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. 2009 Oct;49(2):174-80.
doi: 10.1016/j.ymeth.2009.04.005. Epub 2009 May 4.

Molecular dynamics flexible fitting: a practical guide to combine cryo-electron microscopy and X-ray crystallography

Leonardo G Trabuco  1 , Elizabeth Villa, Eduard Schreiner, Christopher B Harrison, Klaus Schulten
Affiliations

Molecular dynamics flexible fitting: a practical guide to combine cryo-electron microscopy and X-ray crystallography

Leonardo G Trabuco et al. Methods. 2009 Oct.

Abstract

Hybrid computational methods for combining structural data from different sources and resolutions are becoming an essential part of structural biology, especially as the field moves toward the study of large macromolecular assemblies. We have developed the molecular dynamics flexible fitting (MDFF) method for combining high-resolution atomic structures with cryo-electron microscopy (cryo-EM) maps, that results in atomic models representing the conformational state captured by cryo-EM. The method has been applied successfully to the ribosome, a ribonucleoprotein complex responsible for protein synthesis. MDFF involves a molecular dynamics simulation in which a guiding potential, based on the cryo-EM map, is added to the standard force field. Forces proportional to the gradient of the density map guide an atomic structure, available from X-ray crystallography, into high-density regions of a cryo-EM map. In this paper we describe the necessary steps to set up, run, and analyze MDFF simulations and the software packages that implement the corresponding functionalities.

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Figures

Figure 1
Figure 1
Flexible fitting of a tRNA into a cryo-EM map. An atomic model of the ribosome was obtained by applying MDFF to a 6.7-Å cryo-EM map [21, 22]. For illustration purposes, we show only a single tRNA and the corresponding density segmented from the cryo-EM map. The top panel contains the initial tRNA structure after rigid-body docking, whereas the bottom panel contains the fitted structure obtained with MDFF.
Figure 2
Figure 2
Potential derived from the cryo-EM map. (A) Surface representation of a 6.7-Å cryo-EM map of the ribosome [46, 22]. (B) Section of a 2-D slice of the potential generated from the map shown in A, represented as a contour plot. Arrows represent forces calculated using Eq. (3) for each grid point, driving the atomic structure toward high-density regions. In an actual MDFF simulation, the force applied to each atom is calculated by interpolating between the on-lattice forces depicted in the figure.
Figure 3
Figure 3
Restraints applied to base-paired RNA residues. (A) RNA interaction edges for both pyrimidines (cytosine is shown on the left) and purines (guanine is shown on the right), according to [47]; (B) Dihedral angles and the two interatomic distances to which restraints are applied (see Section 2.3.2).
Figure 4
Figure 4
Multistep flexible fitting protocol applied to ribosome cryo-EM maps [21] (see Section 2.4.3 for details).
Figure 5
Figure 5
Convergence of MDFF simulation steps. For each MDFF step, both the (A) RMSD (shown in blue) and (B) global and local CCC (shown in red and black, respectively) reach a plateau. In the figure, the local CCC was calculated with a threshold of 0.2σ above the mean (see Section 3 for details). The insets show the corresponding full-scale plots. The multistep MDFF simulation presented corresponds to flexible fitting applied to a 6.7-Å cryo-EM map of the ribosome [21].
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