A toolbox for ab initio 3-D reconstructions in single-particle electron microscopy

Abstract

Structure determination of a novel macromolecular complex via single-particle electron microscopy depends upon overcoming the challenge of establishing a reliable 3-D reconstruction using only 2-D images. There are a variety of strategies that deal with this issue, but not all of them are readily accessible and straightforward to use. We have developed a "toolbox" of ab initio reconstruction techniques that provide several options for calculating 3-D volumes in an easily managed and tightly controlled work-flow that adheres to standard conventions and formats. This toolbox is designed to streamline the reconstruction process by removing the necessity for bookkeeping, while facilitating transparent data transfer between different software packages. It currently includes procedures for calculating ab initio reconstructions via random or orthogonal tilt geometry, tomograms, and common lines, all of which have been tested using the 50S ribosomal subunit. Our goal is that the accessibility of multiple independent reconstruction algorithms via this toolbox will improve the ease with which models can be generated, and provide a means of evaluating the confidence and reliability of the final reconstructed map.

Figure 1

The alignment and classification pipeline. (A) Routines from different packages are available for raw particle alignment. (B) Summary page showing output for a stack of raw particles aligned with IMAGIC multi-reference alignment. The total sum from the aligned stack and a graphical correlation distribution are additionally displayed. (C) An aligned stack can be subjected to one of four feature analyses, as implemented in each of the processing packages. (D) A package-specific feature analysis summary form is displayed along with the further option of particle clustering into class averages.

Figure 2

Ab Initio reconstructions and initial models within Appion: Each methodology for acquiring an initial 3-D structure, as implemented within the Appion framework, is illustrated with a step-by-step scheme, wherein each box represents the major stage at which user intervention is required. Colored boxes are specific to the particular methodology employed, whereas white boxes represent generic procedures in single-particle image analysis.

Figure 3

Initial models and final reconstructions of the 50S ribosomal subcomplex. (A) EMDB ribosomal model showing relative rotations for each view with an emphasis on defining features such as the L1 and L11 protein arms, the central protuberance (CP), and the tRNA cleft with the protein transferase center (PTC). (B-F) Rotated views of the ribosomal subunit are shown for each initial model (left) and final reconstruction (right). The best resolution is given by FSC_0.5 and RMeasure, respectively. All parameters are summarized within Supplementary Information.

Figure 4

Innacurate reconstructions from initial models. All parameters for refinement were identical to those used to generate the reconstructions in Figure 3. (A) EMDB model as in Figure 3A. Iterative projection-matching refinement was performed on (B) a hemisphere, (C) symmetric Gaussian sphere, (D) asymmetric Gaussian sphere, (E) poorly reconstructed model from angular reconstitution using a preferred orientation dataset, and (F) cross common lines using a non-preferred orientation dataset. Convergence was not achieved for any model.

Figure 5

Influence of particle alignment on a common lines Euler search. All parameters for refinement were identical to those used to generate the reconstructions in Figure 3. Refined and iteratively aligned class averages created from the 5^th iteration of angular reconstitution batch refinement were input into a cross common lines euler search to obtain an initial model which readily converged to an accurate structure (top). Both initial model and final reconstruction are compared to those from Figure 4F (bottom), which did not converge.

Figure 6

Undesired features found in the models generated by the various methods for ab initio 3-D reconstructions. (A) Side view of the 50S ribosomal subunit with elongated density is shown, created using automated 3d0 construction with angular reconstitution. (B) Frontal view showing the generally lower resolution of tomography, obtained using only 22 particles from an earlier attempt. (C) Side view of the RCT reconstruction showing the carbon grain upon which the ribosome lies in its preferred orientation on negatively-stained grids.