(A) Heterogeneous refinement (K = 6, Cryosparc) of all the particles used to calculate the 4.1 Å map was performed to determine if other BP stoichiometric or conformational states were present in our EM sample. This calculation produced a single class (colored in pale green and indicated with a red square) that could be attributed to the BP complex. The structure in this class corresponds to the DnaB6-λP5.entity; no other states were found. The volumes in Classes 1, 2 and 6 appear to be an open, planar form of DnaB. Classes 4 and 5 appear to be a distorted, open, non-planar DnaB, with no λP present. The percentage of particles in each class are indicated below the volumes. (B) Homogenous refinement (K = 6, Cryosparc) of all the particles used to calculate the 4.1 Å map. As with the heterogeneous refinement, only a single class with a volume corresponding to the BP complex was obtained (Class 3, shown in bright orange and indicated with a red square). This volume represents density corresponding to the DnaB6-λP5 species. The volumes produced by particles in Classes 4 and 5 show a DnaB architecture reminiscent of the BP complex, but they lacked density for λP. Classes 1, 2 and 6 yielded volumes that feature a distorted DnaB. (C) 2D classification (Cryosparc) of the particles used to calculate the 4.1 Å map. A small percentage (<1.4%) of these particles appeared to correspond to uncomplexed DnaB. Removal of these particles from our data set, followed by another round of refinement (Cryosparc), did not appreciably improve our EM map. The gold standard FSC curve (right) shows half-sets that converge at 4.1 Å. (D) Heterogenous refinement (using a single ab initio starting model) of all template-picked BP particles (~267,000) in Cryosparc yielded two classes of interest, both of which corresponded to DnaB6-λP5. One class, comprised of 29,734 particles, produced a map with a resolution of 8.02 Å, and a second class, with 84,647 particles, produced a map with a resolution of 7.25 Å. Although of lower resolution, these maps display comparable features as our 4.1 Å EM map.