Computational protocol: Cryo-EM structure of Mcm2-7 double hexamer on DNA suggests a lagging-strand DNA extrusion model

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Protocol publication

[…] We collected ∼3,000 raw movie micrographs. The movie frames were first aligned and superimposed by the program MotionCorr 2.0 (). Contrast transfer function parameters of each aligned micrograph were calculated using the program CTFFIND4 (). All the remaining steps, including particle auto selection, 2D classification, 3D classification, 3D refinement, and density map postprocessing, were performed using RELION 2.0 (). The template for automatic picking was generated from a 2D average of about ∼10,000 manually picked particles in different views. Automatic particle selection was performed for the entire dataset, and 312,403 particles were initially picked. We then carefully inspected the selected particles, removed “bad” ones that were broken or aggregated particles, repicked some initially missed “good” ones with expected size and shape, and sorted the remaining good particles by similarity to the 2D references; the bottom 10% of the particles with the lowest z-scores were removed from the particle pool. 2D classification of all good particles was performed and particles in the classes with features unrecognizable by visual inspection were removed. A total of 163,255 particles was used for further 3D classification. We derived five 3D models from the dataset and chose the two best models with strong DNA density for final refinement; the other three models were distorted or had weak DNA density, and those particles were discarded. The final dataset having 58,772 particles was used for further 3D refinement with application of the expected C2 symmetry, resulting in the 3.9-Å 3D density map. The resolution of the map was estimated by the gold-standard Fourier shell correlation at a correlation cutoff value of 0.143. The 3D density map was sharpened by applying a negative B-factor of −168 Å2. We also refined the 3D map without applying symmetry (C1) and obtained a map at 4.2-Å resolution. After atomic modeling and careful analysis of the two maps, we found the two hexamer structures and how they interact with the DNA are virtually the same. We therefore focused our analysis on the twofold symmetric 3.9-Å 3D map. [...] The cryo-EM structure of the yeast Mcm2–7 double hexamer [Protein Data Bank (PDB) ID code 3JA8] was used as the initial model directly docking into the EM map. Each Mcm protein was then split into two parts, an N-terminal domain and a C-terminal domain, for subsequent rigid body-fitting into the 3D density map with COOT and Chimera. The dsDNA was then manually built into the long density that ran through the two Mcm2-7 hexameric structures in the program COOT. The DNA sequence used in preparation of the double hexamer is an origin DNA (ARS1) that had been multimerized. However, when the DNA is cut by the restriction enzyme to release the multimers of the double hexamer on DNA, it is possible that the DNA is not registered the same way relative to the origin in the double hexamers. For this reason we did not try to register the DNA sequence. We modeled the dsDNA in a random sequence, and the twofold symmetry was not enforced either in the DNA sequence during modeling or in refinement. The entire Mcm2–7 double-hexamer DNA model was first refined by rigid body refinement of individual chains in the PHENIX program and subsequently was adjusted manually in COOT. Finally, the atomic model was validated using MolProbity. Structural figures were prepared in Chimera and PyMOL (https://pymol.org/2/).The final model was cross-validated using a method described previously (). Briefly, we randomly added 0.1 Å noise to the coordinates of the final model using the PDB tools in Phenix and then refined the noise-added against the first half map (Half1) that was produced from one half of the particle dataset during refinement by RELION. We performed one round of coordinate refinement, followed by a B-factor refinement. The refined model was then correlated with the 3D maps of the two half maps (Half1 and Half2) in Fourier space to produce two forward scatter (FSC) curves: FSCwork (the model versus the Half1 map) and FSCfree (the model versus the Half2 map), respectively. A third FSC curve was calculated between the refined model and the final 3.9-Å resolution density map produced from all particles. The general agreement of these curves was taken as an indication that the model was not overfitted.The map coordinates for the Mcm2-7 double hexamer on DNA were deposited as EMD-9400, PDB ID code 5BK4. […]

Pipeline specifications

Software tools MotionCorr, CTFFIND, RELION, MolProbity, PyMOL
Applications cryo-EM, Protein structure analysis
Diseases Fused Teeth
Chemicals Zinc