Consensus among flexible fitting approaches improves the interpretation of cryo-EM data

Abstract

Cryo-elecron microscopy (cryo-EM) can provide important structural information of large macromolecular assemblies in different conformational states. Recent years have seen an increase in structures deposited in the Protein Data Bank (PDB) by fitting a high-resolution structure into its low-resolution cryo-EM map. A commonly used protocol for accommodating the conformational changes between the X-ray structure and the cryo-EM map is rigid body fitting of individual domains. With the emergence of different flexible fitting approaches, there is a need to compare and revise these different protocols for the fitting. We have applied three diverse automated flexible fitting approaches on a protein dataset for which rigid domain fitting (RDF) models have been deposited in the PDB. In general, a consensus is observed in the conformations, which indicates a convergence from these theoretically different approaches to the most probable solution corresponding to the cryo-EM map. However, the result shows that the convergence might not be observed for proteins with complex conformational changes or with missing densities in cryo-EM map. In contrast, RDF structures deposited in the PDB can represent conformations that not only differ from the consensus obtained by flexible fitting but also from X-ray crystallography. Thus, this study emphasizes that a "consensus" achieved by the use of several automated flexible fitting approaches can provide a higher level of confidence in the modeled configurations. Following this protocol not only increases the confidence level of fitting, but also highlights protein regions with uncertain fitting. Hence, this protocol can lead to better interpretation of cryo-EM data.

Figure 1

RMSD between different RDF/flexible-fitted structures of the four studied proteins obtained from different flexible-fitting approaches (MDfit, YUP.SCX, and NMFF) and from the PDB database fitted using rigid -body fitting (RDF) into Cryo-EM maps. For RF2, bars with * on the top have RMSD values > 10 Å (i.e., 14.6, 15.6, 17.8 and 11.6 Å between RDF and MDfit, RDF and NMFF, RDF and YUP, and MDfit and YUP, respectively).

Figure 2

A comparison of rigidly fitted (RDF, red, PDB: 2O0F) and flexibly fitted (MDfit, green; NMFF, blue; YUP.CSX, cyan) structures of release factor 3 (RF3) after aligning domain I with the RDF structure.

Figure 3

A comparison of initial (yellow, PDB: 1GQE), rigidly fitted (RDF, red, PDB: 1MI6) and flexibly fitted (MDfit, green) structures of release factor 2 (RF2). A closed-to-open transition of ~17 Å is required to fit the initial structure into the cryo-EM map (12.8 Å resolution) such that the domain III is peeled away from domains II & IV and the domain I is also considerably shifted away, according to the RDF model. This complex transition could not be achieved using any of the three flexible fitting approaches.

Figure 4

A comparison of the rigidly fitted structure (RDF, red, PDB: 1PN6), the flexibly fitted structures (MDfit, green; NMFF, blue; YUP.CSX, cyan), and the X-ray structure (PDB: 2WRI, magenta) of elongation factor G (EFG) after aligning domains I–II with the X-ray structure. (a) Flexible-fitted structures show a consensus in the fitting, which is significantly different from the RDF structure. (b) The flexibly fitted structures are also near to the crystal structure in comparison to the RDF structure. Domain V of EFG, which shows the largest orientation differences between the flexibly fitted and the RDF structures, is zoomed in on the right.