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- EMDB-72257: Cryo-EM structure of a tmRNA-rescue complex imaged in a cell lysate -

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Basic information

Entry
Database: EMDB / ID: EMD-72257
TitleCryo-EM structure of a tmRNA-rescue complex imaged in a cell lysate
Map data
Sample
  • Organelle or cellular component: Crude cell extract
Keywordsribosome rescue / bacterial ribosome / RIBOSOME
Biological speciesEscherichia coli K-12 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.56 Å
AuthorsMay MB / Lopez-Perez GS / Davis JH
Funding support United States, 2 items
OrganizationGrant numberCountry
National Science Foundation (NSF, United States)NSF-CAREER grant 2046778 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01-GM144542 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2026
Title: Capturing ribosomal structures in cellular extracts with cryoPRISM: A purification-free cryoEM approach reveals novel structural states.
Authors: Mira B May / Gabriella S Lopez-Perez / Joseph H Davis /
Abstract: Structural analyses of ribosomes by single particle cryogenic electron microscopy (cryoEM) have traditionally relied on purified or reconstituted samples, with particles often trapped in desired ...Structural analyses of ribosomes by single particle cryogenic electron microscopy (cryoEM) have traditionally relied on purified or reconstituted samples, with particles often trapped in desired states using genetic, pharmacological, or biochemical perturbations. While informative, such in vitro methods often fail to capture the full diversity of structural states and associated protein factors present in cells. In contrast, in situ cryoelectron tomography preserves cellular context but is limited by low throughput and modest resolution. Here, we present cryoPRISM (purification-free ribosome imaging from subcellular mixtures), a rapid ex vivo workflow encompassing cell lysis, vitrification, and image analysis methods for high-resolution analyses of ribosomal structures directly from cell lysates. Applying cryoPRISM in , we resolved more than 20 distinct ribosomal states spanning assembly, translation initiation, elongation, trans-translation, and quiescence, including a novel configuration of EF-G bound to idle ribosomes with the ribosome hibernation factor ribosome-associated inhibitor A. Given its speed, accessibility, and ability to preserve native interactions and structural heterogeneity, we anticipate that cryoPRISM will be broadly applicable for uncovering ribosomal biology across diverse organisms and conditions.
History
DepositionAug 21, 2025-
Header (metadata) releaseMar 11, 2026-
Map releaseMar 11, 2026-
UpdateMar 11, 2026-
Current statusMar 11, 2026Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_72257.png

Downloads & links

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Map

FileDownload / File: emd_72257.map.gz / Format: CCP4 / Size: 325 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

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AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 440 pix.
= 466.4 Å		1.06 Å/pix.
x 440 pix.
= 466.4 Å		1.06 Å/pix.
x 440 pix.
= 466.4 Å

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.06 Å
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Contour LevelBy AUTHOR: 0.219
Minimum - Maximum-0.3658447 - 1.0568367
Average (Standard dev.)0.0030042087 (±0.06187898)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions440440440
Spacing440440440
CellA=B=C: 466.39996 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_72257_msk_1.map
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Half map: #1

Fileemd_72257_half_map_1.map
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Half map: #2

Fileemd_72257_half_map_2.map
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Sample components

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Entire : Crude cell extract

EntireName: Crude cell extract
Components
  • Organelle or cellular component: Crude cell extract

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Supramolecule #1: Crude cell extract

SupramoleculeName: Crude cell extract / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Details: Frozen cell pellets were lysed under cryogenic conditions using a cryomill. Frozen lysate was resuspended and clarified via centrifugation at 21,000g. DNase I (33 U/mL) was added and crude ...Details: Frozen cell pellets were lysed under cryogenic conditions using a cryomill. Frozen lysate was resuspended and clarified via centrifugation at 21,000g. DNase I (33 U/mL) was added and crude cell extract was plunge-frozen.
Source (natural)Organism: Escherichia coli K-12 (bacteria) / Strain: NCM3722

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
Component:
ConcentrationFormulaName
28.0 mMC4H13Cl2NOTris(hydroxymethyl)aminomethane hydrochloride pH 7.5
90.0 mMNH4ClAmmonium chloride
0.45 mMC10H16N2O8Ethylenediaminetetraacetic Acid
5.4 mMC2H6OS2-Mercaptoethanol
0.5 mMCaCl2Calcium chloride

Details: 33 U/mL DNase I (New England Biolabs #M0303L) was added in addition.
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 200 / Support film - #0 - Film type ID: 1 / Support film - #0 - Material: CARBON / Support film - #0 - topology: HOLEY / Support film - #1 - Film type ID: 2 / Support film - #1 - Material: GRAPHENE / Support film - #1 - topology: CONTINUOUS
Details: Grid was coated with monolayer graphene following (Grassetti et al, JOVE 2023) and treated with UV/ozone for 10 minutes using a Bioforce PC440 UV/ozone cleaner
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 45.1838 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.03 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1503396
CTF correctionSoftware - Name: Warp (ver. 1.0.9) / Type: NONE
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.56 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.5.3) / Number images used: 3398
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.5.3)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.5.3)
Final 3D classificationSoftware: (Name: cryoDRGN (ver. 2.2), cryoSPARC (ver. 4.5.3))
FSC plot (resolution estimation)

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