Escaping the symmetry trap in helical reconstruction

Abstract

Helical reconstruction is the method of choice for obtaining 3D structures of filaments from electron cryo-microscopy (cryoEM) projections. This approach relies on applying helical symmetry parameters deduced from Fourier-Bessel or real space analysis, such as sub-tomogram averaging. While helical reconstruction continues to provide invaluable structural insights into filaments, its inherent dependence on imposing a pre-defined helical symmetry can also introduce bias. The applied helical symmetry produces structures that are infinitely straight along the filament's axis and can average out biologically important heterogeneities. Here, we describe a simple workflow aimed at overcoming these drawbacks in order to provide truer representations of filamentous structures.

Fig. 1. Helical reconstruction of archaella from M. villosus. (a) Examples of 2D classification of the M. villosus archaellum obtained in Relion 3.1. (b) Layer line profiles of the M. villosus (Mvi, top) and M. hungatei (Mhu, bottom [re-used from ref. with permission]) filaments. (c) Examples of 3D classifications obtained in Relion 3.1. Classes 1 and 4, which show typical features of archaella (central α-helix bundles (blue) and peripheral globular domains (mauve)), were selected for further processing. Scale bar, 50 Å.

Fig. 2. Relaxing the symmetry reveals an improved map. (a and b) CryoEM map viewed from outside (a) and its cross-section (b) of the M. villosus archaellum filament solved imposing a helical symmetry with 108° twist and 5.5 Å rise. The map is coloured in petrol blue–light yellow–magenta from the core to the periphery of the filament. (c) CryoEM density forming the asymmetric unit of the filament in (a) and (b). (d) Close-ups of the cryoEM map in (c) showing large side chains densities in mesh and backbone tracing in orange. (e) CryoEM map from (c) as blue mesh with atomic model of ArlB2 as orange ribbon. The two arrows highlight areas in which the cryoEM map is fragmented. (f) Close-ups of the fragmented cryoEM areas. (g and h) CryoEM map (g) and cross-section (h) of the filament after symmetry relaxation. The colour scheme is the same as in (a). (i) CryoEM density of the two subunits forming the filament in (g). (j) CryoEM maps of the two subunits in (i) in blue mesh with atomic models in ribbon representation. ArlB1, orange; ArlB2, purple. (k) Close-ups of the areas in (j) in which the cryoEM map supports the atomic models of ArlB1 and ArlB2. The arrows in (j) and (k) point at the same areas as in (e) and (f). Comparison between (e), (f), (j) and (k) shows how the map improved through symmetry relaxation and contains additional information, such as glycan densities (* in (j) and (k)). Scale bar in (a, b, g, h), 50 Å; in (c, e, i, j), 10 Å.

Fig. 3. Refining helical parameters after symmetry relaxation. (a) CryoEM map of the M. villosus archaellum filament solved with relaxed helical symmetry showing ArlB1 in orange and ArlB2 in purple. (b) Close-up of the head domains of ArlB1 (orange) and ArlB2 (purple). (c) Helical net diagram showing the positions of ArlB1 (orange dots) and ArlB2 (purple dots) in a two-dimensional plot. Solid black lines show various component helices. (d) CryoEM map of the archaellum filament solved imposing helical symmetry with refined parameters of −71.8° twist and 33.4 Å rise. The map is coloured in petrol blue–light yellow–magenta from the core to the periphery of the filament. (e) Superposition of the cryoEM map obtained relaxing the helical symmetry (solid grey) and cryoEM map obtained applying refined helical parameters (mesh magenta). Two close-ups show the peripheral areas best resolved using the refined helical parameters. (f) Fourier shell correlation curves comparison between the cryoEM maps obtained with initial helical parameters (108° twist and 5.5 Å rise; orange), relaxed helical symmetry (grey), refined helical parameters (−71.8° twist and 33.4 Å rise; red). Scale bar in (a, d, e), 50 Å; in (b), 10 Å.

Fig. 4. Flow chart for helical processing including a symmetry relaxation step.