Biology subjects

Kopylov, M.

Publications and source records attributed to Kopylov, M..

17 recordsLinked to original sources

bioRxiv2026

Imaging large fields-of-view at high resolution in cryo-ET with square beam montaging

Visualizing macromolecules within their native cellular context by cryo-electron tomography (cryo-ET) is fundamentally limited by the trade-off between field of view and resolution: Capturing high-resolution information about biomolecules requires high magnification, which restricts the field of view and obscures the cellular context in which those biomolecules function. Collecting montage data by tiling the electron beam over the region of interest offers one solution, although traditional round electron beams cause excessive radiation damage across overlapping regions. We previously made electron beams square in shape, enabling montage collection with minimal overlap and thereby reducing excessive exposure and loss of high-resolution information. Here, we create a pipeline for collecting and processing montage cryo-ET data with square electron beams. We show that square beam montages retain high-resolution information by reconstructing virus-like particles to 3.5 [A] resolution using sub-tomogram averaging, and apply the workflow to imaging a glial cell and malaria parasite lamellae over fields of view up to 65 m2. We also provide a comprehensive protocol to make square beams accessible to the community.

molecular biology↗

bioRxiv2026

Language Modeling Materializes a World Model of Protein Biology

Proteins are fundamental to life. The full extent of their biology is beyond our ability to characterize with experimental approaches in the physical laboratory. Accurate digital representations could accelerate the discovery of protein biology through virtual experiments. We propose language modeling to learn unified and general representations that can be scaled to all of protein biology. Building on these representations, we develop a structure prediction model that exceeds the performance of established methods for biomolecular complex prediction across benchmarks, including for the interactions of antibodies with their targets. A simple search procedure yields high experimental success rates for the discovery of proteins with nanomolar binding affinities for both miniproteins and single-chain antibodies, a modality critical for therapeutic design. Study of the concepts in the language models representation space reveals a systematic organization aligned with the reductionist understanding of proteins developed through empirical science. Leveraging this organization, we generate a comprehensive map of protein biology encompassing over 6.8 billion sequences and 1.1 billion predicted structures, identifying connections across known and unknown biology. As a whole, this shows language modeling as a powerful substrate for representing the biology of proteins, operating across scales from the prediction and design of protein interactions at the atomic level, to identifying properties of proteins at different levels of granularity and abstraction, to the scale of mapping connections between proteins across billions of years of evolution.

bioinformatics↗

bioRxiv2026

Characterization Standard for In-situ Cryo-electron Tomography

Standardized biological specimens are essential for optimizing cryoEM workflows and benchmarking instrument performance. While apoferritin fulfills this role for single-particle analysis, no equivalent exists for cryo-electron tomography. Ribosomes are frequently used but require large datasets due to C1 symmetry and structural heterogeneity, limiting rapid optimization and standardized comparison of workflows. Here, we present PP7 virus-like particles (VLPs) overexpressed in E. coli as a scalable in situ benchmark. VLPs have high orders of symmetry enabling rapid, high-resolution validation of tomographic pipelines from minimal datasets, while their distinct structural features across low to high resolutions provide a practical resolution metric.

cell biology↗

bioRxiv2025

Overcoming air-water interface induced artifacts in Cryo-EM with protein nanocrates

Contact with the air-water interface can bias the orientation of macromolecules during cryo-EM sample preparation, leading to uneven sample distribution, preferred orientation, and damage to the molecules of interest. To prevent this, we describe a method to encapsulate target proteins within highly hydrophilic, structurally homogeneous, and stable protein shells, which we refer to as "nanocrates" for this purpose. Here, we describe packaging, data acquisition, and reconstruction of three proof-of-principle examples, each illuminating a different aspect of the method: apoferritin (ApoF, demonstrating high-resolution), thyroglobulin (Tg, solving a known preferred orientation problem), and 7,8-dihydroneopterin aldolase (DHNA, a structure previously uncharacterized by cryo-EM).

bioengineering↗

bioRxiv2025

The Inaugural Flatiron Institute Cryo-EM Conformational Heterogeneity Challenge

Despite the rise of single particle cryo-electron microscopy (cryo-EM) as a premier method for resolving macromolecular structures at atomic resolution, methods to address molecular heterogeneity in vitrified samples have yet to reach maturity. With an increasing number of new methods to analyze the multitude of heterogeneous states captured in single particle images, a systematic approach to validation in this field is needed. With this motivation, we issued a challenge to the community to analyze two cryo-EM particle image sets of thyroglobulin that exhibit continuous conformational heterogeneity. The first dataset was experimental and the second was generated with a simulator, allowing control over the distribution of molecular structures and enabled direct comparison between participants submissions and the ground truth molecular structures and distributions. Participants were asked to submit 80 volumes representing the heterogeneous ensemble and estimate their respective populations in the image sets provided. Participation of the research community in the challenge was strong, with submissions from nearly all developers of heterogeneity methods, resulting in 41 submissions across both datasets. Submissions qualitatively exceeded expectations, with the molecular motions identified by methods resembling both each other and the ground truth motion. However, quantitatively assessing these similarities was a challenge in and of itself. In the process of assessing the submissions, we developed several validation metrics, most of which require reference to the underlying ground truth volumes. However, we have also explored the use of metrics that do not necessarily reference ground truth. This is particularly apt for experimental datasets where ground truth is inaccessible. These approaches allowed us to assess the similarity and accuracy in volume quality, molecular motions, and conformational distribution of di!erent submissions. These metrics and the e!orts of all participants help chart a path forward for the improvements of heterogeneity methods for cryo-EM and for future challenges to validate these new methods as they continue to be developed by the community.

biophysics↗

bioRxiv2024

Identification and cryoEM structure determination of Escherichia phage YDC107 tail found in a bacteria-contaminated buffer

Cryo-electron microscopy data analysis can yield multiple structures from a single heterogeneous dataset. Here, we show a workflow we used for the identification of a contaminant from a cryoEM grid without prior knowledge of protein sequence. We determined the tail structure of Escherichia phage YDC107 from only several thousand particles. The workflow combines high-resolution single-particle data processing with de novo model determination using ML-based methods. Structural analysis revealed that the central part of the phage tail has a C6 symmetry, however the overall symmetry of each segment is C3 due to dimerization of a flexible domain. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=67 SRC="FIGDIR/small/627647v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1444ae3org.highwire.dtl.DTLVardef@907ec1org.highwire.dtl.DTLVardef@71eebdorg.highwire.dtl.DTLVardef@1f0e6a1_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2024

Multi-species cryoEM calibration and workflow verification standard

Cryogenic electron microscopy (cryoEM) is a rapidly growing structural biology modality that has been successful in revealing molecular details of biological systems. However, unlike established biophysical and analytical techniques with calibration standards, cryoEM has lacked comprehensive biological test samples. We introduce a cryoEM calibration sample that is a mixture of compatible macromolecules that can be used not only for resolution optimization but also provides multiple reference points for evaluating instrument performance, data quality, and image processing workflows in a single experiment. This combined test specimen provides researchers a reference point for validating their cryoEM pipeline, benchmarking their methodologies, and testing new algorithms.

biochemistry↗

bioRxiv2024

Cryo-ET reveals the in situ architecture of the polar tube invasion apparatus from microsporidian parasites

Microsporidia are divergent fungal pathogens that employ a harpoon-like apparatus called the polar tube (PT) to invade host cells. The PT architecture and its association with neighboring organelles remain poorly understood. Here, we use cryo-electron tomography to investigate the structural cell biology of the PT in dormant spores from the human-infecting microsporidian species, Encephalitozoon intestinalis. Segmentation and subtomogram averaging of the PT reveal at least four layers: two protein-based layers surrounded by a membrane, and filled with a dense core. Regularly spaced protein filaments form the structural skeleton of the PT. Combining cryo-electron tomography with cellular modeling, we propose a model for the 3-dimensional organization of the polaroplast, an organelle that is continuous with the membrane layer that envelops the PT. Our results reveal the ultrastructure of the microsporidian invasion apparatus in situ, laying the foundation for understanding infection mechanisms.

microbiology↗

bioRxiv2024

Environmental differences impact Acinetobacter baumannii phage isolation and infectivity

With the global rise of antimicrobial resistance, phage therapy is increasingly re-gaining traction as a strategy to treat bacterial infections. For phage therapy to be successful however, we first need to isolate appropriate candidate phages for both clinical and experimental research. Acinetobacter baumannii is an opportunistic pathogen known for its ability to rapidly evolve resistance to antibiotics, making it a prime target for phage therapy. Yet phage isolation is often hampered by A. baumanniis ability to rapidly switch between capsular states. Here, we report the discovery and structural characterisation of a novel lytic phage, Mystique. This phage was initially isolated against the wild-type AB5075: a commonly used clinical model strain against which no phage has previously been readily available for the capsulated form. When screening Mystique on 103 highly diverse isolates of A. baumannii, we found that it has a broad host range, being able to infect 85.4% of all tested strains when tested on bacterial lawns - a host range which expanded to 91.3% when tested in liquid culture. This variation between solid and liquid environments on phage infectivity was also observed for several other phages in our collection that were assumed unable to infect AB5075, and capsule negative mutants that initially seemed completely resistant to Mystique proved susceptible when assayed in liquid. Overall, through the discovery of a novel phage we demonstrate how environmental differences can drastically impact phage infectivity with important consequences for phage isolation and characterisation efforts. Author summaryBacterial infections caused by Acinetobacter baumannii are a major global health concern due to high antibiotic resistance, earning it a critical priority pathogen ranking by the WHO. Phage therapy is resurging as a treatment option, with some success against A. baumannii. However, the wild-type clinical model strain used to assess new therapies lacks an available phage, and isolating phages for A. baumannii is challenging due to its complex capsule. Here, we report the discovery of a novel lytic phage, Mystique, which exhibits a broad host range, infecting 94 out of 103 tested A. baumannii strains. We conducted genomic sequencing and structural analysis to fully characterise Mystique. Additionally, we found that the testing environment significantly impacts results; some phages that do not form plaques on bacterial lawns can still infect and amplify in liquid cultures of the same strain. Moreover, mutants resistant to Mystique based on plaque assays were susceptible in liquid culture assays. This work underscores the necessity of a multifaceted approach for phage isolation and characterisation, as traditional phage assays may not be sufficient for studying bacteria-phage dynamics in certain bacteria such as A. baumannii.

microbiology↗

bioRxiv2024

Dose-Efficient Cryo-Electron Microscopy for Thick Samples using Tilt-Corrected Scanning Transmission Electron Microscopy, Demonstrated on Cells and Single Particles

Cryo-EM is a powerful tool in structural biology, providing insights through techniques like single-particle analysis (SPA) and cryogenic electron tomography (cryo-ET). In thick specimens, challenges arise as an exponentially larger fraction of the transmitted electrons lose energy from inelastic scattering and can no longer be properly focused as a result of chromatic aberrations in the post-specimen optics. Rather than filtering out the inelastic scattering at the price of reducing potential signal, as is done in energy-filtered transmission electron microscopy (EFTEM), we show how a dose-efficient and unfiltered image can be rapidly obtained using tilt-corrected bright-field scanning-TEM (tcBF-STEM) data collected on a pixelated detector. Enhanced contrast and a 3-5x improvement in collection efficiency are observed for 2D images of intact bacterial cells and large organelles using tcBF-STEM compared to EFTEM for thicknesses beyond 500 nm. As a proof of concept for the techniques performance in structural determination, we present an SPA map at subnanometer resolution for a highly symmetric virus-like particle (VLP) with 789 particles. These findings suggest applications for tcBF-STEM in cryo-EM of thicker cellular volumes where current approaches struggle.

cell biology↗

bioRxiv2024

Heterologous Prime-Boost with Immunologically Orthogonal Protein Nanoparticles for Peptide Immunofocusing

Protein nanoparticles are effective platforms for antigen presentation and targeting effector immune cells in vaccine development. Encapsulins are a class of protein-based microbial nanocompartments that self-assemble into icosahedral structures with external diameters ranging from 24 to 42 nm. Encapsulins from Mxyococcus xanthus were designed to package bacterial RNA when produced in E. coli and were shown to have immunogenic and self-adjuvanting properties enhanced by this RNA. We genetically incorporated a 20-mer peptide derived from a mutant strain of the SARS-CoV-2 receptor binding domain (RBD) into the encapsulin protomeric coat protein for presentation on the exterior surface of the particle. This immunogen elicited conformationally-relevant humoral responses to the SARS-CoV-2 RBD. Immunological recognition was enhanced when the same peptide was presented in a heterologous prime/boost vaccination strategy using the engineered encapsulin and a previously reported variant of the PP7 virus-like particle, leading to the development of a selective antibody response against a SARS-CoV-2 RBD point mutant. While generating epitope-focused antibody responses is an interplay between inherent vaccine properties and B/T cells, here we demonstrate the use of orthogonal nanoparticles to fine-tune the control of epitope focusing. Table of Contents graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=121 SRC="FIGDIR/small/581861v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@53407corg.highwire.dtl.DTLVardef@1ae8d0eorg.highwire.dtl.DTLVardef@aec021org.highwire.dtl.DTLVardef@c7e0ef_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2023

Square beams for optimal tiling in TEM

Imaging large fields of view at a high magnification requires tiling. Transmission electron microscopes typically have round beam profiles; therefore, tiling across a large area is either imperfect or results in uneven exposures, a problem on dose-sensitive samples. Here, we introduce a square electron beam that can be easily retrofitted in existing microscopes and demonstrate its application, showing it can tile nearly perfectly and deliver cryo-EM imaging with a resolution comparable to conventional setups.

biophysics↗

bioRxiv2020

Characterization of the SARS-CoV-2 S Protein: Biophysical, Biochemical, Structural, and Antigenic Analysis

Coronavirus disease 2019 (COVID-19) is a global health crisis caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and there is a critical need to produce large quantities of high-quality SARS-CoV-2 Spike (S) protein for use in both clinical and basic science settings. To address this need, we have evaluated the expression and purification of two previously reported S protein constructs in Expi293F and ExpiCHO-S cells, two different cell lines selected for increased expression of secreted glycoproteins. We show that ExpiCHO-S cells produce enhanced yields of both SARS-CoV-2 S proteins. Biochemical, biophysical, and structural (cryo-EM) characterization of the SARS-CoV-2 S proteins produced in both cell lines demonstrate that the reported purification strategy yields high quality S protein (non-aggregated, uniform material with appropriate biochemical and biophysical properties). Importantly, we show that multiple preparations of these two recombinant S proteins from either cell line exhibit identical behavior in two different serology assays. We also evaluate the specificity of S protein-mediated host cell binding by examining interactions with proposed binding partners in the human secretome. In addition, the antigenicity of these proteins is demonstrated by standard ELISAs, and in a flexible protein microarray format. Collectively, we establish an array of metrics for ensuring the production of high-quality S protein to support clinical, biological, biochemical, structural and mechanistic studies to combat the global pandemic caused by SARS-CoV-2.

biochemistry↗

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↗

bioRxiv2019

Ribosome dimerization prevents loss of essential ribosomal proteins during quiescence

The formation of ribosome dimers during periods of quiescence is widespread among bacteria and some higher eukaryotes. However, the mechanistic importance of dimerization is not well understood. In bacteria ribosome dimerization is mediated by the Hibernation Promoting Factor (HPF). Here, we report that HPF from the Gram-positive bacterium Bacillus subtilis preserves active ribosomes by preventing the loss of essential ribosomal proteins. Ribosomes isolated from strains either lacking HPF ({Delta}hpf) or encoding a mutant allele of HPF that binds the ribosome but does not mediate dimerization were substantially depleted of the small subunit proteins S2 and S3. Strikingly, these proteins are located at the ribosome dimer interface. We used single particle cryo-EM to further characterize ribosomes isolated from a {Delta}hpf mutant strain and observed that many were missing S2, S3, or both. These data support a model in which the ribosome dimerization activity of HPF evolved to protect labile proteins that are essential for ribosome function.\n\nSignificance StatementWhen nutrients become scarce, many bacterial species enter an extended state of quiescence. A major challenge of this state is how to attenuate protein synthesis, the most energy consuming cellular process, while preserving ribosomes for the return to favorable conditions. Here, we show that the ribosome-binding protein HPF which dimerizes ribosomes functions to protect essential ribosomal proteins at the dimer interface. HPF is almost universally conserved in bacteria and HPF deletions in diverse species exhibit decreased viability under nutrient limitation. Our data provide mechanistic insight into this phenotype and establish a role for HPF in maintaining translationally competent ribosomes during quiescence.

microbiology↗

bioRxiv2018

The impact of data reduction and lossy image formats on electron microscope images

Recent advances in instrumentation and software for cryoEM have increased the applicability and utility of this method. Coupled with the adoption of automated pipelines, significant infrastructure support is required to sustain high throughput workflows. In particular, data generation rates may outpace the ability to deploy data storage and archival solutions. We have investigated what effects data compression and conversion to different file formats have on the ability to obtain high resolution cryoEM reconstructions. Standard lossless data compression strategies have a high impact on reducing the size of direct detector electron counting movie stacks, but provide more modest gains for aligned summed images. We show that EM images can be compressed using standard lossy methods to reduce file storage to 5-10% of the size of the original aligned sum or movie stack file and yet still retain enough information such that modern image processing pipelines will provide sub-2[A] reconstructions from the compressed data.

biophysics↗

bioRxiv2018

Benchmarking cryo-EM single particle analysis workflow

Cryo electron microscopy facilities running multiple instruments and serving users with varying skill levels need a robust and reliable method for benchmarking both the hardware and software components of their single particle analysis workflow. The workflow is complex, with many bottlenecks existing at the specimen preparation, data collection and image analysis steps; the samples and grid preparation can be of unpredictable quality, there are many different protocols for microscope and camera settings, and there is a myriad of software programs for analysis that can depend on dozens of settings chosen by the user. For this reason, we believe it is important to benchmark the entire workflow, using a standard sample and standard operating procedures, on a regular basis. This provides confidence that all aspects of the pipeline are capable of producing maps to high resolution. Here we describe benchmarking procedures using a test sample, rabbit muscle aldolase.

molecular biology↗