iU-ExM: nanoscopy of organelles and tissues with iterative ultrastructure expansion microscopy

Nature Communications, Год журнала: 2023, Номер 14(1)

Опубликована: Ноя. 30, 2023

Expansion microscopy (ExM) is a highly effective technique for super-resolution fluorescence that enables imaging of biological samples beyond the diffraction limit with conventional microscopes. Despite development several enhanced protocols, ExM has not yet demonstrated ability to achieve precision nanoscopy techniques such as Single Molecule Localization Microscopy (SMLM). Here, address this limitation, we have developed an iterative ultrastructure expansion (iU-ExM) approach achieves SMLM-level resolution. With iU-ExM, it now possible visualize molecular architecture gold-standard samples, eight-fold symmetry nuclear pores or organization conoid in Apicomplexa. its wide-ranging applications, from isolated organelles cells and tissue, iU-ExM opens new avenues scientists studying structures functions.

Язык: Английский

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Integrating cellular electron microscopy with multimodal data to explore biology across space and time

Cell, Год журнала: 2024, Номер 187(3), С. 563 - 584

Опубликована: Фев. 1, 2024

Biology spans a continuum of length and time scales. Individual experimental methods only glimpse discrete pieces this spectrum but can be combined to construct more holistic view. In Review, we detail the latest advancements in volume electron microscopy (vEM) cryo-electron tomography (cryo-ET), which together visualize biological complexity across scales from organization cells large tissues molecular details inside native cellular environments. addition, discuss emerging methodologies for integrating three-dimensional (3DEM) imaging with multimodal data, including fluorescence microscopy, mass spectrometry, single-particle analysis, AI-based structure prediction. This multifaceted approach fills gaps continuum, providing functional context, spatial organization, identity, interactions. We conclude perspective on incorporating diverse data into computational simulations that further bridge extend while dimension time.

Язык: Английский

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

Nature Communications, Год журнала: 2024, Номер 15(1)

Опубликована: Май 11, 2024

Abstract Visual proteomics attempts to build atlases of the molecular content cells but automated annotation cryo electron tomograms remains challenging. Template matching (TM) and methods based on machine learning detect structural signatures macromolecules. However, their applicability limited in terms both abundance size targets. Here we show that performance TM is greatly improved by using template-specific search parameter optimization including higher-resolution information. We establish a pipeline with systematically tuned parameters for automated, objective comprehensive identification structures confidence 10 100-fold above noise level. demonstrate high-fidelity high-confidence localizations nuclear pore complexes, vaults, ribosomes, proteasomes, fatty acid synthases, lipid membranes microtubules, individual subunits inside crowded eukaryotic cells. provide software tools generic implementation our method broadly applicable towards realizing visual proteomics.

Язык: Английский

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The molecular architecture of the nuclear basket

Cell, Год журнала: 2024, Номер 187(19), С. 5267 - 5281.e13

Опубликована: Авг. 9, 2024

The nuclear pore complex (NPC) is the sole mediator of nucleocytoplasmic transport. Despite great advances in understanding its conserved core architecture, peripheral regions can exhibit considerable variation within and between species. One such structure cage-like basket. crucial roles mRNA surveillance chromatin organization, an architectural has remained elusive. Using in-cell cryo-electron tomography subtomogram analysis, we explored NPC's structural variations basket across fungi (yeast; S. cerevisiae), mammals (mouse; M. musculus), protozoa (T. gondii). integrative modeling, computed a model yeast that revealed how hub nucleoporins (Nups) ring binds to basket-forming Mlp/Tpr proteins: coiled-coil domains form struts basket, while their unstructured termini constitute distal densities, which potentially serve as docking site for preprocessing before

Язык: Английский

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Easy and accurate protein structure prediction using ColabFold

Nature Protocols, Год журнала: 2024, Номер unknown

Опубликована: Окт. 14, 2024

Язык: Английский

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