Symmetry-Restrained Flexible Fitting for Symmetric EM Maps

Abstract

Summary Many large biological macromolecules have inherent structural symmetry, being composed of a few distinct subunits, repeated in a symmetric array. These complexes are often not amenable to traditional high-resolution structural determination methods, but can be imaged in functionally relevant states using cryo-electron microscopy (cryo-EM). A number of methods for fitting atomic-scale structures into cryo-EM maps have been developed, including the molecular dynamics flexible fitting (MDFF) method. However, quality and resolution of the cryo-EM map are the major determinants of a method’s success. In order to incorporate knowledge of structural symmetry into the fitting procedure, we developed the symmetry-restrained MDFF method. The new method adds to the cryo-EM map-derived potential further restraints on the allowed conformations of a complex during fitting, thereby improving the quality of the resultant structure. The benefit of using symmetry-based restraints during fitting, particularly for medium to low-resolution data, is demonstrated for three different systems.

Architecture of the Human Mitochondrial Iron-Sulfur Cluster Assembly Machinery

“…The dashed red line shows where the FSC curve crosses the correlation value of 0.143. atoms involved (Ͼ100,000). However, we were able to subject one-half of the structure, consisting of six NFS1 dimers docked on top of four [FXN ] 3 ⅐[ISCU] 3 sub-complexes, to mild simulation using Molecular Dynamics Flexible Fitting (47,48) as described under "Experimental Procedures." Prior to the simulation, residues 42-98 in the N-terminal region of FXN were repositioned guided by cross-linking data.…”

“…Prior to the simulation, residues 42-98 in the N-terminal region of FXN were repositioned guided by cross-linking data. In addition, the N-terminal flexible portions of ISCU (residues [35][36][37][38][39][40][41][42][43][44][45][46][47][48][49] and NFS1 (residues 55-66) were removed to avoid steric clashes during the simulation. The simulation improved the fitting of the structure into the EM density map, as the cross-correlation coefficient improved from 0.46 to 0.62.…”

Architecture of the Yeast Mitochondrial Iron-Sulfur Cluster Assembly Machinery

“…The EM map was then converted to Situs format to allow rigid-body docking of the entire complex structure, followed by molecular dynamics simulations and energy minimizations using nanoscale molecular dynamics, NAMD 2.10 (43). The Molecular Dynamics Flexible Fitting method used NAMD to perfect the complex structure while simultaneously improving its fit to the EM map (44,45). The CHARMM22 force field was used in vacuum with a dielectric constant of 80, a constant simulation temperature of 50 K, and Langevin dynamics coupled with a damping constant of 5 ps Ϫ1 .…”

“…The CHARMM22 force field was used in vacuum with a dielectric constant of 80, a constant simulation temperature of 50 K, and Langevin dynamics coupled with a damping constant of 5 ps Ϫ1 . Harmonic restraints for amine N, carbonyl C, and ␣-C atoms were applied to maintain the model 4, 3, 2 symmetry with a symmetry force constant of 20 for 100 ps (200 steps) (45). Harmonic restraints were also applied to maintain chirality and secondary structure.…”

“…It has been shown that EM density maps with resolution between 3.3 and 35 Å are suitable to attempt docking of protein structures (45,56,57). We therefore attempted docking of Yfh1 and Isu1 structures in the EM map of the refined three-dimensional model.…”