Flexible fitting of atomic structures into electron microscopy maps using molecular dynamics

Abstract

A novel method to flexibly fit atomic structures into electron microscopy (EM) maps using molecular dynamics simulations is presented. The simulations incorporate the EM data as an external potential added to the molecular dynamics force field, allowing all internal features present in the EM map to be used in the fitting process, while the model remains fully flexible and stereochemically correct. The molecular dynamics flexible fitting (MDFF) method is validated for available crystal structures of protein and RNA in different conformations; measures to assess and monitor the fitting process are introduced. The MDFF method is then used to obtain high-resolution structures of the E. coli ribosome in different functional states imaged by cryo-EM.

Figure 1

Reconstruction of the E. coli ribosome from cryo-EM data at ∼12.8 Å resolution (K.M., L.G.T., E.V., A. Zavialov, M. Ehrenberg, K.S., and J.F., in preparation). (A) A density histogram shows two distinct peaks pertaining to the solvent and macromolecule; (B) 2-D slice of the density; (C) 2-D slice of the density after clamping values below the average, thus homogenizing the density corresponding to the solvent surrounding the macromolecule and the bulk solvent.

Figure 2

Harmonic restraints applied to base-paired RNA residues. (A) RNA interaction edges for both purines (adenine is shown of the left) and pyrimidines (cytosine is shown on the right), according to Leontis and Westhof (1998); (B) dihedral angles, and the two interatomic distances (dashed lines) to which harmonic restraints are applied.

Figure 3

Validation of the MDFF method using X-ray structures in two conformations. (A) Acethyl-CoA synthase; (B) 16S rRNA (only the head is shown for clarity, since it is the only region where the two conformations differ significantly). The target structures and simulated maps are shown in gray, whereas the initial and final fitted structures are shown in green (top) and colored by backbone RMSD per residue with respect to the target structures (bottom; color scales in Å). The final structures correspond to fittings into 10-Å simulated maps generated from the target structures. Movies of the fittings are included in Supplemental Data S4.

Figure 4

Fitting into the TC-bound ribosome cryo-EM map at 6.7-Å resolution by means of MDFF. (A) Overview of the all-atom ribosome structure fitted into the 6.7-Å map, with a close view into the decoding center (inset). (B) Conformation of tRNA in the A/T site. The crystal structure from the free TC used as a starting point for the fitting (PDB 1OB2, unpublished data) is shown in red; the A/T tRNA model obtained by applying the MDFF method to the 6.7-Å map is shown in blue; the A/T tRNA model previously obtained using a 9.0-Å map constructed by interpolating two manual fittings of tRNA (PDB 1OB2) is shown in green (Valle et al., 2003). (C) Conformation of tRNA in the A/T site (blue) compared to a partial crystal structure of the A-site tRNA (Selmer et al., 2006) (red). The crystal structure from the free TC used as a starting point for the fitting (PDB 1OB2, unpublished data) is shown on the left; the A/T tRNA model obtained by applying the MDFF method to the 6.7-Å map is shown on the right. (D) Conformational dynamics of the GTPase-associated center. Shown are differences in the conformation of the GTPase-associated center between the TC-bound ribosome (EM map at 6.7-Å resolution, top), and the accommodated ribosome (EM map at 9-Å resolution, bottom). Rigid-body docked structures into the corresponding maps, used as initial coordinates for flexible fitting, are shown on the left; flexibly fitted structures are shown on the right.