Automated batch fiducial-less tilt-series alignment in Appion using Protomo

Abstract

The field of electron tomography has benefited greatly from manual and semi-automated approaches to marker-based tilt-series alignment that have allowed for the structural determination of multitudes of in situ cellular structures as well as macromolecular structures of individual protein complexes. The emergence of complementary metal-oxide semiconductor detectors capable of detecting individual electrons has enabled the collection of low dose, high contrast images, opening the door for reliable correlation-based tilt-series alignment. Here we present a set of automated, correlation-based tilt-series alignment, contrast transfer function (CTF) correction, and reconstruction workflows for use in conjunction with the Appion/Leginon package that are primarily targeted at automating structure determination with cryogenic electron microscopy.

Keywords: Automated processing; Cryo-electron tomography; Marker-free tilt-series alignment; Protomo; Single particle tomography; Transmission electron microscopy.

Fig. 1

Beam-induced bead motion and bead aggregation. (A) A tilt-series exposure taken at a tilt angle of 51 degrees with a dose of 2.34 e⁻/Ų after a cumulative dose of 60 e⁻/Ų, where (left) is before frame alignment while (right) is after. Some beam-induced beam motion can be observed. (B) A zero degree, frame uncorrected tilt exposure with a dose of 57.5 e⁻/Ų. Clear anisotropic beam-induced bead motion on the order of 1 to 10 nanometers can be observed. (C) Hole exposure showing nearly complete gold bead aggregation outside of holes. The central hole is (A). Gold colloidal beads are 10nm in diameter in all images.

Fig. 2

Quality assessment plots. (A) A plot of the CCMS(shift) values vs. iteration number for a real tilt-series. CCMS plots include a convergence baseline (dotted). The spikes in the CCMS plots happen after switching from higher to lower image sampling. This often causes the subsequent one or two iterations to over-estimate the shift, rotation and/or scaling values while the angle refinements are converging to their new and more accurate estimates. (B) Plots of the x and y correction factors for iteration 29 in (A) together with their mean and standard deviations. This tilt-series with a CCMS value of 1.26% is well aligned by the criteria displayed here. (C) Plots of the x and y correction factors for iteration 27 for comparison. Notice that the high tilt angle images on the right of the Correction Factor (x) plot are well above 1%, causing the standard deviation to produce an unacceptable CCMS(shift) value of 2.27%. Correction factor plots include color-coded mean and standard deviation lines (solid and dotted lines, respectively) where the color represents whether the value is above or below pre-defined convergence values (red or green, respectively). Tilt-series and reconstruction videos for iterations (B) and (C) are shown in Movies S1 and S2, respectively.

Fig. 3

Defocus estimation and interpolation for the tilt-series in Fig. 2. The inset images show the estimated CTF curves and defocus values for a select number of tilt angle images, where yellow inner rings indicate a confidently estimated defocus value. Images for which the defocus could not be confidently estimated (e.g. inset, left) have their defocus values interpolated based on the confidently estimated defocus values over the entire tilt-series (blue line). Defocus values were estimated using ACE (Mallick et al., 2005).

Fig. 4

AAV-DJ reconstructions. (A) SPT reconstruction of AAV-DJ from 229 particles from a central tilt axis slice of a single tilt-series. (B) Single particle cryo-EM reconstruction of AAV-DJ lowpass filtered to 30 Å. UCSF Chimera was used for visualization (Pettersen et al., 2004).

Fig. 5

Full CCMS plot and geometry model refinement plots for the Protomo alignment of the first axis of the eTomo tutorial tilt-series. The CCMS values and their scaled sum for each iteration are shown together with recommended values as dotted lines (left). Lowpass (lp) units are angstroms. Angle refinement plots for the tilt azimuth, tilt elevation, and stage orientation are shown with convergence in later iterations (right).

Fig. 6

Similarly located x, y, and z slice-throughs of the eTomo fiducial alignment (left) and the Protomo non-fiducial correlation-based alignment (right). The resolution and level of detail in each reconstruction is comparable. Note that the grid of this particular sample was tilted and/or bent causing the slab in the reconstruction to be positioned at slight angles as seen in the x and y slice-throughs on the right. 3dmod was used for visualization (Kremer et al., 1996).