Biology subjects

Bulkley, D.

Publications and source records attributed to Bulkley, D..

5 recordsLinked to original sources

bioRxiv2026

A tripartite mechanism licenses RNA polymerase import into the phage nucleus

Nucleus-forming jumbo phages import a phage-encoded non-virion RNA polymerase (nvRNAP) into a proteinaceous nucleus, but how it is selected for import is unknown. Here, we identify an import pathway that couples nvRNAP subunit interactions with phage-encoded import factors. Maximal nvRNAP import requires a novel factor Imp7 (gp166) and the essential import factor Imp1. Imp7 is essential at environmental temperatures (20 C), where impaired nvRNAP import causes a profound loss of middle and late transcription and blocks phage DNA replication. Import of individual nvRNAP subunits also depends on other subunits, consistent with import licensing at the level of an assembled complex. In the absence of middle transcription and DNA replication, the phage nucleus surprisingly still assembles and segregates from the cytoplasm, demonstrating that nvRNAP nuclear localization and activity are not a critical checkpoint for nucleus assembly. Together, these findings define a specialized pathway for nuclear import of the multi-subunit RNAP and reveal that nuclear compartment assembly precedes the transcriptional and replicative programs required for its maturation.

microbiology↗

bioRxiv2024

High-resolution single particle imaging at 100-200 keV with the Gatan Alpine direct electron detector.

Developments in direct electron detector technology have played a pivotal role in enabling high-resolution structural studies by cryo-EM at 200 and 300 keV. Yet, theory and recent experiments indicate advantages to imaging at 100 keV, energies for which the current detectors have not been optimized. In this study, we evaluated the Gatan Alpine detector, designed for operation at 100 and 200 keV. Compared to the Gatan K3, Alpine demonstrated a significant DQE improvement at these voltages, specifically a ~4-fold improvement at Nyquist at 100 keV. In single-particle cryo-EM experiments, Alpine datasets yielded better than 2 [A] resolution reconstructions of apoferritin at 120 and 200 keV on a ThermoFisher Scientific (TFS) Glacios microscope. We also achieved a ~3.2 [A] resolution reconstruction for a 115 kDa asymmetric protein complex, proving its effectiveness with complex biological samples. In-depth analysis revealed that Alpine reconstructions are comparable to K3 reconstructions at 200 keV, and remarkably, reconstruction from Alpine at 120 keV on a TFS Glacios surpassed all but the 300 keV data from a TFS Titan Krios with GIF/K3. Additionally, we show Alpines capability for high-resolution data acquisition and screening on lower-end systems by obtaining ~3 [A] resolution reconstructions of apoferritin and aldolase at 100 keV and detailed 2D averages of a 55 kDa sample using a side-entry cryo holder. Overall, we show that Gatan Alpine performs well with the standard 200 keV imaging systems and may potentially capture the benefits of lower accelerating voltages, possibly bringing smaller sized particles within the scope of cryo-EM.

biophysics↗

bioRxiv2020

Bivalent binding of a fully human IgG to the SARS-CoV-2 spike proteins reveals mechanisms of potent neutralization

In vitro antibody selection against pathogens from naive combinatorial libraries can yield various classes of antigen-specific binders that are distinct from those evolved from natural infection1-4. Also, rapid neutralizing antibody discovery can be made possible by a strategy that selects for those interfering with pathogen and host interaction5. Here we report the discovery of antibodies that neutralize SARS-CoV-2, the virus responsible for the COVID-19 pandemic, from a highly diverse naive human Fab library. Lead antibody 5A6 blocks the receptor binding domain (RBD) of the viral spike from binding to the host receptor angiotensin converting enzyme 2 (ACE2), neutralizes SARS-CoV-2 infection of Vero E6 cells, and reduces viral replication in reconstituted human nasal and bronchial epithelium models. 5A6 has a high occupancy on the viral surface and exerts its neutralization activity via a bivalent binding mode to the tip of two neighbouring RBDs at the ACE2 interaction interface, one in the "up" and the other in the "down" position, explaining its superior neutralization capacity. Furthermore, 5A6 is insensitive to several spike mutations identified in clinical isolates, including the D614G mutant that has become dominant worldwide. Our results suggest that 5A6 could be an effective prophylactic and therapeutic treatment of COVID-19.

microbiology↗

bioRxiv2019

Hedgehog pathway activation through conformational blockade of the Patched sterol conduit

Activation of the Hedgehog pathway may have therapeutic value for improved bone healing, taste receptor cell regeneration, and alleviation of colitis or other conditions. Systemic pathway activation, however, may be detrimental and therapeutic application has been difficult for lack of agents amenable to tissue targeting. We have developed a novel agonist, a conformation-specific nanobody against the Hedgehog receptor Patched1. This nanobody potently activates the Hedgehog pathway in vitro and in vivo by stabilizing an alternative conformation of a Patched1 \"switch helix\", as revealed by cryo-EM structure determination. Although this conformation likely constitutes part of the transport cycle, nanobody-trapping disrupts the cycle and prevents substrate movement through the Patched1 sterol conduit. Our conformation-selective nanobody approach provides a new route to the development of transporter-related pharmacologic agents and may be generally applicable to the study of other transporters.

biophysics↗

bioRxiv2018

A simple and robust procedure for preparing graphene-oxide cryo-EM grids

Graphene oxide (GO) sheets have been used successfully as a supporting substrate film in several recent cryogenic electron-microscopy (cryo-EM) studies of challenging biological macromolecules. However, difficulties in preparing GO-covered holey carbon EM grids have limited its widespread use. Here, we report a simple and robust method for covering holey carbon EM grids with GO sheets and demonstrate that these grids are suitable for high-resolution single particle cryo-EM. GO substrates adhere macromolecules, allowing cryo-EM grid preparation with lower specimen concentrations and providing partial protection from the air-water interface. Additionally, the signal from images of the GO lattice beneath the frozen-hydrated specimen can be discerned in many motion-corrected micrographs, providing a high-resolution fiducial for evaluating beam-induced motion correction.

biophysics↗