Biology subjects

Grigorieff, N.

Publications and source records attributed to Grigorieff, N..

At least 19 recordsLinked to original sources

bioRxiv2025

Structural basis for non-AUG translation regulation by 5MPs

The cellular proteome is regulated by translation initiation on AUG or non-canonical (non-AUG) start codons1-3. Non-AUG initiation remodels proteome during stress and is implicated in cancer and other diseases4-6. The eIF5-mimic proteins (5MPs) restrict non-AUG start codon usage and thereby reprogram proteoform expression from mRNAs with alternative start sites, such as the oncogenic c-Myc7-10. The mechanism by which 5MPs induce such translational reprogramming remains unknown. Here, using in extracto cryo-electron microscopy (cryo-EM) and biochemical assays, we report that translational repression by 5MP strongly depends on the sequence context near the AUG or non-AUG codons. Cryo-EM structures of 5MP-bound 48S pre-initiation complexes (PICs) from native cell extracts reveal that 5MP binds at the A site of the small ribosomal subunit, stabilizing an expanded open-head conformation of the PIC scanning along mRNA. The N-terminal region of 5MP blocks the A site, whereas the C-terminal domain docks at eIF2{beta} and the initiator tRNAMet outside the P site (i.e., Pout). These findings indicate that 5MP protein directly biases the initiating 48S complexes toward the open conformation, promoting mRNA scanning and inhibiting initiation at suboptimal start codons.

biophysics↗

bioRxiv2025

Tolerance to the antifungal drug fluconazole is mediated by tuning cytoplasmic fluidity

Treatment failure rates for fungal infections cannot be explained simply by increased rates of drug resistance. Antifungal drug tolerance, the ability of a susceptible isolate to grow in the presence of inhibitory drug concentrations, relies upon a broad set of stress response pathways and can contribute to antifungal drug treatment failures. As the physical properties of the cytoplasm are critical for diverse cellular processes, we investigated whether cytoplasmic mesoscale fluidity is linked to antifungal tolerance, taking advantage of a fluorescent microrheological probe. Here, we show either using fluconazole, a common drug that inhibits ergosterol biosynthesis, or mutants in the ergosterol biosynthesis pathway, that cytoplasmic fluidity decreased and could be reversed upon drug removal. Reducing ribosome concentration decreased drug tolerance and restored cytoplasmic fluidity, highlighting a link between tolerance and cytoplasmic crowding and/or viscosity. However, growth in fluconazole did not increase ribosome concentrations; rather growth in fluconazole increased the number of dormant, hibernating ribosomes, cytoplasmic protein concentration, viscosity and condensate formation. Specifically, growth in fluconazole resulted in a substantial increase in processing bodies (P-bodies), whose presence correlated with azole tolerance. Furthermore, we found a substantial increase in cell-cell heterogeneity in all biophysical, biochemical and molecular outputs analyzed. Together, our results reveal that changes in the physical properties of the cytoplasm occur in response to antifungal drug and suggest that these changes, as well as increased cell-cell heterogeneity, are crucial for survival in fungistatic drugs.

microbiology↗

bioRxiv2025

In extracto cryo-EM reveals eEF2 as a major hibernation factor on 60S and 80S particles

Cryogenic electron microscopy (cryo-EM) made impressive progress in resolving cellular macromolecules and their detailed interactions. Single-particle cryo-EM traditionally relies on purified macromolecules and lacks the complexity of cellular environments, whereas in situ cryo-EM or cryo-ET require extensive sample preparation and data acquisition, presenting challenges in achieving high resolution. We describe cryo-EM of cellular lysates--in extracto cryo-EM--allowing the flexibility and high-resolution of cryo-EM in the context of cellular components. High-resolution 2D template matching (2DTM) yields [~]2.2 [A] maps of the mammalian translational apparatus. Elongating ribosome abundances in primate cell lines (MCF-7 and BSC-1) and rabbit reticulocyte lysates range from [~]70% to [~]10%, reflecting translational stress responses. Non-translating (hibernating) ribosomes carrying no mRNA, feature numerous proteins shielding ribosomal functional centers. Elongation factor 2 (eEF2) is the most abundant hibernation factor bound to >95% of ribosomes and, unexpectedly, to 60S subunits. eEF2*GDP is stabilized by interactions with the sarcin-ricin loop and protein uL14. Hibernating ribosomes also feature LARP1 involved in initiation and mTOR signaling; eIF5A implicated in elongation and termination; and other factors, exposing the variety of hibernation scenarios. Our work underscores the efficiency and potential of in extracto cryo-EM to discover native cellular complexes and mechanisms at near-atomic resolution.

molecular biology↗

bioRxiv2025

Improved cryo-EM reconstruction of sub-50 kDa complexes using 2D template matching

Visualizing the structures of small proteins and complexes has been a longstanding challenge in single-particle cryo-EM. Some of these targets have been successfully resolved by binding to antibody fragments (Fabs) or fusing with external scaffolds to increase their size. Recent advances in conventional single-particle techniques have enabled the determination of an increasing number of structures smaller than 100 kDa, achieving resolutions relevant to drug research. Compared to X-ray crystallography, cryo-EM preserves the near-native states of biomolecules, can resolve structural heterogeneity, and has the potential to apply to a wide range of targets. In this work, we demonstrate that the alignment and reconstruction of small macromolecular complexes can be improved using high-resolution structures as priors combined with 2D template matching. Using this method, we reconstructed a previously intractable [~] 43 kDa protein kinase and improved the density of its ligand-binding site. Our theoretical analysis predicts that this method can further extend single-particle cryo-EM to important drug-binding complexes well below 50 kDa. Cryo-EM | 3D reconstruction | signal detection | template matching 61

biophysics↗

bioRxiv2025

Unbend: Correction of local beam-induced sample motion in cryo-EM images using a 3D spline model

The exposure of frozen biological samples to the high-energy electron beam in a cryo-electron microscope commonly leads to beam-induced sample motion and distortions. Previously, we described Unblur, which is part of our cisTEM software to correct for beam-induced motion based on the alignment of full frames in a movie collected during the beam exposure (Grant et al., 2015). However, Unblur cannot accommodate motion due to more localized sample bending and distortions. Here, we present Unbend, extending Unblur by incorporating local motion correction using a three-dimensional cubic spline model. The 3D spline model is constructed using cubic B-splines along the exposure time axis, and bicubic B-splines within movie frames. Unbend is integrated into our cisTEM software with a new local motion visualization panel within the cisTEM graphical user interface. We processed movie frames from various in-situ sample types, including whole cells, lamellae, and cell lysates, to analyze motion behavior across different specimen types. To quantify the improvement in high-resolution signal, we utilized the 2D template matching method, which operates independently of the motion correction process, to search large ribosomal subunits from the motion-corrected micrographs. Overall, the signal-to-noise ratio of detected particles improved by 3-8% across different samples compared with full-frame aligned micrographs, while the number of detected target particles increased by up to [~]300%. The total and Von Mises equivalent strain shows a deformation scale of less than 1% in most of the samples, confirming that our model induces minimal additional distortion. Furthermore, we processed micrograph montages to study motion patterns across an entire sample, revealing considerable variance in distortion scale within the same sample, suggesting a complex underlying mechanism.

biophysics↗

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↗

bioRxiv2025

In-Situ High-Resolution Cryo-EM Reconstructions from CEMOVIS

Cryo-electron microscopy can be used to image cells and tissue at high resolution. To ensure electron transparency, sample thickness must not exceed 500 nm. Focused-ion-beam (FIB) milling has become the standard method to prepare thin samples (lamellae), however, the material removed by the milling process is lost, the imageable area is usually limited to a few square microns, and the surface layers sustain damage from the ion beam. We have examined cryo-electron microscopy of vitreous sections (CEMOVIS), a preparation technique based on cutting thin sections with a knife, as an alternative to FIB-milling. CEMOVIS sections also sustain damage, including compression, shearing and cracks. However, samples can be sectioned in series, producing many orders of magnitude more imageable area compared to lamellae making CEMOVIS an alternative to FIB-milling with distinct advantages. Using 2-dimensional template matching on images of CEMOVIS sections of Saccharomyces cerevisiae cells, we reconstructed the 60S ribosomal subunit at near-atomic resolution, demonstrating that, in many regions of the sections, the molecular structure of these subunits is largely intact, comparable to FIB-milled lamellae.

biophysics↗

bioRxiv2024

Dynamic cytoplasmic fluidity during morphogenesis in a human fungal pathogen

The molecular crowding of the cytoplasm impacts a range of cellular processes. Using a fluorescent microrheological probe (GEMs), we observed a striking decrease in molecular crowding during the yeast to filamentous growth transition in the human fungal pathogen Candida albicans. This decrease in crowding is due to a decrease in ribosome concentration that results in part from an inhibition of ribosome biogenesis, combined with an increase in cytoplasmic volume; leading to a dilution of the major cytoplasmic crowder. Moreover, our results suggest that inhibition of ribosome biogenesis is a trigger for C. albicans morphogenesis.

cell biology↗

bioRxiv2024

Virion-associated influenza hemagglutinin clusters upon sialic acid binding visualized by cryo-electron tomography

Influenza viruses are enveloped, negative sense single-stranded RNA viruses covered in a dense layer of glycoproteins. Hemagglutinin (HA) accounts for 80-90% of influenza glycoprotein and plays a role in host cell binding and membrane fusion. While previous studies have characterized structures of receptor-free and receptor-bound HA in vitro, the effect of receptor binding on HA organization and structure on virions remains unknown. Here, we used cryo-electron tomography (cryoET) to visualize influenza virions bound to a sialic acid receptor mimic. Overall, receptor binding did not result in significant changes in viral morphology; however, we observed rearrangements of HA trimer organization and orientation. Compared to the even inter-glycoprotein spacing of unliganded HA trimers, receptor binding promotes HA trimer clustering and formation of a triplet of trimers. Subtomogram averaging and refinement yielded 8-10 [A] reconstructions that allowed us to visualize specific contacts between HAs from neighboring trimers and identify molecular features that mediate clustering. Taken together, we present new structural evidence that receptor binding triggers clustering of HA trimers, revealing an additional layer of HA dynamics and plasticity.

biophysics↗

bioRxiv2024

New Statistical Metric for Robust Target Detection in Cryo-EM Using 2DTM

2D template matching (2DTM) can be used to detect molecules and their assemblies in cellular cryo-EM images with high positional and orientational accuracy. While 2DTM successfully detects spherical targets such as large ribosomal subunits, challenges remain in detecting smaller and more aspherical targets in various environments. In this work, a novel 2DTM metric, referred to as the 2DTM p-value, is developed to extend the 2DTM framework to more complex applications. The 2DTM p-value combines information from two previously used 2DTM metrics, namely the 2DTM signal-to-noise ratio (SNR) and z-score, which are derived from the cross-correlation coefficient between the target and the template. The 2DTM p-value demonstrates robust detection accuracies under various imaging and sample conditions and outperforms the 2DTM SNR and z-score alone. Specifically, the 2DTM p-value improves the detection of aspherical targets such as a modified artificial tubulin patch particle (500 kDa) and a much smaller clathrin monomer (193 kDa) in simulated data. It also accurately recovers mature 60S ribosomes in yeast lamellae samples, even under conditions of increased Gaussian noise. The new metric will enable the detection of a wider variety of targets in both purified and cellular samples through 2DTM.

biophysics↗

bioRxiv2024

Amortized template-matching of molecular conformations from cryo-electron microscopy images using simulation-based inference

AO_SCPLOWBSTRACTC_SCPLOWBiomolecules undergo conformational changes to perform their function. Cryo-electron microscopy (cryo-EM) can capture snapshots of biomolecules in various conformations. However, these images are noisy and display the molecule in unknown orientations, making it difficult to separate conformational differences from differences due to noise or projection directions. Here, we introduce cryo-EM simulation-based inference (cryoSBI) to infer the conformations of biomolecules and the uncertainties associated with the inference from individual cryo-EM images. CryoSBI builds on simulation-based inference, a combination of physics-based simulations and probabilistic deep learning, allowing us to use Bayesian inference even when likelihoods are too expensive to calculate. We begin with an ensemble of conformations, which can be templates from molecular simulations or modelling, and use them as structural hypotheses. We train a neural network approximating the Bayesian posterior using simulated images from these templates, and then use it to accurately infer the conformations of biomolecules from experimental images. Training is only done once, and after that, it takes just a few milliseconds to make inference on an image, making cryoSBI suitable for arbitrarily large datasets. CryoSBI eliminates the need to estimate particle pose and imaging parameters, significantly enhancing the computational speed in comparison to explicit likelihood methods. We illustrate and benchmark cryoSBI on synthetic data and showcase its promise on experimental single-particle cryo-EM data.

biophysics↗

bioRxiv2024

CTFFIND5 provides improved insight into quality, tilt and thickness of TEM samples

Images taken by transmission electron microscopes are usually affected by lens aberrations and image defocus, among other factors. These distortions can be modeled in reciprocal space using the contrast transfer function (CTF). Accurate estimation and correction of the CTF is essential for restoring the high-resolution signal in cryogenic electron microscopy (cryoEM). Previously, we described the implementation of algorithms for this task in the cisTEM software package (Grant et al., 2018). Here we show that taking sample characteristics, such as thickness and tilt, into account can improve CTF estimation. This is particularly important when imaging cellular samples, where measurement of sample thickness and geometry derived from accurate modeling of the Thon ring pattern helps judging the quality of the sample. This improved CTF estimation has been implemented in CTFFIND5, a new version of the cisTEM program CTFFIND. We evaluated the accuracy of these estimates using images of tilted aquaporin crystals and eukaryotic cells thinned by focused ion beam milling. We estimate that with micrographs of sufficient quality CTFFIND5 can measure sample tilt with an accuracy of 3{degrees} and sample thickness with an accuracy of 5 nm.

biophysics↗

bioRxiv2023

Baited reconstruction with 2D template matching for high-resolution structure determination in vitro and in vivo without template bias

Cryogenic electron microscopy (cryo-EM) has revolutionized structural biology, rapidly increasing the number of available molecular structures. Because of this, as well as advances in structure prediction, the focus of structural biology has begun to shift to studying macromolecular structures in their native cellular environment. A dominant feature of cryo-EM images is shot noise, making the identification of small particles of interest difficult. This is further compounded by structural noise if these particles are imaged against a background of other molecules, such as inside a cell. 2D template matching (2DTM) can be used to localize complexes with high precision, even in the presence of cellular background. Once localized, these particles may be averaged together in 3D reconstructions; however, regions included in the template may suffer from template bias, leading to inflated resolution estimates and making the interpretation of high-resolution features unreliable. We evaluate conditions that minimize template bias and show that molecular features not present in the template can be reconstructed at high resolution from targets found by 2DTM, extending prior work at low-resolution. Moreover, we present a quantitative metric for template bias to aid the interpretation of 3D reconstructions calculated with particles localized using high-resolution templates and fine angular sampling.

biophysics↗

bioRxiv2023

Quantification of gallium cryo-FIB milling damage in biological lamella

Cryogenic electron microscopy (cryo-EM) has the potential to reveal the molecular details of biological processes in their native, cellular environment at atomic resolution. However, few cells are sufficiently thin to permit imaging with cryo-EM. Thinning of frozen cells to <500 nm lamellae by cryogenic focused ion beam (FIB) milling has enabled visualization of cellular structures with cryo-EM. FIB-milling represents a significant advance over prior approaches because of its ease of use, scalability, and lack of large-scale sample distortions. However, the amount of damage caused by FIB-milling to the generated thin cell section has not yet been determined. We recently described a new approach for detecting and identifying single molecules in cryo-EM images of cells using 2D template matching (2DTM). 2DTM is sensitive to small differences between a molecular model (template) and the detected structure (target). Here we use 2DTM to demonstrate that under the standard conditions used for machining lamellae of biological samples, FIB-milling introduces a layer of variable damage that extends to a depth of 60 nm from each lamella surface. This thickness exceeds previous estimates and limits the recovery of information for in situ structural biology. We find that the mechanism of FIB-milling damage is distinct from radiation damage during cryo-EM imaging. By accounting for both electron scattering and FIB-milling damage, we find that FIB-milling damage will negate the potential improvements from lamella thinning beyond 90 nm. SignificanceThe molecular mechanisms of biological macromolecules and their assemblies is often studied using purified material. However, the composition, conformation and function of most macromolecules depend on their cellular context, and therefore, must also be studied inside cells. Focused ion beam (FIB) milling enables cryogenic electron microscopy to visualize macromolecules in cells at close to atomic resolution by generating thin sections of frozen cells. However, the extent of FIB-milling damage to frozen cells is unknown. Here we show that Ga+ FIB-milling introduces damage to a depth of [~]60 nm from each lamella surface, leading to a loss of recoverable information of up to 20% in 100 nm samples. FIB-milling with Ga+ therefore presents both an opportunity and an obstacle for structural cell biology.

biophysics↗

bioRxiv2022

From primordial clocks to circadian oscillators

Circadian rhythms play an essential role in many biological processes and surprisingly only three prokaryotic proteins are required to constitute a true post-translational circadian oscillator. The evolutionary history of the three Kai proteins indicates that KaiC is the oldest member and central component of the clock, with subsequent additions of KaiB and KaiA to regulate its phosphorylation state for time synchronization. The canonical KaiABC system in cyanobacteria is well understood, but little is known about more ancient systems that possess just KaiBC, except for reports that they might exhibit a basic, hourglass-like timekeeping mechanism. Here, we investigate the primordial circadian clock in Rhodobacter sphaeroides (RS) that contains only KaiBC to elucidate its inner workings despite the missing KaiA. Using a combination X-ray crystallography and cryo-EM we find a novel dodecameric fold for KaiCRS where two hexamers are held together by a coiled-coil bundle of 12 helices. This interaction is formed by the C-terminal extension of KaiCRS and serves as an ancient regulatory moiety later superseded by KaiA. A coiled-coil register shift between daytime- and nighttime-conformations is connected to the phosphorylation sites through a long-range allosteric network that spans over 160 [A]. Our kinetic data identify the difference in ATP-to-ADP ratio between day and night as the environmental cue that drives the clock and further unravels mechanistic details that shed light on the evolution of self-sustained oscillators.

biochemistry↗

bioRxiv2022

Defocus Corrected Large Area Cryo-EM (DeCo-LACE) for Label-Free Detection of Molecules across Entire Cell Sections

A major goal of biological imaging is localization of biomolecules inside a cell. Fluorescence microscopy can lo-calize biomolecules inside whole cells and tissues, but its ability to count biomolecules and accuracy of the spatial coordinates is limited by the wavelength of visible light. Cryo-electron microscopy (cryo-EM) provides highly accurate position and orientation information of biomolecules but is often confined to small fields of view inside a cell, limiting biological context. In this study we use a new data-acquisition scheme called "Defocus-Corrected Large-Area cryo-EM" (DeCo-LACE) to collect high-resolution images of entire sections (100 - 200 nm thick lamel-lae) of neutrophil-like mouse cells, representing 1-2% of the total cellular volume. We use 2D template matching (2DTM) to determine localization and orientation of the large ribosomal subunit in these sections. These data provide "maps" of ribosomes across entire sections of mammalian cells. This high-throughput cryo-EM data collection approach together with 2DTM will advance visual proteomics and provide biological insight that cannot be obtained by other methods.

cell biology↗

bioRxiv2022

In situ single particle classification reveals distinct 60S maturation intermediates in cells

Electron cryo-microscopy (cryo-EM) can generate high-resolution views of cells with faithful preservation of molecular structure. In situ cryo-EM, therefore, has enormous potential to reveal the atomic details of biological processes in their native context. However, in practice, the utility of in situ cryo-EM is limited by the difficulty of reliably locating and confidently identifying molecular targets (particles) and their conformational states in the crowded cellular environment. We recently showed that 2DTM, a fine-grained template-based search applied to cryo-EM micrographs, can localize particles in two-dimensional views of cells with high precision. Here we demonstrate that the signal-to-noise ratio (SNR) observed with 2DTM can be used to differentiate related complexes in focused ion beam (FIB)-milled cell sections. We apply this method in two contexts to locate and classify related intermediate states of 60S ribosome biogenesis in the Saccharomyces cerevisiae cell nucleus. In the first, we separate the nuclear pre-60S population from the cytoplasmic mature 60S population, using the subcellular localization to validate assignment. In the second, we show that relative 2DTM SNRs can be used to separate mixed populations of nuclear pre-60S that are not visually separable. We use a maximum likelihood approach to define the probability of each particle belonging to each class, thereby establishing a statistic to describe the confidence of our classification. Without the need to generate 3D reconstructions, 2DTM can be applied even when only a few target particles exist in a cell.

biophysics↗

bioRxiv2021

Locating Macromolecular Assemblies in Cells by 2D Template Matching with cisTEM

Over the last decade, single-particle electron cryo-microscopy has become one of the main techniques contributing to the growing library of high-resolution structures of macromolecules and their assemblies. For a full understanding of molecular mechanisms, however, it is important to place them into the broader context of a cell. Traditionally, this context can be visualized in 3D by electron cryo-tomography, and more recently, has also been studied by template matching of 2D images of cells and viruses. A current limitation of the latter approach is the high computational cost that limits the throughput and widespread adoption of this method. We describe here a GPU-accelerated implementation of 2D template matching in the image processing software cisTEM that allows for easy scaling and improves the accessibility of this approach. We apply 2D template matching to identify ribosomes in images of frozen-hydrated Mycoplasma pneumoniae cells and demonstrate that it can function as a versatile tool for in situ visual proteomics and in situ structure determination. We compare the results with 3D template matching of tomograms acquired on identical sample locations. We identify strengths and weaknesses of both techniques which offer complementary information about target localization and identity.

cell biology↗