Single particle cryo-electron microscopy (cryoEM) is often performed under the assumption that particles are not adsorbed to the air-water interfaces and in thin, vitreous ice. In this study, we performed fiducial-less tomography on over 50 different cryoEM grid/sample preparations to determine the particle distribution within the ice and the overall geometry of the ice in grid holes. Surprisingly, by studying particles in holes in 3D from over 1000 tomograms, we have determined that the vast majority of particles (approximately 90%) are adsorbed to an air-water interface. The implications of this observation are wide-ranging, with potential ramifications regarding protein denaturation, conformational change, and preferred orientation. We also show that fiducial-less cryo-electron tomography on single particle grids may be used to determine ice thickness, optimal single particle collection areas and strategies, particle heterogeneity, and de novo models for template picking and single particle alignment.

Single particle cryo-electron microscopy (cryoEM) is often performed under the assumption that particles are freely floating away from the air-water interfaces and in thin, vitreous ice. In this study, we performed fiducial-less tomography on over 50 different cryoEM grid/sample preparations to determine the particle distribution within the ice and the overall geometry of the ice in grid holes. Surprisingly, by studying particles in holes in 3D from over 1,000 tomograms, we have determined that the vast majority of particles (approximately 90%) are adsorbed to an air-water interface. The implications of this observation are wide-ranging, with potential ramifications regarding protein denaturation, conformational change, and preferred orientation. We also show that fiducial-less cryo-electron tomography on single particle grids may be used to determine ice thickness, optimal single particle collection areas and strategies, particle heterogeneity, and de novo models for template picking and single particle alignment.Contributions:

Assembly of bacterial ring-shaped hexameric replicative helicases on single-stranded (ss) DNA requires specialized loading factors. However, mechanisms implemented by these factors during opening and closing of the helicase, which enable and restrict access to an internal chamber, are not known. Here, we investigate these mechanisms in the Escherichia coli DnaB helicase•bacteriophage λ helicase loader (λP) complex. We show that five copies of λP bind at DnaB subunit interfaces and reconfigure the helicase into an open spiral conformation that is intermediate to previously observed closed ring and closed spiral forms; reconfiguration also produces openings large enough to admit ssDNA into the inner chamber. The helicase is also observed in a restrained inactive configuration that poises it to close on activating signal, and transition to the translocation state. Our findings provide insights into helicase opening, delivery to the origin and ssDNA entry, and closing in preparation for translocation.

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Replicative helicases are closed protein rings and require loader proteins for assembly on DNA at origins of DNA replication. Multiple copies of the bacterial DnaC or the phage lambda P loader proteins bind to the closed planar ring of the DnaB replicative helicase and trigger its reconfiguration into an open spiral conformation wherein an internal chamber becomes accessible for entry to physiologically produced single stranded DNA at the replication origin. Although a great deal was learned from a previously determined cryo-EM structure of the E. coli DnaB helicase bound to the phage helicase loader lambda P (BP), the pentameric lambda P ensemble was not well resolved and this stymied deeper insights. We have revised the BP structure using X-ray and AlphaFold determined structures to interpret a 2.8 angstrom cryo-EM density map. We find that the lambda P ensemble adopts a profoundly asymmetric configuration; one copy of lambda P, which is visualized in full, binds at the top and bottom of the open DnaB spiral; presence of a single breach in the DnaB open spiral means that the remaining four copies of lambda P must adopt distinct, and currently unknown, conformations. Although DnaBs internal chamber remains accessible to entry of ssDNA, the lambda P protomer whose binding site spans the breach effectively blocks the path into the inner chamber and gives rise to an autoinhibited configuration for the BP complex. Comparisons of the lambda P and DnaC bound complexes of the DnaB helicase shed new light on how the two loaders, though unrelated in sequence or structure, converged on the same ring-opening mechanism. The autoinhibited conformation of the BP complex suggests structural changes that must accompany recruitment to the initiator protein complex at the replication origin.

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