Biology subjects

Hall, L. N.

Publications and source records attributed to Hall, L. N..

5 recordsLinked to original sources

bioRxiv2026

Optimized cryo-FIB milling strategy to generate thin, minimally damaged biological lamellae

Focused ion beam (FIB)-milling has been adapted to thin frozen cells for visualization of macromolecular structures in situ with cryogenic electron microscopy. However, only a few large and abundant complexes have been annotated to date. FIB-milling introduces damage which limits the recoverable information from cellular sections. Here, we present Nilas, a low-energy milling strategy optimized to minimize damage and produce thin lamellae. Nilas-milled lamellae show minimal FIB-milling damage, contain areas at or below 50 nm and produce higher resolution in situ 3D reconstructions. Nilas improves the recovery of ribosomal subunits and reduces the predicted minimal detectable molecular mass with two-dimensional template matching (2DTM) to 220 kDa. Consistently, we recover additional non-ribosomal complexes including RNA polymerase III with 2DTM in Nilas-milled lamellae. Nilas is compatible with common milling hardware, making it accessible to diverse users. By extending the size limit for in situ structural biology we bring visual proteomics closer to reality.

biophysics↗

bioRxiv2025

Leopard-EM: An extensible 2DTM package to accelerate in situ structural biology

The ability to generate high-resolution views of cells with cryogenic electron microscopy (cryo-EM) can reveal the molecular mechanisms of biological processes in their native cellular context. The revolutionary impact of this strategy is limited by the difficulty of accurately annotating structures within these images. 2D template matching (2DTM), in which high-resolution structural models are used as computational probes to locate and orient molecular complexes with high precision, has shown initial promise in annotating single molecules in cellular cryo-EM images. While the scientific community works to identify best practices for applying 2DTM to specific biological questions and to maximize sensitivity and throughput, a modular and extensible software architecture would support the rapid development of novel methodological approaches, thus accelerating innovation within the field. To achieve this, we developed Leopard-EM (Location & oriEntatiOn of PARticles found using two-Dimensional tEmplate Matching), a modular Python-based 2DTM implementation built to be easily customizable. We implemented an automated pixel size refinement procedure and find that 2DTM is sensitive to pixel size to within 0.001[A]. To demon-strate the flexibility of the Leopard-EM architecture, we developed a constrained search protocol that improved small ribosomal subunit (SSU) detection by approximately eightfold by using initial locations and orientations determined for the large ribosomal subunit (LSU). Using this strategy, we captured a distribution of ribosome rotation states within a living cell at single-molecule resolution. We envision that Leopard-EM can be used as a platform for development of in situ cryo-EM data processing workflows, facilitating the rapid development of this field. Leopard-EM is available at https://github.com/Lucaslab-Berkeley/Leopard-EM.

biophysics↗

bioRxiv2025

Structures reveal how the Cas1-2/3 integrase captures, delivers, and integrates foreign DNA into CRISPR loci

Cas1 and Cas2 are the hallmark proteins of prokaryotic adaptive immunity. However, these two proteins are often fused to other proteins and the functional association of these fusions often remain poorly understood. Here we purify Cas1 and the Cas2/3 fusion protein from Pseudomonas aeruginosa. We determine multiple structures of the Cas1-2/3 complex at distinct stages of CRISPR adaptation. Collectively, these structures reveal a prominent, positively charged channel on one face of the integration complex that captures short fragments of foreign DNA. Foreign DNA binding triggers conformational changes in Cas2/3 that expose new DNA binding surfaces necessary for homing the DNA-bound integrase to specific CRISPR loci. The length of the foreign DNA substrate determines if Cas1-2/3 docks completely onto the CRISPR repeat to successfully catalyze two sequential transesterification reactions required for integration. Taken together, these structures clarify how the Cas1-2/3 proteins orchestrate foreign DNA capture, site-specific delivery, and integration of new DNA into the bacterial genome. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/658980v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@11ad39borg.highwire.dtl.DTLVardef@3a8474org.highwire.dtl.DTLVardef@1cdf25borg.highwire.dtl.DTLVardef@1e24ff5_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTS- A positively charged channel on the Cas1-2/3 complex captures fragments of DNA - A loop in the RecA1 domain controls access to the Cas3 nuclease active site - Foreign DNA binding allosterically regulates access to additional DNA binding sites - Distortion of the CRISPR repeat sequence licenses complete foreign DNA integration

molecular biology↗

bioRxiv2023

Colloidal aggregation confounds cell-based Covid-19 antiviral screens

Colloidal aggregation is one of the largest contributors to false-positives in early drug discovery and chemical biology. Much work has focused on its impact on pure-protein screens; here we consider aggregations role in cell-based infectivity assays in Covid-19 drug repurposing. We began by investigating the potential aggregation of 41 drug candidates reported as SARs-CoV-2 entry inhibitors. Of these, 17 formed colloidal-particles by dynamic light scattering and exhibited detergent-dependent enzyme inhibition. To evaluate antiviral efficacy of the drugs in cells we used spike pseudotyped lentivirus and pre-saturation of the colloids with BSA. The antiviral potency of the aggregators was diminished by at least 10-fold and often entirely eliminated in the presence of BSA, suggesting antiviral activity can be attributed to the non-specific nature of the colloids. In confocal microscopy, the aggregates induced fluorescent puncta of labeled spike protein, consistent with sequestration of the protein on the colloidal particles. Addition of either non-ionic detergent or of BSA disrupted these puncta. These observations suggest that colloidal aggregation is common among cell-based anti-viral drug repurposing, and perhaps cell-based assays more broadly, and offers rapid counter-screens to detect and eliminate these artifacts, allowing the community invest resources in compounds with true potential as a Covid-19 therapeutic.

biochemistry↗

bioRxiv2023

Mapping the biochemical landscape of rubisco

Rubisco is the primary CO2 fixing enzyme of the biosphere yet has slow kinetics. The roles of evolution and chemical mechanism in constraining the sequence landscape of rubisco remain debated. In order to map sequence to function, we developed a massively parallel assay for rubisco using an engineered E. coli where enzyme function is coupled to growth. By assaying >99% of single amino acid mutants across CO2 concentrations, we inferred enzyme velocity and CO2 affinity for thousands of substitutions. We identified many highly conserved positions that tolerate mutation and rare mutations that improve CO2 affinity. These data suggest that non-trivial kinetic improvements are readily accessible and provide a comprehensive sequence-to-function mapping for enzyme engineering efforts.

biochemistry↗