Multi-species cryoEM calibration and workflow verification standard

Abstract

Cryogenic electron microscopy (cryoEM) is a rapidly growing structural biology modality that has been successful in revealing molecular details of biological systems. However, unlike established biophysical and analytical techniques with calibration standards, cryoEM has lacked comprehensive biological test samples. Here, a cryoEM calibration sample consisting of a mixture of compatible macromolecules is introduced that can not only be used for resolution optimization, but also provides multiple reference points for evaluating instrument performance, data quality and image-processing workflows in a single experiment. This combined test specimen provides researchers with a reference point for validating their cryoEM pipeline, benchmarking their methodologies and testing new algorithms.

Keywords: 3D reconstruction and image processing; benchmarking; calibration; cryo-electron microscopy; education; resolution; single-particle analysis; single-particle cryoEM; structure determination; training; workflow standard.

Figure 1

CryoSPARC processing parameters for one-shot parallel processing. Gray rows, processing common to all four species; blue rows, apoferritin-specific steps; red rows, β-gal-specific steps; yellow rows, PP7-specific steps; green rows, TMV-specific steps. See also Supplementary Fig. S7.

Figure 2

Multi-species data-set overview. (a) An exemplar micrograph with four types of particles boxed out (ApoF, blue; β-gal, red; PP7, yellow; TMV, green). (b) Typical processing results: individual extracted particles, 2D class averages, xy slice through the center of ab initio reconstruction and 3D map visualized in ChimeraX.

Figure 3

One-shot processing strategy in cryoSPARC. (a) Diagnostic images from two rounds of particle picking and 2D classification. Blob picking was first used, and templates were selected for the second round of picking using a template picker. (b) Particles belonging to classes corresponding to ApoF, β-gal, PP7 or TMV were selected and used as input for individual ab initio jobs with K = 1. (c) Output of 3D refinement jobs for all four species. Step-by-step information on jobs and settings for one-shot processing in cryoSPARC is described in Fig. 1 ▸.

Figure 4

CryoEM reconstructions from a multi-species data set. (a) Isosurface representations of (left to right) ApoF, β-gal, PP7 and TMV. (b) PDB model fitted into a mesh map of (left to right) ApoF (PDB entry 1fha chain A residues 14–42), β-gal (PDB entry 6x1q chain A residues 429–448), PP7 (PDB entry 1dwn chain A residues 96–121) and TMV (PDB entry 6r7m chain A residues 107–136. (c) Histogram and directional FSC plot with sphericity representation and transparent isosurface view of (left to right) ApoF at a global resolution of 2.47 Å with 164 200 particles and a sphericity of 0.985, β-gal at a global resolution of 2.74 Å with 37 617 particles and a sphericity of 0.976, PP7 at a global resolution of 3.37 Å with 1803 particles and a sphericity of 0.98 and TMV at a global resolution of 2.46 Å with 12 038 particles and a sphericity of 0.977.