title: Cryo-EM of dynamic protein complexes in eukaryotic DNA replication.
abstract: DNA replication in Eukaryotes is a highly dynamic process that involves several dozens of proteins. Some of these proteins form stable complexes that are amenable to high-resolution structure determination by cryo-EM, thanks to the recent advent of the direct electron detector and powerful image analysis algorithm. But many of these proteins associate only transiently and flexibly, precluding traditional biochemical purification. We found that direct mixing of the component proteins followed by 2D and 3D image sorting can capture some very weakly interacting complexes. Even at 2D average level and at low resolution, EM images of these flexible complexes can provide important biological insights. It is often necessary to positively identify the feature-of-interest in a low resolution EM structure. We found that systematically fusing or inserting maltose binding protein (MBP) to selected proteins is highly effective in these situations. In this chapter, we describe the EM studies of several protein complexes involved in the eukaryotic DNA replication over the past decade or so. We suggest that some of the approaches used in these studies may be applicable to structural analysis of other biological systems.
authors: Cryo-EM Structural Biology Laboratory, Van Andel Research Institute, Grand Rapids, Michigan, 49503.
authors: Jingchuan
authors: J
authors: Sun
authors: Cryo-EM Structural Biology Laboratory, Van Andel Research Institute, Grand Rapids, Michigan, 49503.|The Biochemistry and Structural Biology Program, Stony Brook University, Stony Brook, New York, 11794.
authors: Zuanning
authors: Z
authors: Yuan
authors: Cryo-EM Structural Biology Laboratory, Van Andel Research Institute, Grand Rapids, Michigan, 49503.
authors: Lin
authors: L
authors: Bai
authors: Cryo-EM Structural Biology Laboratory, Van Andel Research Institute, Grand Rapids, Michigan, 49503.
authors: Huilin
authors: H
authors: Li
mesh_terms: D000465:Algorithms; D000818:Animals; D020285:Cryoelectron Microscopy / Q000379:methods*; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism / Q000648:ultrastructure*; D005057:Eukaryotic Cells / Q000502:physiology / Q000648:ultrastructure*; D006801:Humans; D021621:Imaging, Three-Dimensional / Q000379:methods*; D009097:Multienzyme Complexes / Q000378:metabolism / Q000648:ultrastructure*; D000099272:Replisomes
chemical_list: D004259:DNA-Directed DNA Polymerase; D009097:Multienzyme Complexes; D000099272:Replisomes
keywords: Cryo-EM; DNA replication; eukaryotic DNA replication; origin recognition complex; replicative helicase