Biology subjects

Mendez, J. H.

Publications and source records attributed to Mendez, J. H..

9 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↗

bioRxiv2023

Signal Transduction in an Enzymatic Photoreceptor Revealed by Cryo-Electron Microscopy

Phytochromes are essential photoreceptor proteins in plants with homologs in bacteria and fungi that regulate a variety of important environmental responses. They display a reversible photocycle between two distinct states, the red-light absorbing Pr and the far-red light absorbing Pfr, each with its own structure. The reversible Pr to Pfr photoconversion requires covalently bound bilin chromophore and regulates the activity of a C-terminal enzymatic domain, which is usually a histidine kinase (HK). In plants, phytochromes translocate to nucleus where the C-terminal effector domain interacts with protein interaction factors (PIFs) to induce gene expression. In bacteria, the HK phosphorylates a response-regulator (RR) protein triggering downstream gene expression through a two-component signaling pathway. Although plant and bacterial phytochromes share similar structural composition, they have contrasting activity in the presence of light with most BphPs being active in the dark. The molecular mechanism that explains bacterial and plant phytochrome signaling has not been well understood due to limited structures of full-length phytochromes with enzymatic domain resolved at or near atomic resolution in both Pr and Pfr states. Here, we report the first Cryo-EM structures of a wild-type bacterial phytochrome with a HK enzymatic domain, determined in both Pr and Pfr states, between 3.75 and 4.13 [A] resolution, respectively. Furthermore, we capture a distinct Pr/Pfr heterodimer of the same protein as potential signal transduction intermediate at 3.75 [A] resolution. Our three Cryo-EM structures of the distinct signaling states of BphPs are further reinforced by Cryo-EM structures of the truncated PCM of the same protein determined for the Pr/Pfr heterodimer as well as Pfr state. These structures provide insight into the different light-signaling mechanisms that could explain how bacteria and plants see the light.

molecular biology↗

bioRxiv2023

Smart Leginon enables cryoEM screening automation across multiple grids

SummaryCryoEM multi-grid screening is often a tedious process that demands hours of attention. Here, this protocol shows how to set up standard Leginon collection and Smart Leginon Autoscreen to automate this process. This protocol can be applied to the majority of cryoEM holey foil grids. Advancements in cryo-electron microscopy (cryoEM) techniques over the past decade have allowed structural biologists to routinely resolve macromolecular protein complexes to near-atomic resolution. The general workflow of the entire cryoEM pipeline involves iterating between sample preparation, cryoEM grid preparation, and sample/grid screening before moving on to high-resolution data collection. Iterating between sample/grid preparation and screening is typically a major bottleneck for researchers, as every iterative experiment must optimize for sample concentration, buffer conditions, grid material, grid hole size, ice thickness, and protein particle behavior in the ice, amongst other variables. Furthermore, once these variables are satisfactorily determined, grids prepared under identical conditions vary widely in whether they are ready for data collection, so additional screening sessions prior to selecting optimal grids for high-resolution data collection are recommended. This sample/grid preparation and screening process often consumes several dozen grids and days of operator time at the microscope. Furthermore, the screening process is limited to operator/microscope availability and microscope accessibility. Here, we demonstrate how to use Leginon and Smart Leginon Autoscreen to automate the majority of cryoEM grid screening. Autoscreen combines machine learning, computer vision algorithms, and microscope-handling algorithms to remove the need for constant manual operator input. Autoscreen can autonomously load and image grids with multi-scale imaging using an automated specimen-exchange cassette system, resulting in unattended grid screening for an entire cassette. As a result, operator time for screening 12 grids may be reduced to [~]10 minutes with Autoscreen compared to [~]6 hours using previous methods which are hampered by their inability to account for high variability between grids. This protocol and video tutorial first introduces basic Leginon setup and functionality, then demonstrates Autoscreen functionality step-by-step from the creation of a template session to the end of a 12 grid automated screening session.

molecular biology↗

bioRxiv2022

Characterizing the Resolution and Throughput of the Apollo Direct Electron Detector

Advances in electron detection have been essential to the success of high-resolution cryo-EM structure determination. A new generation of direct electron detector called the Apollo, has been developed by Direct Electron. The Apollo uses a novel event-based MAPS detector custom designed for ultra-fast electron counting. We have evaluated this new camera, finding that it delivers high detective quantum efficiency (DQE) and low coincidence loss, enabling high-quality electron counting data acquisition at up to nearly 80 input electrons per pixel per second. We further characterized the performance of Apollo for single particle cryo-EM on real biological samples. Using mouse apoferritin, Apollo yielded better than 1.9 [A] resolution reconstructions at all three tested dose rates from a half-day data collection session each. With longer collection time and improved specimen preparation, mouse apoferritin was reconstructed to 1.66 [A] resolution. Applied to a more challenging small protein aldolase, we obtained a 2.24 [A] resolution reconstruction. The high quality of the map indicates that the Apollo has sufficiently high DQE to reconstruct smaller proteins and complexes with high-fidelity. Our results demonstrate that the Apollo camera performs well across a broad range of dose rates and is capable of capturing high quality data that produce high-resolution reconstructions for large and small single particle samples.

biochemistry↗

bioRxiv2022

Fully Automated Multi-Grid Cryo-EM Screening using Smart Leginon

Single particle cryo-electron microscopy (cryoEM) is a swiftly growing method for understanding protein structure. With increasing demand for high-throughput, high-resolution cryoEM services comes greater demand for rapid and automated cryoEM grid and sample screening. During screening, optimal grids and sample conditions are identified for subsequent high-resolution data collection. Screening is a major bottleneck for new cryoEM projects because grids must be optimized over several factors, including grid type, grid hole size, sample concentration, buffer conditions, ice thickness, and particle behaviors. Even for mature projects, multiple grids are commonly screened to select a subset for high-resolution data collection. Here, machine learning and novel, purpose-built image processing and microscope-handling algorithms are incorporated into the automated data collection software, Leginon, to provide an open-source solution for fully automated, high-throughput grid screening. This new version, broadly called Smart Leginon, emulates the actions of an operator in identifying areas on the grid to explore as potentially useful for data collection. Smart Leginon Autoscreen sequentially loads and examines grids from an automated specimen exchange system to provide completely unattended grid screening across a set of grids. Comparisons between a multi-grid Autoscreen session and conventional manual screening by five expert microscope operators are presented. On average, Autoscreen reduces operator time from [~]6 hours to <10 minutes and provides a comparable percentage of suitable images for evaluation as the best operator. Smart Leginons ability to target holes that are particularly difficult to identify is analyzed. Finally, Smart Leginons utility is illustrated with three real-world multi-grid user screening/collection sessions, demonstrating the efficiency and flexibility of the software package. Smart Leginons fully automated functionality significantly reduces the burden on operator screening time, improves the throughput of screening, and recovers idle microscope time, thereby improving availability of cryoEM services.

biophysics↗

bioRxiv2022

Structural basis for substrate selection by the SARS-CoV-2 replicase

The SARS-CoV-2 RNA-dependent RNA polymerase coordinates viral RNA synthesis as part of an assembly known as the replication-transcription complex (RTC)1. Accordingly, the RTC is a target for clinically approved antiviral nucleoside analogs, including remdesivir2. Faithful synthesis of viral RNAs by the RTC requires recognition of the correct nucleotide triphosphate (NTP) for incorporation into the nascent RNA. To be effective inhibitors, antiviral nucleoside analogs must compete with the natural NTPs for incorporation. How the SARS-CoV-2 RTC discriminates between the natural NTPs, and how antiviral nucleoside analogs compete, has not been discerned in detail. Here, we use cryo-electron microscopy to visualize the RTC bound to each of the natural NTPs in states poised for incorporation. Furthermore, we investigate the RTC with the active metabolite of remdesivir, remdesivir triphosphate (RDV-TP), highlighting the structural basis for the selective incorporation of RDV-TP over its natural counterpart ATP3,4. Our results elucidate the suite of interactions required for NTP recognition, informing the rational design of antivirals. Our analysis also yields insights into nucleotide recognition by the nsp12 NiRAN, an enigmatic catalytic domain essential for viral propagation5. The NiRAN selectively binds GTP, strengthening proposals for the role of this domain in the formation of the 5 RNA cap6.

biophysics↗

bioRxiv2020

The structures of natively assembled clathrin coated vesicles

Vesicle trafficking by clathrin coated vesicles (CCVs) is one of the major mechanisms by which proteins and nutrients are absorbed by the cell and transported between organelles. The individual proteins comprising the coated vesicles include clathrin heavy chain, clathrin light chain, and a variety of adaptor protein complexes. Much is known about the structures of the individual CCV components, but data are lacking about the structures of the fully assembled complexes together with membrane and in complex with cargo. Here we determined the structures of natively assembled CCVs in a variety of geometries. We show that the adaptor {beta}2-appendages crosslink adjacent CHC {beta}-propellers and that the appendage densities reside almost exclusively in CCV hexagonal faces. We resolve how AP2 and other associated factors in hexagonal faces form an assembly hub with an extensive web of interactions between neighboring {beta}-propellers and propose a structural model that explains how adaptor binding can direct the formation of pentagonal and hexagonal faces.

biophysics↗

bioRxiv2019

Throughput and Resolution with a Next Generation Direct Electron Detector

Direct electron detectors (DEDs) have revolutionized cryo-electron microscopy by facilitating correction of beam-induced motion and radiation damage and by providing high-resolution image capture. A new generation of DEDs has been developed by Direct Electron, the DE64, that has good performance in both integrating and counting modes. Integrating mode is superior in throughput while counting mode is superior in image quality. We show that despite being ~10x slower in throughput, counting mode is superior in terms of reconstruction resolution per unit time of data collection.

biophysics↗