A viral genome packaging motor transitions between cyclic and helical symmetry to translocate dsDNA

Molecular segregation and biopolymer manipulation require the action of molecular motors to do work by applying directional forces to macromolecules. The additional strand conserved E (ASCE) ring motors are an ancient family of molecular motors responsible for diverse biological polymer manipulation tasks. Viruses use ASCE segregation motors to package their genomes into their protein capsids and provide accessible experimental systems due to their relative simplicity. We show by cryo-EM–focused image reconstruction that ASCE ATPases in viral double-stranded DNA (dsDNA) packaging motors adopt helical symmetry complementary to their dsDNA substrates. Together with previous data, our results suggest that these motors cycle between helical and planar configurations, providing a possible mechanism for directional translocation of DNA. Similar changes in quaternary structure have been observed for proteasome and helicase motors, suggesting an ancient and common mechanism of force generation that has been adapted for specific tasks over the course of evolution.

Dynamic and asymmetric fluctuations in the microtubule wall captured by high-resolution cryoelectron microscopy

Microtubules are tubular polymers with essential roles in numerous cellular activities. Structures of microtubules have been captured at increasing resolution by cryo-EM. However, dynamic properties of the microtubule are key to its function, and this behavior has proved difficult to characterize at a structural level due to limitations in existing structure determination methods. We developed a high-resolution cryo-EM refinement method that divides an imaged microtubule into its constituent protofilaments, enabling deviations from helicity and other sources of heterogeneity to be quantified and corrected for at the single-subunit level. We demonstrate that this method improves the resolution of microtubule 3D reconstructions and substantially reduces anisotropic blurring artifacts, compared with methods that utilize helical symmetry averaging. Moreover, we identified an unexpected, discrete behavior of the m-loop, which mediates lateral interactions between neighboring protofilaments and acts as a flexible hinge between them. The hinge angle adopts preferred values corresponding to distinct conformations of the m-loop that are incompatible with helical symmetry. These hinge angles fluctuate in a stochastic manner, and perfectly cylindrical microtubule conformations are thus energetically and entropically penalized. The hinge angle can diverge further from helical symmetry at the microtubule seam, generating a subpopulation of highly distorted microtubules. However, the seam-distorted subpopulation disappears in the presence of Taxol, a microtubule stabilizing agent. These observations provide clues into the structural origins of microtubule flexibility and dynamics and highlight the role of structural polymorphism in defining microtubule behavior.

A viral genome packaging motor transitions between cyclic and helical symmetry to translocate dsDNA

SUMMARYMolecular segregation and biopolymer manipulation require the action of molecular motors to do work by applying directional forces to macromolecules. The additional strand conserved E (ASCE) ring motors are an ancient family of molecular motors responsible for diverse tasks ranging from biological polymer manipulation (e.g. protein degradation and chromosome segregation) to establishing and maintaining proton gradients across mitochondrial membranes. Viruses also utilize ASCE segregation motors to package their genomes into their protein capsids and serve as accessible experimental systems due to their relative simplicity. We show by CryoEM focused image reconstruction that ASCE ATPases in viral dsDNA packaging motors adopt helical symmetry complementary to their dsDNA substrates. Together with previous data, including structural results showing these ATPases in planar ring conformations, our results suggest that these motors cycle between helical and planar cyclical symmetry, providing a possible mechanism for directional translocation of DNA. We further note that similar changes in quaternary structure have been observed for proteasome and helicase motors, suggesting an ancient and common mechanism of force generation that has been adapted for specific tasks over the course of evolution.