Structure Determination of Small Macromolecular Complexes by Cryo-Electron Microscopy

Application of emerging methods in single particle cryo-electron microscopy (cryo-EM) has led to the determination of the structures of a variety of macromolecular complexes at sub-nanometer resolutions. The overwhelming majority of these are large complexes, mostly with high symmetry. Extending cryo-EM methods to routinely determine the structures of small macromolecular complexes with sizes of < 500 kDa to sub-nanometer resolution still remains an exciting frontier in modern structural biology. Working with small protein complexes has certain inherent challenges such as conformational flexibility and beam induced movement during exposure to electrons which can significantly reduce resolution. Further, during image processing, errors can be introduced from model-bias, over-refinement, and inaccuracy in orientation determination. Although these errors are problematic for all reconstructions, they are particularly detrimental for structural analysis of complexes that are small and have either low or no internal symmetry. We show a step-wise systematic workflow for structure determination by cryo-EM at ∼ 6 A resolution of s-galactosidase, a 464 kDa protein, whose structure has previously been determined at atomic resolution by X-ray crystallography. Our approach employs a number of recent developments in cryo-EM technology that include the use of direct electron detectors, the ability to fractionate the dose to use only those portions of the exposure that are recorded before there is significant radiation damage, and the use of image processing procedures that can track and correct for the movement of single molecules during the course of the exposure.