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- PDB-9pyc: Cryo-EM structure of EF-G and RaiA simultaneously bound to an E. ... -

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Basic information

Entry
Database: PDB / ID: 9pyc
TitleCryo-EM structure of EF-G and RaiA simultaneously bound to an E. coli ribosome imaged in a cell lysate
Components
  • Elongation factor G
  • Ribosome-associated inhibitor A
KeywordsRIBOSOMAL PROTEIN / hibernation / elongation factor / bacterial ribosome
Function / homology
Function and homology information


dormancy process / negative regulation of translational elongation / ribosome disassembly / guanosine tetraphosphate binding / ribosomal small subunit binding / translational elongation / translation elongation factor activity / negative regulation of translational initiation / response to cold / cytosolic small ribosomal subunit ...dormancy process / negative regulation of translational elongation / ribosome disassembly / guanosine tetraphosphate binding / ribosomal small subunit binding / translational elongation / translation elongation factor activity / negative regulation of translational initiation / response to cold / cytosolic small ribosomal subunit / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / rRNA binding / GTPase activity / GTP binding / cytoplasm / cytosol
Similarity search - Function
: / Ribosome hibernation promoting factor/RaiA / Ribosome hibernation promotion factor-like / Sigma 54 modulation protein / S30EA ribosomal protein / Translation elongation factor EFG/EF2 / : / Elongation factor G, domain III / EFG, domain V / Elongation Factor G, domain II / Elongation Factor G, domain III ...: / Ribosome hibernation promoting factor/RaiA / Ribosome hibernation promotion factor-like / Sigma 54 modulation protein / S30EA ribosomal protein / Translation elongation factor EFG/EF2 / : / Elongation factor G, domain III / EFG, domain V / Elongation Factor G, domain II / Elongation Factor G, domain III / Translation elongation factor EFG/EF2, domain IV / Elongation factor G, domain IV / Elongation factor G, domain IV / Elongation factor G C-terminus / Elongation factor EFG, domain V-like / Elongation factor G C-terminus / EF-G domain III/V-like / Tr-type G domain, conserved site / Translational (tr)-type guanine nucleotide-binding (G) domain signature. / Translation elongation factor EFTu-like, domain 2 / Elongation factor Tu domain 2 / Translational (tr)-type GTP-binding domain / Elongation factor Tu GTP binding domain / Translational (tr)-type guanine nucleotide-binding (G) domain profile. / Small GTP-binding protein domain / Translation protein, beta-barrel domain superfamily / Ribosomal protein S5 domain 2-type fold, subgroup / Ribosomal protein S5 domain 2-type fold / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Elongation factor G / Ribosome-associated inhibitor A
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsMay, M.B. / Davis, J.H.
Funding support United States, 2items
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 7, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 18, 2026Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Elongation factor G
F: Ribosome-associated inhibitor A


Theoretical massNumber of molelcules
Total (without water)90,4802
Polymers90,4802
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Elongation factor G / EF-G


Mass: 77676.227 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / Strain: NCM3722
References: UniProt: P0A6M8, Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement
#2: Protein Ribosome-associated inhibitor A / Protein Y / pY / Ribosome associated factor Y / Spot Y


Mass: 12803.583 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / Strain: NCM3722 / References: UniProt: P0AD49
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Crude cell extract / Type: ORGANELLE OR CELLULAR COMPONENT
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.
Entity ID: all / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Escherichia coli K-12 (bacteria) / Strain: NCM3722
Buffer solutionpH: 7.5
Details: 33 U/mL DNase I (New England Biolabs #M0303L) was added in addition.
Buffer component
IDConc.NameFormulaBuffer-ID
128 mMTris(hydroxymethyl)aminomethane hydrochloride pH 7.5C4H13Cl2NO1
290 mMAmmonium chlorideNH4Cl1
30.45 mMEthylenediaminetetraacetic AcidC10H16N2O81
45.4 mM2-MercaptoethanolC2H6OS1
50.5 mMCalcium chlorideCaCl21
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 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
Grid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 283 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 30 nm
Image recordingElectron dose: 45.1838 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1Warp1.0.9particle selection
4Warp1.0.9CTF correction
9cryoSPARC4.5.3initial Euler assignment
10cryoSPARC4.5.3final Euler assignment
11cryoDRGN2.2classification
12cryoSPARC4.5.3classification
13cryoSPARC4.5.33D reconstruction
14PHENIX1.21.2-5419model refinement
CTF correctionType: NONE
Particle selectionNum. of particles selected: 1503396
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 21018 / Symmetry type: POINT

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