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. We introduce a cryoEM calibration sample that is a mixture of compatible macromolecules that can be used not only 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 a reference point for validating their cryoEM pipeline, benchmarking their methodologies, and testing new algorithms.

Figure 1.. Multi-species dataset overview.

A) An exemplar micrograph with four types of particles boxed out. B) Typical processing results: individual extracted particles, 2D class averages, x-y slice through the center of ab-initio reconstruction, 3D map visualized in ChimeraX.

Figure 2.. CryoEM reconstructions from multi-species dataset.

A) Isosurface representations of (from left to right): ApoF, β-gal, TMV, and PP7. B) PDB model fit to map in mesh of (from left to right): ApoF 1FHA chain A: 14–42, β-gal 6X1Q chain A: 429–448, TMV 6R7M chain A: 107–136, and PP7 1DWN chain A: 96–121. C) Histogram and directional FSC plot with sphericity representation and transparent isosurface view (from left to right): ApoF at a global resolution of 2.47 Å with 164,200 particles and sphericity of 0.985, β-gal at a global resolution of 2.74 Å with 37,617 particles and sphericity of 0.976, TMV at a global resolution of 2.46 Å with 12,038 particles and sphericity of 0.977, and PP7 at a global resolution of 3.37 Å with 1,803 particles and sphericity of 0.980.

Figure 3.. Representative images from trials of multi-protein mixtures.

A through E: negative stain micrographs of mixes 1 through 5. F through I: cryo micrographs of mixes 6 through 9. A=mix 1, B=mix 2, C=mix 3, D=mix 4, E=mix 5, F=mix 6, G=mix 7, H=mix 8, I=mix 9. Exact mix compositions are shown in Table 2.