures in the entire EMDB This is due to the fact that only a limited number of proteins form arrays of helical symmetry. The most widely used approach is the implementation of the iterative helical real-space reconstruction (IHRSR) based on the SPIDER package (Frank et al, 1996) and additional tools for helical symmetry determination and imposition (Egelman, 2000). SPRING contains programs that determine the microscope parameters, analyze and classify the segmented helices, explore helical symmetry at low resolution, refine high-resolution symmetry and determine the 3D structure
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