Biology subjects

Howes, A. C.

Publications and source records attributed to Howes, A. C..

2 recordsLinked to original sources

bioRxiv2026

CryoSHIELDs: tunable amphipathic peptide additives for cryo-EM

While cryogenic electron microscopy (cryo-EM) has transformed structural biology, sample preparation remains a major determinant of success. Specifically, the air-water interface (AWI) can damage complexes, impose preferred orientation, and reduce particle density. Here, we introduce cryoSHIELDs, a chemically diverse library of peptide additives designed to passivate the AWI and improve cryo-EM grid preparation. Across 24 protein systems spanning GPCRs, ABC and SLC transporters, soluble and Fab-containing complexes, and enzymes, cryoSHIELDs increased the success rate of structure determination. Individual cryoSHIELDs improved particle density, angular sampling, complex stability, and, for routine targets, map quality and resolution. This toolkit enabled the structural characterization of important targets, including the ABCC6 transporter, revealing unique TMD0-mediated stabilization, and DAMGO-bound -opioid receptor-Gi complex using substantially lower protein concentration. CryoSHIELDs provide a simple, tunable, and broadly applicable platform for more reliable cryo-EM in discovery biology and structure-based drug development.

biophysics↗

bioRxiv2025

Architecture and Function of Holocentric CENP-A-Independent Kinetochores

Kinetochores are essential macromolecular complexes that anchor chromosomes to the mitotic spindle to ensure faithful cell division1. Despite their critical role, the structural organization of kinetochores assembled on centromeres with vastly distinct architectures across diverse species remains poorly understood2,3. To address this question, we determined the cryo-EM structures of the inner kinetochore (CCAN) from the silkmoth Bombyx mori, an insect that lacks the canonical centromere-specifying histone variant CENP-A and exhibits chromosome-wide centromeric activity (holocentric). Our analysis reveals that B. mori CCAN assembles via atypical histone-fold protein dimerization into a self-contained, head-to-head dimer that topologically entraps and loops DNA, creating a point-centromere-like architecture. This structure also incorporates four previously uncharacterized Centromeric Subunit proteins that are evolutionarily repurposed from the outer kinetochore Dam1/DASH complex. Our work establishes this self-contained CCAN dimer as a key structural unit that forms the basis of a holocentric organization and suggests that large-scale centromere architectures can emerge from the modular arrangement of such discrete kinetochore units.

cell biology↗