Biology subjects

Tuijtel, M. W.

Publications and source records attributed to Tuijtel, M. W..

6 recordsLinked to original sources

bioRxiv2025

Optimal tilt-increment for cryo-ET

Cryo-electron tomography (cryo-ET) enables high-resolution, three-dimensional imaging of cellular structures in their native, frozen state. However, image quality is limited by a trade-off between angular sampling and radiation damage. Therefore, the choice of the angular increment during data collection is a critical parameter that affects tomogram quality and downstream analyses. Optimising this increment is challenging due to the high demands on microscope time, storage, and computation. In this study, we systematically evaluated tilt increments of 1{degrees}, 2{degrees}, 3{degrees}, 5{degrees}, and 10{degrees} using lamellae from Dictyostelium discoideum cells. We found that at a constant total electron dose, finer tilt increments (1-3{degrees}) produced better-aligned tomograms with higher signal-to-noise ratios and improved outcomes in template matching and subtomogram averaging. A 3{degrees} increment emerged as the optimal balance between data quality, alignment accuracy, dose per image, and processing efficiency. This practical recommendation supports both high-throughput and high-resolution structural studies and can guide future cryo-ET data acquisition strategies.

biophysics↗

bioRxiv2024

cryoTIGER: Deep-Learning Based Tilt Interpolation Generator for Enhanced Reconstruction in Cryo Electron Tomography

Cryo-electron tomography enables the visualization of macromolecular complexes within native cellular environments, but is limited by incomplete angular sampling and the maximal electron dose that biological specimen can be exposed to. Here, we developed cryoTIGER (Tilt Interpolation Generator for Enhanced Reconstruction), a computational workflow leveraging a deep learning-based frame interpolation to generate intermediate tilt images. By interpolating between tilt series projections, cryoTIGER improves angular sampling, leading to enhanced 3D reconstructions, more refined particle localization, and improved segmentation of cellular structures. We evaluated our interpolation workflow on diverse datasets and compared its performance against non-interpolated data. Our results demonstrate that deep learning-based interpolation improves image quality and structural recovery. The presented cryoTIGER framework offers a computational alternative to denser sampling during tilt series acquisition, paving the way for enhanced cryo-ET workflows and advancing structural biology research.

molecular biology↗

bioRxiv2023

High-confidence 3D template matching for cryo-electron tomography

Cryo-electron tomography (CryoET) resolves individual macromolecules inside living cells. However, the complex composition and high density of cells challenge the faithful identification of features in tomograms. Here, we capitalize on recent advances in electron tomography and demonstrate that 3D template matching (TM) localizes a wide range of structures inside crowded eukaryotic cells with confidence 10 to 100-fold above the noise level. We establish a TM pipeline with systematically tuned parameters for automated, objective and comprehensive feature identification. High-fidelity and high-confidence localizations of nuclear pore complexes, vaults, ribosomes, proteasomes, lipid membranes and microtubules, and individual subunits, demonstrate that TM is generic. We resolve [~]100-kDa proteins, connect the functional states of complexes to their cellular localization, and capture vaults carrying ribosomal cargo in situ. By capturing individual molecular events inside living cells with defined statistical confidence, high-confidence TM greatly speeds up the CryoET workflow and sets the stage for visual proteomics.

molecular biology↗

bioRxiv2023

Thinner is not always better: Optimising cryo lamellae for subtomogram averaging

Cryo-electron tomography (cryo-ET) is a powerful method to elucidate subcellular architecture and to structurally analyse biomolecules in situ by subtomogram averaging (STA). Specimen thickness is a key factor affecting cryo-ET data quality. Cells that are too thick for transmission imaging can be thinned by cryo-focused-ion-beam (cryo-FIB) milling. However, optimal specimen thickness for cryo-ET on lamellae has not been systematically investigated. Furthermore, the ions used to ablate material can cause damage in the lamellae, thereby reducing STA resolution. Here, we systematically benchmark the resolution depending on lamella thickness and the depth of the particles within the sample. Up to ca. 180 nm, lamella thickness does not negatively impact resolution. This shows that there is no need to generate very thin lamellae and thickness can be chosen such that it captures major cellular features. Furthermore, we show that gallium-ion-induced damage extends to depths of up to 30 nm from either lamella surface.

biophysics↗

bioRxiv2023

Histone divergence in Trypanosoma brucei results in unique alterations to nucleosome structure

Eukaryotes have a multitude of diverse mechanisms for organising and using their genomes, but the histones that make up chromatin are highly conserved. Unusually, histones from kinetoplastids are highly divergent. The structural and functional consequences of this variation are unknown. Here, we have biochemically and structurally characterised nucleosome core particles (NCPs) from the kinetoplastid parasite Trypanosoma brucei. A structure of the T. brucei NCP reveals that global histone architecture is conserved, but specific sequence alterations lead to distinct DNA and protein interaction interfaces. The T. brucei NCP is unstable and has weakened overall DNA binding. However, dramatic changes at the H2A-H2B interface introduce local reinforcement of DNA contacts. The T. brucei acidic patch has altered topology and is refractory to known binders, indicating that the nature of chromatin interactions in T. brucei may be unique. Overall, our results provide a detailed molecular basis for understanding evolutionary divergence in chromatin structure.

biochemistry↗

bioRxiv2022

Selecting optimal support grids for super-resolution cryogenic correlated light and electron microscopy

Cryogenic transmission electron microscopy (cryo-TEM) and super-resolution fluorescence microscopy (FM) are two popular and ever improving methods for high-resolution imaging of biological samples. In recent years, the combination of these two techniques into one correlated workflow has gained attention as a promising route towards contextualizing and enriching cryo-TEM imagery. A problem that is often encountered in the combination of these methods is that of light-induced damage to the sample during fluorescence imaging that renders the sample structure unsuitable for TEM imaging. In this paper, we describe how absorption of light by TEM sample support grids leads to sample damage, and we systematically explore the importance of parameters of grid design. We explain how, by changing the grid geometry and materials, one can increase the maximum illumination power density in fluorescence microscopy by up to an order of magnitude, and demonstrate the significant improvements in super-resolution image quality that are enabled by the selection of support grids that are optimally suited for correlated microscopy.

molecular biology↗