Biology subjects

Evans, J. E.

Publications and source records attributed to Evans, J. E..

At least 19 recordsLinked to original sources

bioRxiv2026

Five Rotational Registers Define Modular Portal-Capsid Assembly in Cyanophage P-SSP7

The virus P-SSP7 infects the cyanobacterium Prochlorococcus marinus, one of the most abundant photosynthetic microbes in the ocean, making this pairing a useful model for studying host-virus interactions. Infection proceeds through a portal-tail complex that attaches to the viral protein shell at a single specialized vertex, where the two structures have mismatched symmetries. Previous studies described this region only at coarse resolution which continues to limit understanding of how the virus assembles and injects its genome. Using cryo-electron microscopy of high-quality samples, we determined the first near-atomic-resolution structure of the complete P-SSP7 virion and its portal-tail complex. We captured 3D classes where the shell and portal-tail complex connect in five distinct arrangements. Fitting and modeling 2 of these states helped resolve and establish modular attachment in five rotational registers as the basis for capsid-portal symmetry mismatch in P-SSP7 assembly.

microbiology↗

bioRxiv2026

Coherent Structured Illumination Microscopy with Enhanced Optical Sectioning

Coherent structured illumination microscopy (c-SIM) is a synthetic aperture optical technique for sub-diffraction limit imaging that extends the utility of traditional SIM to non-fluorescent samples. Here, we present a complementary 5-beam implementation of c-SIM that provides enhanced optical sectioning compared to conventional quadrupolar illumination. Since our approach detects intensity images due to coherent light scattering, it avoids the complications associated with detecting complex fields. Through comparative measurements on calibration samples and live microalgae, we show that 5-beam c-SIM effectively suppresses coherent defocus effects, improving image quality while simultaneously providing a 2-fold lateral resolution improvement.

bioengineering↗

bioRxiv2026

Visual exoproteomics of Clostridium thermocellum during anaerobic biomass-degradation identifies functional spirosomes

Visual proteomics enables the study of low-abundance proteins and identification of unknown complexes from heterogeneous samples by complementing high-resolution cryogenic electron microscopy (cryoEM) with external inputs on protein identity such as mass spectrometry. Using this approach, we interrogated the exoproteome of the anaerobic cellulose-degrading bacterium Clostridium thermocellum as it carried out biomass degradation. Mass spectrometry indicated a broad exoproteome composition, including cellulose degrading machinery CelA and CipA. A focus on large exoproteome assemblies revealed abundant protein filaments and pleomorphic vesicular structures. Analysis of the most abundant protein filaments yielded an [~]4 {square} resolution native structure that, aided by mass spectrometry, de novo modeling, and structural searching, was found to be the aldehyde-alcohol dehydrogenase (AdhE) spirosome. AdhE contained both NAD+ and Fe in their expected binding sites and biochemical and structural analyses of enriched spirosome preparations indicated they were functional. Altered NADH solution concentrations triggered conformational changes in the exoproteomic spirosomes, and the constituent AdhE remained capable of ethanol production. Although the basis for functional extracellular spirosome accumulation in live anaerobic C. thermocellum cultures remains unclear, their abundance in crude exoproteomes suggests their presence could influence biomass fueled C. thermocellum growth.

biochemistry↗

bioRxiv2026

Spatiotemporal 4D Whole-cell Modeling of a Minimal Autotroph Reveals Central Carbon Metabolism Regulated Locally by Protein Megacomplexes via Post-translational Modifications under Light Disturbance

Photosynthetic microorganisms rely on multiple pathways in central carbon metabolism to adapt to fluctuating light and energy availability across diel cycles. Mechanistic insight into the regulatory dynamics of this adaptation requires integrating processes spanning disparate timescales, from rapid redox-dependent post-translational modifications (PTMs) to slower changes in protein expression and metabolic pathway usage. To address this complexity beyond genome-based inference and traditional modeling, we develop a whole-cell four-dimensional (3D + time) model of the marine cyanobacterium Prochlorococcus marinus MED4 that explicitly represents the spatial organization of enzymatic and molecular processes in central carbon metabolism under light perturbation. We employ a perturbation-based research design to experimentally generate time-series, multi-omics measurements that provide molecular descriptors and cryo-ET images as constraints for this dynamic 4D framework. The integration of experiments and modeling across defined light regimes enables quantitative validation of system-level responses and forecasting under distinct light disturbances. We test the hypothesis that light-dependent redox PTMs regulating the structural assembly of a protein megacomplex, the "dark complex," modulate metabolic flux at a conserved regulatory node of the Calvin-Benson cycle (CBC) in cyanobacteria. Our model shows that subcellular spatial organization buffers rapid light-induced changes in thylakoid reaction rates, which are followed by redox-PTM-mediated sequestration or release of CBC enzymes in the dark complex, ultimately impacting carbon fixation dynamics within carboxysomes. Comparison with an equivalently parameterized well-mixed stochastic model demonstrates that post-translational regulation not only buffers transcriptional noise and diffusion-driven fluctuations but also stabilizes phenotypic outcomes, underscoring the importance of spatial heterogeneity in phenotypic robustness. This ability to probe adaptive, spatiotemporally resolved mechanisms in photosynthetic machinery and central carbon metabolism addresses a critical gap in genotype-to-phenotype inference and expands modeling and design capabilities for understudied or genetically intractable autotrophs such as P. marinus MED4. Significance StatementThis work advances 4D whole-cell modeling by presenting the first spatiotemporal simulation of a photosynthetic autotroph using the Lattice Microbes platform. Using Prochlorococcus marinus MED4, we show that subcellular spatial organization of organelles, diffusion constraints, and redox regulation collectively shape central carbon metabolism across orders of magnitude in space and time. Through a perturbation-based strategy that generates multi-omics data sets over time, we construct and validate a spatially and temporally resolved model of MED4, constrained by high-resolution (10 nm) cryo-electron tomography. Our results highlight the importance of localized biochemical reactions and redox-dependent post-translational modification of enzymes in regulating carbon fixation in a noisy environment under light disturbance. This study establishes a spatiotemporal, whole-cell physiology modeling framework as a transformative tool for uncovering multiscale regulatory responses to environmental gradients.

systems biology↗

bioRxiv2026

Systematic Scale-Up and Enhanced Purification of Marine Cyanophage P-SSP7

Cyanophages represent important models for understanding virus-host interactions, yet high-resolution structural studies remain relatively few due to challenges with preparing enough sample of sufficient quality for cryo-EM and functional multi-omics studies. Here we developed an integrated methodology for scaling production of the model cyanophage P-SSP7 from laboratory maintenance volumes (5-100 mL) to production scales (up to 40 L) while dramatically improving the quality of phage preparation for structural applications. Our systematic approach integrates host cultivation using adaptation to local seawater to reduce production costs, optimized infection protocols to maximize infectious titer yields, and multi-stage purification workflows specifically designed for cryo-EM quality requirements. The final methodology consistently produces infectious phage titers exceeding 3x1012 units/mL with recoverable yields of 1013 total infectious units and >95% purity validated by cryo-electron microscopy at each optimization step. Most significantly, this approach achieves a 60-fold reduction in cryo-EM data collection time by increasing usable particles per field of view for single particle analysis. Overall, our final preparations demonstrate robust phage stability, retaining 68% infectivity after 3 months and 23% after 6 months at 4{degrees}C. This workflow moves cyanophage culturing and downstream structural studies from specialized, resource-intensive endeavors toward routine research capability and establishes an adaptable framework for scaling production that can be applied to other host-virus systems.

microbiology↗

bioRxiv2025

Cryo-EM protein structure without purification

Cryo-electron microscopy (cryo-EM) is a powerful technique for macromolecular structure determination. While several aspects of the workflow are either semi- or fully automated, the beginning of the workflow related to the generation of macromolecular samples remains a bottleneck limiting full automation. In most cases, the production of the initial sample still takes days to months. Initial sample production often takes days to months. Here, we show that cryo-EM can solve structures of proteins directly from cell-free expression complex mixtures with minimal or no purification while also reducing reagent volumes and overall labor and time. Using a cell-free reaction volume of just 25 microliters, we achieve rapid protein synthesis and structural characterization in under 24 hours at resolutions better than 3[A]. These results highlight a lower cost and rapid gene to structure workflow with minimal perturbation satisfies the foundational requirements needed to facilitate future improvements in throughput via full workflow automation.

biochemistry↗

bioRxiv2024

Synergy and antagonism in a genome-scale model of metabolic hijacking by bacteriophage

Bacteriophage auxiliary metabolic genes (AMGs) alter host metabolism by hijacking reactions. Previous studies used functional annotations to infer AMG impacts but neglected propagation effects on global metabolism and phage production. We demonstrate the first integration of AMGs and phage assembly into a genome-scale metabolic model, using a general method applied to the infection of Prochloroccocus marinus MED4 by P-HM2. We experimentally validate our approach to predicting AMG impact on growth using cp12 mutations in Syne-chococcus elongatus. We predict that 17 directly hijacked reactions substantially impact over 30% of the metabolism, including carbon fixation, photosynthesis, and nucleotide synthesis. We find that indirect impacts are synergistically and antagonistically coupled and are either phage-aligned--shifting feasible reaction velocities in accordance with maximal phage production--or phage-antialigned. Pareto optimization reveals that phage-aligned reactions limit host growth, while phage-antialigned reactions do not. We provide systems-level insight into AMG perturbations, highlighting how nontrivial cascading effects shape microbial functions.

systems biology↗

bioRxiv2024

Improving the visualization of viruses in soil

Viruses are numerically the most abundant forms on Earth, and most are present in soil. Even though viruses are highly abundant in soil and critical to rhizosphere function, visualizing the diverse morphotypes within soil has been challenging. The difficulty is primarily due to the heterogenous nature of isolated suspensions that typically contain nanometer to micron scale debris which renders protein crystallography for structural studies unfeasible and hinders cryo-electron microscopy due to ice thickness and contrast issues. Here we employed and compared a simple spin filtration method to cleanup solutions of extracted viruses for direct observation with cryo-electron microscopy. Although relatively simple, the method employs common physical biochemical separation steps to remove large and small debris which dramatically improves image quality and preservation of structural features to permit visualizing morphotypes not typically seen with conventional negative stain approaches. In addition to tailed and non-tailed polyhedral phages, several under reported or novel morphotypes of soil viruses are directly visualized as a particle library with both 2D and 3D information.

microbiology↗

bioRxiv2024

Illuminating microbial mat assembly: Cyanobacteria and Chloroflexota cooperate to structure light-responsive biofilms

Microbial mats are stratified communities often dominated by unicellular and filamentous phototrophs within an exopolymer matrix. It is challenging to quantify the dynamic responses of community members in situ as they experience steep gradients and rapid fluctuations of light. To address this, we developed a binary consortium using two representative isolates from hot spring mats: the unicellular oxygenic phototrophic cyanobacterium Synechococcus OS-B (Syn OS-B) and the filamentous anoxygenic phototroph Chloroflexus MS-CIW-1 (Chfl MS-1). We quantified the motility of individual cells and entire colonies and demonstrated that Chfl MS-1 formed bundles of filaments that moved in all directions with no directional bias to light. Syn OS- B was slightly less motile but exhibited positive phototaxis. This binary consortium displayed cooperative behavior by moving further than either species alone and formed ordered arrays where both species aligned with the light source. No cooperative motility was observed when a non-motile pilB mutant of Syn OS-B was used instead of Syn OS-B. The binary consortium also produced more adherent biofilm than individual species, consistent with the close interspecies association revealed by electron microscopy. We propose that cyanobacteria and Chloroflexota cooperate in forming natural microbial mats, by colonizing new niches and building robust biofilms. SignificanceMicrobial mats are dense, layered communities with ancient origins and widespread occurrence, but how they assemble is not well understood. To investigate how microbial motility, physical interactions, and responses to light affect mat assembly, we developed a binary consortium from representative hot spring mat isolates. Individually, the Cyanobacteria and Chloroflexota isolates displayed significant differences in motility and biofilm formation. When combined, the consortium exhibited enhanced motility towards light and formed more robust biofilms. This model consortium approach complements in situ studies by directly testing the role of motility and physical cooperation in shaping microbial mats, and could inform biofilm applications in industrial settings.

microbiology↗

bioRxiv2024

Electric charge controls plasmodesma conductivity

While plant cells are enclosed by rigid cell walls that counteract intracellular hydrostatic pressure1, their plasma membrane, cytosol, and endoplasmic reticulum (ER) remain connected through plasmodesmata, nanoscopic cell wall pores2. Plasmodesmal cell-to-cell transport occurs in the cytosolic sleeve between the plasma membrane and the ER membrane3-5, and is generally thought to be limited by the size of the moving particle alone6. Given that biological membranes carry negative electric surface charges7-9, this steric notion conflicts with physical theory of ion diffusion in nanometer-sized pores with charged walls10. Quantifying the movements of differently sized and charged fluorescent dyes in Tradescantia stamen hairs, we found that anionic fluorophores of up to 1 kDa traversed plasmodesmata whereas much smaller cationic ones did not. While this agrees with theoretical expectations of different size exclusion limits for cations and anions, it questions current dogma concerning plasmodesma function and also structure, as it implies positively rather than negatively charged surfaces within plasmodesmal pores. Our findings call for re-evaluations of current models of symplasmic transport, especially of charged molecules like the phytohormone auxin (indole-acetic acid) and certain amino acids.

cell biology↗

bioRxiv2024

Cell-free expression and biochemical characterization of polysaccharide-synthesizing glycosyltransferases

Polysaccharides are a major class of natural polymers found abundantly across all major life forms and play a critical role as structural, metabolic, or functional components in biomolecular processes. Some polysaccharides like cellulose and hyaluronan are synthesized by membrane-bound family-2 glycosyltransferases (GTs). Despite the fact that the GT-2 family has the maximum number of deposited sequences, the biochemistry of GT-2 family enzymes is still poorly understood due to difficulties associated with GT membrane protein expression, purification, and reconstitution in lipid carriers. Here, we chose Populus tremula x tremuloides cellulose synthase 8 (PttCesA8) and Streptococcus equisimilis hyaluronan synthase (SeHas) as putative family-2-GTs to be expressed in a wheat-germ-based cell-free expression (CFE) system as proteoliposomes. The cell-free products were obtained as reconstituted liposomes directly from CFE reactions at high yields and short processing times compared to other approaches. GT enzymes expression was confirmed using SDS-PAGE and immunoblotting, and the integration of GTs in lipid layers was observed using cryogenic electron microscopy. Both GTs tested were catalytically active when incubated with their respective substrates and cofactors. The Michalis-Menten kinetic constants, Km for PttCesA8, was 295.8 {micro}M, and SeHas was 321.51 {micro}M (toward UDP N-Acetyl Glucosamine) and 207.88 {micro}M (toward UDP Glucuronic Acid), respectively. UDP was found to actively inhibit both these GTs with apparent inhibition constants of 10.08 {micro}M and 24.38 {micro}M. Mutation of specific conserved residues in structure-deficit SeHas confirmed the importance of lysine-139, glutamine-248, and threonine-283 residues in hyaluronan biosynthesis. In summary, wheat-germ-based CFE can be used to express functionally active and liposome-reconstituted family-2 GTs at high yields with relative ease to enable classical enzymology assays and will also enable more detailed structural studies in the near future.

bioengineering↗

bioRxiv2022

Structure Of Eukaryotic Type III Glutamine Synthetase Lacking Ring-Ring Quaternary Contacts

There are three major classes of the enzyme Glutamine Synthetase (GS), denoted as GSI, GSII and GSIII in both prokaryotes and eukaryotes, that share widely conserved active-site residues but vary in average protein length, oligomeric assembly state and cofactor requirements. Key structures have been determined for all prokaryotic and eukaryotic GS classes except the GSIII class of eukaryotic origin, where even basic questions about its functional oligomeric state are unresolved. Here, we report the 3.2[A] cryo-EM structure of eukaryotic GSIII from the ancient organism of green algal lineage Ostreococcus tauri. Using a combined structural and biochemical approach, we demonstrate that O. tauri GSIII self-assembles and functions exclusively as a single hexameric ring. This is unlike most GSs in other classes, where they form double-stacked ring assemblies instead. Major structural differences in the ring-ring stacking across the classes suggest evolutionary pressure may have favored higher-order interactions that might be beneficial but are not necessarily required for the catalytic GS performance.

biochemistry↗

bioRxiv2022

Cryo-EM structure of the diapause chaperone artemin

The protein artemin constitutes over 10% of all protein in Artemia cysts during diapause and acts as both an RNA and protein chaperone. However, its mechanistic details remain elusive since no high-resolution structure of artemin exists. Here we report the full-length structure of artemin at 2.04 [A] resolution. The cryo-EM map contains density for an intramolecular disulfide bond between Cys22-Cys61 and resolves the entire C-terminus extending into the core of the assembled protein cage. We also provide data supporting the role of C-terminal helix F towards stabilizing the dimer form that is believed to be important for its chaperoning activity. We were able to destabilize this effect by placing a tag at the C-terminus to fully pack the internal cavity and cause limited steric hindrance.

biochemistry↗

bioRxiv2021

AutoMicroED: A semi-automated MicroED processing pipeline

Despite rapid adaptation of micro-electron diffraction (MicroED) for protein and small molecule structure determination to sub-angstrom resolution, the lack of automation tools for easy MicroED data processing remains a challenge for expanding to the broader scientific community. In particular, automation tools, which are novice user friendly, compatible with heterogenous datasets and can be run in unison with data collection to judge the quality of incoming data (similar to cryosparc LIVE for single particle cryoEM) do not exist. Here, we present AutoMicroED, a cohesive and semi-automatic MicroED data processing pipeline that runs through image conversion, indexing, integration and scaling of data, followed by merging of successful datasets that are pushed through phasing and final structure determination. AutoMicroED is compatible with both small molecule and protein datasets and creates a straightforward and reproducible method to solve single structures from pure samples, or multiple structures from mixed populations. The immediate feedback on data quality, data completeness and more parameters, aids users to identify whether they have collected enough data for their needs. Overall, AutoMicroED permits efficient structure elucidation for both novice and experienced users with comparable results to more laborious manual processing.

biophysics↗

bioRxiv2019

The tunable intrahexamer heteroassembly mechanism of pseudoenzyme/enzyme pair, PDX1.2/PDX1.3, with its molecular impact on vitamin B6 regulation

Pseudoenzymes have emerged as key regulatory elements in all kingdoms of life despite being catalytically non-active. Yet many factors defining why one protein is active while its homolog is inactive remain uncertain. For pseudoenzyme-enzyme pairs, the similarity of both subunits can often hinder conventional characterization approaches. In plants, a pseudoenzyme PDX1.2 positively regulates vitamin B6 production by association with its active catalytic homologs such as PDX1.3 through an unknown mechanism. Here we used an integrative experimental approach to learn that such pseudoenzyme-enzyme pair associations result in hetero-complexes of variable stoichiometry, which are unexpectedly tunable. We also present the atomic structure of the PDX1.2 pseudoenzyme as well as the population averaged PDX1.2-PDX1.3 pseudoenzyme-enzyme pair. Finally, we dissected hetero-dodecamers of each stoichiometry to understand the arrangement of monomers in the hetero-complexes and identified symmetry- imposed preferences in PDX1.2-PDX1.3 interactions. Our results provide a new model of pseudoenzyme-enzyme interactions and their native heterogeneity.

molecular biology↗

bioRxiv2018

Considerations for imaging thick, low contrast, and beam sensitive samples with liquid cell transmission electron microscopy

Transmission electron microscopy of whole cells is hindered by the inherently large thickness and low atomic contrast intrinsic of cellular material. Liquid cell transmission electron microscopy allows samples to remain in their native hydrated state and may permit visualizing cellular dynamics in-situ. However, imaging biological cells with this approach remains challenging and identifying an optimal imaging regime using empirical data would help foster new advancements in the field. Recent questions about the role of the electron beam inducing morphological changes or damaging cellular structure and function necessitates further investigation of electron beam-cell interactions, but is complicated by variability in imaging techniques used across various studies currently present in literature. The necessity for using low electron fluxes for imaging biological samples requires finding an imaging strategy which produces the strongest contrast and signal to noise ratio for the electron flux used. Here, we experimentally measure and evaluate signal to noise ratios and damage mechanisms between liquid and cryogenic samples for cells using multiple electron imaging modalities all on the same instrument and with equivalent beam parameters to standardize the comparison. We also discuss considerations for optimal electron microscopy imaging conditions for future studies on whole cells within liquid environments.

cell biology↗

bioRxiv2018

Protein Structural Biology Using Cell-Free Platform from Wheat Germ

One of the biggest bottlenecks for structural analysis of proteins remains the creation of high yield and high purity samples of the target protein. Cell-free protein synthesis technologies are powerful and customizable platforms for obtaining functional proteins of interest in short timeframes while avoiding potential toxicity issues and permitting high-throughput screening. These methods have benefited many areas of genomic and proteomics research, therapeutics, vaccine development and protein chip constructions. In this work, we demonstrate a versatile and multistage eukaryotic wheat-germ cell-free protein expression pipeline to generate functional proteins of different sizes from multiple host organism and DNA source origins. We also developed a robust purification procedure, which can produce highly-pure (>98%) proteins with no specialized equipment required and minimal time invested. This pipeline successfully produced and analyzed proteins in all three major geometry formats used for structural biology including single particle analysis, and both two-dimensional and three-dimensional protein crystallography. The flexibility of the wheat germ system in combination with the multiscale pipeline described here provides a new workflow for rapid generation of samples for structural characterization that may not be amenable to other recombinant approaches.

molecular biology↗

bioRxiv2018

Cell-free production of full-length ice nucleating protein InaZ

Unlike inorganic or other synthetic alternatives, ice nucleating proteins (INPs) remain the most efficient ice nuclei today. Their potential applications in cryo-preservation, biomedicine, food industry and in the modulation of climate are widespread. Nevertheless, over several decades, cell-based recombinant methods have experienced multiple difficulties expressing these large proteins in full-length and in necessary yields while retaining functionality. As a result, our understanding of the structure and ice nucleation mechanism for this class of proteins is incomplete, and, most importantly, the full extent of possible applications unrealized. Using a wheat-germ cell-free expression pipeline, we successfully expressed and purified full-length ice nucleating protein InaZ from Pseudomonas syringae, known as a model INP. High protein yield and solubility has been achieved using this system. Ice nucleation experiments inside a dynamic environmental scanning electron microscope (ESEM) confirmed that the produced InaZ products remain functional. Preliminary structural assessments of these proteins using Transmission Electron Microscopy (TEM) showed experimental evidence for their structural organization as fibrils. We believe that the current platform will be suitable for expressing other INPs of interest and can be further employed as new engineering system either for industrial or scientific needs.

synthetic biology↗