pp. 2335·30. decembar 2023.· Issue No. 1
Cryo-electron tomography in native cells: atomic resolution in context
author-image
Juliana Barbosa CardosoORCID 0009-0003-0316-7930
DOI: https://doi.org/10.65932/CAR-2023-1-2Creative Commons BY-NC-ND 4.0 CC BY-NC-ND 4.0
Preuzmi PDF
Tip: PDFVeličina: 0.34 MB
Preuzmi JATS XML
Tip: XMLVeličina: 3.44 KB
Cryo-electron tomography in native cells: atomic resolution in context
Between 2016 and 2022, cryo-electron tomography (cryo-ET) underwent a generational transition from a method that produced informative but resolution-limited images of vitrified cells to one that produced, on a growing subset of targets, atomic-resolution structures inside intact bacterial and eukaryotic cells without recourse to purification. The transition was the consequence of four converging technical developments: routine cryo-focused-ion-beam (cryoFIB) milling that produced electron-transparent lamellae of 100-300 nm thickness from vitrified cells, the Volta phase plate that improved low-defocus image contrast, direct-detection cameras whose dose-fractionated frames preserved high-resolution information, and a suite of imageprocessing developments (Warp, M, emClarity, AreTomo, SPHIRE-crYOLO) that enabled tiltseries alignment, subtomogram averaging and multi-particle refinement at sub-nanometer resolutions. The cumulative empirical demonstration that ribosomes inside intact bacterial cells can be resolved to 3.5 Å — the Tegunov-Xue-Cramer-Mahamid 2021 multi-particle-M result — established that the field's long-standing goal of “atomic resolution in context” was achievable, not merely aspirational. The accelerating pace of in situ structural studies has, however, made cross-study comparison increasingly difficult: the relevant figure of merit is not resolution alone, but resolution-in-context, and no single metric currently captures the tradeoff between achievable resolution, preservation of native cellular context, and spatial localisation specificity. In this article I review the technical landscape of in situ cryo-ET from 2016 to 2022 and propose, as the original contribution, the In Situ Resolution-in-Context Index (IRiCI) — a single normalised composite metric, bounded on [0,1], that integrates five performance dimensions (achieved resolution, native-context preservation, spatial localisation specificity, throughput per tomogram, and intercell reproducibility) and returns a quantitative ranking of in situ cryo-ET studies on a metric explicitly designed to reward atomic resolution and intact cellular context simultaneously. Applied to ten landmark studies from the 2016-2022 window, IRiCI returns a ranking that identifies the Tegunov et al. (2021) in-cell ribosome-antibiotic structure and the Allegretti et al. (2020) in-cell nuclear pore complex study as the joint leaders, with the Mahamid et al. (2016) nuclear-periphery work as the foundational precursor.

Between 2016 and 2022, cryo-electron tomography (cryo-ET) underwent a generational transition from a method that produced informative but resolution-limited images of vitrified cells to one that produced, on a growing subset of targets, atomic-resolution structures inside intact bacterial and eukaryotic cells without recourse to purification. The transition was the consequence of four converging technical developments: routine cryo-focused-ion-beam (cryoFIB) milling that produced electron-transparent lamellae of 100-300 nm thickness from vitrified cells, the Volta phase plate that improved low-defocus image contrast, direct-detection cameras whose dose-fractionated frames preserved high-resolution information, and a suite of imageprocessing developments (Warp, M, emClarity, AreTomo, SPHIRE-crYOLO) that enabled tiltseries alignment, subtomogram averaging and multi-particle refinement at sub-nanometer resolutions. The cumulative empirical demonstration that ribosomes inside intact bacterial cells can be resolved to 3.5 Å — the Tegunov-Xue-Cramer-Mahamid 2021 multi-particle-M result — established that the field's long-standing goal of “atomic resolution in context” was achievable, not merely aspirational. The accelerating pace of in situ structural studies has, however, made cross-study comparison increasingly difficult: the relevant figure of merit is not resolution alone, but resolution-in-context, and no single metric currently captures the tradeoff between achievable resolution, preservation of native cellular context, and spatial localisation specificity. In this article I review the technical landscape of in situ cryo-ET from 2016 to 2022 and propose, as the original contribution, the In Situ Resolution-in-Context Index (IRiCI) — a single normalised composite metric, bounded on [0,1], that integrates five performance dimensions (achieved resolution, native-context preservation, spatial localisation specificity, throughput per tomogram, and intercell reproducibility) and returns a quantitative ranking of in situ cryo-ET studies on a metric explicitly designed to reward atomic resolution and intact cellular context simultaneously. Applied to ten landmark studies from the 2016-2022 window, IRiCI returns a ranking that identifies the Tegunov et al. (2021) in-cell ribosome-antibiotic structure and the Allegretti et al. (2020) in-cell nuclear pore complex study as the joint leaders, with the Mahamid et al. (2016) nuclear-periphery work as the foundational precursor.

Objavljeno30. decembar 2023.
Stranice2335
AutoriJuliana Barbosa Cardoso