Table 1.

Principal Characteristics of Structural Analysis of Proteins by Cryo-Electron Microscopy and SPA and by Cryo-ET and STA.

Cryo-EM method	Protein	Membrane mimicking model	Images analysis	Examples of protein model and resolution from EMDB data bank.	Comments
SPA	Soluble and transmembrane proteins, filaments, nucleoproteins Viral-like particles Protein > 50–75 Kda complete protein with post-traductional modifications proteins involved in membrane contact sites proteins	Micelles of detergent Amphiphilic polymers (SMA, Amphipols, saponins) Peptidiscs Lipid nanodisc Small lipid vesicles	10, 000- 3 million particles Several free access software including RELION Cryo-SPARC	1.15 Å resolution model of apoferritin. Highest resolution model by SPA In the EMDB data bank, resolution and % of 3D models of protein at xÅ resolution < 2 Å; 0.7% >2 Å and <3 Å; 14.2% >3 Å and < 4 Å; 37.7% 1.15 Å resolution model of apoferritin. Highest resolution model by SPA EMD-11668, (Yip et al., 2020)	For 3D determination at atomic resolution of proteins in different conformation and with activators, inhibitors, or cofactors Developments in deep learning for images analysis, time-resolved cryo-EM, correlation with other structural and dynamics methods SPA is sensitive to conformational heterogeneity and disordered regions
Cryo-ET and Sub-Tomogram Averaging of in vitro systems	Membrane bound and transmembrane proteins Ribosomes Large membrane complexes Viral-like particles In vitro reconstituted membrane contact sites	Lipid vesicles Lipid tubes Proteo-liposomes	Several free access software for 3D reconstruction of cryo-tomograms including IMOD Several free access software for subtomogram averaging including PEET, DYNAMO, EMclarity RELION 1000–100,000 sub-volume to be averaged Need expertise for STA	2.3 Å resolution model of apoferritin EMD-11603, (Tegunov et al., 2021) 2.9 Å resolution model of Equine Virus-like particle, EMD-14031,(Obr et al., 2022) 3.8 Å resolution model of COPII inner coat on tubular membranes, EMD-15949, (Zivanov et al., 2022a) 12 Å resolution model of mouse serotonin receptor in liposomes, EMD-3108, (Kudryashev et al., 2016)	3D architecture of proteins assemblies, proteins assemblies on membrane Analysis of heterogeneous assemblies of proteins 3D view from molecular to micron scale Unambiguous assignment of a function from purified protein. Tuneable parameters in a simpler way than in cell Large number of molecules of proteins to determine 3D models at sub-nanometric resolution by STA
Cryo-ET of purified organelles	Ribosomes on ER-vesicles Ryanodin receptor on ER vesicles Mitofusin on Mitochondria	Purified organelles		4.6 Å resolution model of ribosomes on ER-vesicles, EMD-15872, (Gemmer et al., 2023) 8.9 Å resolution model of Ryanodine receptor in ER vesicles EMD-17272 (Balyschew et al., 2023)	3D architecture of proteins in a purified organelle Large number of molecules of proteins to determine 3D models at sub-nanometric resolution by STA
Cryo-ET edge of cell	Large proteins > 200 Kda Plasma membrane proteins Bacterial secretion systems Viruses entry/egress from cells ER-PM membrane contact sites	Edge of cell Native environment Interior of cell		3.5 Å resolution model of C. crescentus S layer EMD-16183, (Zivanov et al., 2022a) 26.6 Å model of platelet integrins, EMD-12285, (Sorrentino et al., 2021)	3D architecture of protein in a cellular context Need methods for proteins assignment in the cryo-tomograms e.g., cryo-CLEM, deletion of proteins with related function and expression of the proteins of interest, different protein constructs Low number of copies of proteins for STA (< 10 000) Software used to identify target particles in cryo-tomogram often requires specific adaptations to the type of object to be analyzed. Lack of automated tools for particle picking can be problematic for certain target proteins, such as: Tightly stacked filaments Small proteins with weak signals Heterogeneously curved filaments STA is sensitive to conformational heterogeneity and disordered regions
In situ Cryo-ET after Cryo-FIB milling	Large proteins > 200 Kda Ribosomes Filament, microtubules, Nuclear pores Membrane-bound complexes Proteasomes Viruses in cells Membrane contact sites			2.9 Å resolution modele of ribosome (Cheng et al., 2023)* 3.1 Å resolution model of ribosome,, EMD-16721, (Xing et al., 2023) 4.5 Å nebulin bound to actin filament in skeletal sarcomere, EMD-13990, (Wang et al., 2022) 6.5 Å resolution model of mumps virus nucleocapsid in HeLa cells, EMD-13133, (Zhang et al., 2023)

Examples of Proteins and Statistics are Found in the EMDB Data Bank (https://www.ebi.ac.uk/emdb/). SPA: single particle analysis; Cryo-ET: cryo-electron tomography; STA: sub-tomogram averaging; 3D: three-dimensional.

*Article in BioRxiv, 3D models will be released upon publication.