Biology subjects

Budell, W. C.

Publications and source records attributed to Budell, W. C..

4 recordsLinked to original sources

bioRxiv2026

The Signaling Pathway of a Bacterial Two-Component System Determined by cryo-EM

Although two-component systems (TCSs) play ubiquitous roles in sensing signals for bacterial survival and virulence, a detailed structural map of the signal transduction pathway is lacking. Bacteriophytochromes (BphPs) are enzymatic photoreceptors with homologs in plants that regulate bacterial physiological responses via TCS signaling. Here, we employ cryo-EM to elucidate the structural basis of a light-regulated TCS by capturing the full-length BphP histidine kinase (HK) in complex with its downstream response regulator (RR). The RR binds as a dimer to the HK but contacts the enzyme only through one subunit. Light absorption perturbs the entire BphP structure, including the HK domain, which disrupts phosphorylation and phosphotransfer reactions and promote TCS complex dissociation. Thus, light controls signaling through both HK activity and reversible binding of the RR.

biophysics↗

bioRxiv2024

Early intermediates in bacterial RNA polymerase promoter melting visualized by time-resolved cryo-electron microscopy

During formation of the transcription-competent open complex (RPo) by bacterial RNA polymerases (RNAP), transient intermediates pile up before overcoming a rate-limiting step. Structural descriptions of these interconversions in real time are unavailable. To address this gap, time-resolved cryo-electron microscopy (cryo-EM) was used to capture four intermediates populated 120 or 500 milliseconds (ms) after mixing Escherichia coli {sigma}70-RNAP and the PR promoter. Cryo-EM snapshots revealed the upstream edge of the transcription bubble unpairs rapidly, followed by stepwise insertion of two conserved nontemplate strand (nt-strand) bases into RNAP pockets. As nt-strand "read-out" extends, the RNAP clamp closes, expelling an inhibitory {sigma}70 domain from the active-site cleft. The template strand is fully unpaired by 120 ms but remains dynamic, indicating yet unknown conformational changes load it in subsequent steps. Because these events likely describe DNA opening at many bacterial promoters, this study provides needed insights into how DNA sequence regulates steps of RPo formation.

biophysics↗

bioRxiv2024

CryoCycle your grids: Plunge vitrifying and reusing clipped grids to advance cryoEM democratization

CryoEM democratization is hampered by access to costly plunge-freezing supplies. We introduce methods, called CryoCycle, for reliably blotting, vitrifying, and reusing clipped cryoEM grids. We demonstrate that vitreous ice may be produced by plunging clipped grids with purified proteins into liquid ethane and that clipped grids may be reused several times for different protein samples. Furthermore, we demonstrate the vitrification of thin areas of cells prepared on gold-coated, pre-clipped grids.

molecular biology↗

bioRxiv2020

Time-resolved cryoEM using Spotiton

We present an approach for preparing cryoEM grids to study short-lived molecular states. Using piezo electric dispensing, two independent streams of ~50 pL sample drops are deposited within 10 ms of each other onto a nanowire EM grid surface, and the mixing reaction stops when the grid is vitrified in liquid ethane, on the order of ~100 ms later. We demonstrate the utility of this approach for four biological systems where short-lived states are of high interest.

biophysics↗