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The method presented here refines molecular conformations directly against projections of single particles measured by electron microscopy. By optimizing the orientation of the projection at the same time as the conformation, the method is well-suited to two-dimensional class averages from cryoelectron microscopy. Such direct use of two-dimensional images circumvents the need for a three-dimensional density map, which may be difficult to reconstruct from projections due to structural heterogeneity or preferred orientations of the sample on the grid. Our refinement protocol exploits Natural Move Monte Carlo to model a macromolecule as a small number of segments connected by flexible loops, on multiple scales. After tests on artificial data from lysozyme, we applied the method to the Methonococcus maripaludis chaperonin. We successfully refined its conformation from a closed-state initial model to an open-state final model using just one class-averaged projection. We also used Natural Moves to iteratively refine against heterogeneous projection images of Methonococcus maripaludis chaperonin in a mix of open and closed states. Our results suggest a general method for electron microscopy refinement specially suited to macromolecules with significant conformational flexibility. The algorithm is available in the program Methodologies for Optimization and Sampling In Computational Studies.

Original publication




Journal article


Proc Natl Acad Sci U S A

Publication Date





9845 - 9850


Methanococcus, Microscopy, Electron, Muramidase, Protein Conformation