Biology subjects

Stagg, S.

Publications and source records attributed to Stagg, S..

4 recordsLinked to original sources

bioRxiv2025

CryoSift - An accessible and automated CNN-driven tool for cryo-EM 2D class selection

Single-particle cryo-electron microscopy (cryo-EM) has become an essential tool in structural biology. However, automating repetitive tasks remains an ongoing challenge in cryo-EM dataset processing. Here, we present a platform-independent convolutional neural network (CNN) tool for assessing the quality of 2D averages to enable automatic selection of suitable particles for high-resolution reconstructions, termed CryoSift. We integrate CryoSift into a fully automated processing pipeline using the existing cryosparc-tools library. Our integrated and customizable 2D assessment workflow enables high-throughput processing that accommodates experienced to novice cryo-EM users. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=183 SRC="FIGDIR/small/667259v2_ufig1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@1fdac98org.highwire.dtl.DTLVardef@14e3c7corg.highwire.dtl.DTLVardef@16520bforg.highwire.dtl.DTLVardef@48fa0f_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2024

Multiple roles for TFG ring complexes in neuronal cargo trafficking

Pathological variants in Trk-fused gene (TFG) have been implicated in a variety of neurodegenerative conditions. In particular, mutations within its amino-terminal PB1 domain have been suggested to cause hereditary spastic paraplegia (HSP), resulting in progressive lower limb spasticity and weakness. The structural basis for this effect is unknown. Here, we combine X-ray crystallography and cryo-electron microscopy to determine a structural model of TFG, demonstrating the mechanism by which it forms octameric ring complexes. A network of electrostatic and hydrophobic interactions defines the interface between protomers. Moreover, we show that mutations identified previously in HSP patients disrupt this interface, destabilizing octamers, which ultimately leads to axonopathy. Surprisingly, the impacts of these variants are not equivalent in vivo, highlighting the existence of multiple, distinct mechanisms by which TFG mutations contribute to neurodegenerative disease.

biochemistry↗

bioRxiv2024

The Structure of the Drp1 Lattice on Membrane

Mitochondrial health relies on the membrane fission mediated by dynamin-related protein 1 (Drp1). Previous structural studies of Drp1 on remodeled membranes were hampered by heterogeneity, leaving a critical gap in the understanding of the mitochondrial fission mechanisms. Here we present a cryo-electron microscopy structure of full-length human Drp1 decorated on membrane tubules. Using the reconstruction of average subtracted tubular regions (RASTR) technique, we report that Drp1 forms a locally ordered lattice along the tubule without global helical symmetry. The filaments in the lattice are similar to dynamin rungs with conserved stalk interactions. Adjacent filaments are connected by GTPase domain interactions in a novel stacked conformation. We identified two states of the Drp1 lattice among the heterogenous dataset representing conformational changes around hinge 1. Additionally, we observed contact between Drp1 and membrane that can be assigned to the variable domain sequence. Together these structures revealed a putative mechanism by which Drp1 constricts mitochondria membranes in a stepwise, "ratchet" manner. SUMMARYThis study provides new insights into the structure of Drp1 on lipid membranes. A locally ordered Drp1 lattice structure is solved and reveals intermolecular contacts and conformational rearrangements that suggest a mechanism for constriction of mitochondrial membranes.

biochemistry↗

bioRxiv2024

SPOT-RASTR - a cryo-EM specimen preparation technique that overcomes problems with preferred orientation and the air/water interface

In cryogenic electron microscopy (cryo-EM), specimen preparation remains a bottleneck despite recent advancements. Classical plunge freezing methods often result in issues like aggregation and preferred orientations at the air/water interface. Many alternative methods have been proposed, but there remains a lack a universal solution, and multiple techniques are often required for challenging samples. Here, we demonstrate the use of lipid nanotubes with nickel NTA headgroups as a platform for cryo-EM sample preparation. His-tagged specimens of interest are added to the tubules, and they can be frozen by conventional plunge freezing. We show that the nanotubes protect samples from the air/water interface and promote a wider range of orientations. The reconstruction of average subtracted tubular regions (RASTR) method allows for the removal of the nanotubule signal from the cryo-EM images resulting in isolated images of specimens of interest. Testing with {beta}-galactosidase validates the methods ability to capture particles at lower concentrations, overcome preferred orientations, and achieve near-atomic resolution reconstructions. Since the nanotubules can be identified and targeted automatically at low magnification, the method enables fully automated data collection. Furthermore, the particles on the tubes can be automatically identified and centered using 2D classification enabling particle picking without requiring prior information. Altogether, our approach that we call specimen preparation on a tube RASTR (SPOT-RASTR) holds promise for overcoming air-water interface and preferred orientation challenges and offers the potential for fully automated cryo-EM data collection and structure determination.

molecular biology↗