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- EMDB-56368: Escherichia coli ribosome arrested on a chimeric, 110-residue con... -

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Basic information

Entry
Database: EMDB / ID: EMD-56368
TitleEscherichia coli ribosome arrested on a chimeric, 110-residue construct featuring a firefly-luciferase truncation followed by SecM(3W)
Map dataE. coli ribosome featuring a stalled nascent chain corresponding to a truncation of firefly luciferase.
Sample
  • Complex: Escherichia coli ribosome arrested on chimaeric nascent chain composed of truncated firefly luciferase and the SecM(3W) arrest peptide, N=110
    • Protein or peptide: FLuc SecM(3W) N = 110
KeywordsFirefly luciferase / cotranslational folding / Rossmann fold / multi-domain protein / Ribosome-nascent chain / RIBOSOME
Biological speciesEscherichia coli BL21(DE3) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.29 Å
AuthorsWesterfield JM / von Heijne G
Funding support Sweden, Denmark, 3 items
OrganizationGrant numberCountry
Knut and Alice Wallenberg Foundation2022.0241 Sweden
Novo Nordisk FoundationNNF18OC0032828 Denmark
Swedish Research Council2025-03943 Sweden
CitationJournal: bioRxiv / Year: 2026
Title: Quasi-continuous cotranslational compaction and folding of a multidomain protein.
Authors: Spyridoula Mitsikosta / Justin M Westerfield / Fátima Pardo-Avila / Michael Levitt / Gunnar von Heijne / Ane Metola /
Abstract: Most proteins start to fold cotranslationally as they come off the ribosome. So far, studies of cotranslational folding have focused mainly on small, single-domain proteins. Here, we have used Force ...Most proteins start to fold cotranslationally as they come off the ribosome. So far, studies of cotranslational folding have focused mainly on small, single-domain proteins. Here, we have used Force Profile Analysis to study the cotranslational folding of Firefly luciferase, a complex 550-residue protein composed of an N-terminal domain (NTD) encompassing two split Rossmann folds (RF-1, RF-2) and a β-roll, and a flexibly attached C-terminal domain (CTD). The folding process is characterized by a quasi-continuous series of compaction/folding steps that generate intermediate-size pulling forces on the nascent chain, punctuated by a prominent high-force event that represents the folding of the RF-2 domain, and a few low-force instances that likely indicate the formation of distinct folding intermediates. Trigger Factor interacts extensively with the nascent chain when the central part of RF-2 and the early parts of the CTD are synthesized. Our analysis uncovers a cotranslational compaction/folding process that is rich in detail and not just a simple succession of a few distinct, cooperative folding transitions.
History
DepositionJan 14, 2026-
Header (metadata) releaseMar 4, 2026-
Map releaseMar 4, 2026-
UpdateMar 4, 2026-
Current statusMar 4, 2026Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_56368.png

Downloads & links

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Map

FileDownload / File: emd_56368.map.gz / Format: CCP4 / Size: 634.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationE. coli ribosome featuring a stalled nascent chain corresponding to a truncation of firefly luciferase.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 550 pix.
= 453.75 Å		0.83 Å/pix.
x 550 pix.
= 453.75 Å		0.83 Å/pix.
x 550 pix.
= 453.75 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.825 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.18
Minimum - Maximum-0.38110986 - 1.1654705
Average (Standard dev.)0.00013391046 (±0.03437901)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions550550550
Spacing550550550
CellA=B=C: 453.75 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_56368_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_56368_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Escherichia coli ribosome arrested on chimaeric nascent chain com...

EntireName: Escherichia coli ribosome arrested on chimaeric nascent chain composed of truncated firefly luciferase and the SecM(3W) arrest peptide, N=110
Components
  • Complex: Escherichia coli ribosome arrested on chimaeric nascent chain composed of truncated firefly luciferase and the SecM(3W) arrest peptide, N=110
    • Protein or peptide: FLuc SecM(3W) N = 110

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Supramolecule #1: Escherichia coli ribosome arrested on chimaeric nascent chain com...

SupramoleculeName: Escherichia coli ribosome arrested on chimaeric nascent chain composed of truncated firefly luciferase and the SecM(3W) arrest peptide, N=110
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Escherichia coli BL21(DE3) (bacteria)

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Macromolecule #1: FLuc SecM(3W) N = 110

MacromoleculeName: FLuc SecM(3W) N = 110 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli BL21(DE3) (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MHHHHHHEDA KNIKKGPAPF YPLEDGTAGE QLHKAMKRYA LVPGTIAFTD AHIEVDITYA EYFEMSVRLA EAMKRYGLNT NHRIVVCSEN SLQFFMSGSF STPVWIWWWP PIRGSPGSSD KQEGEWPTGL RLSRIGGIH

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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:
ConcentrationName
50.0 mMHEPES-KOH, pH 7.5
150.0 mMKOAc
10.0 mMMg(OAc)2
750.0 mMSucrose
1.0 g/LDDM
GridModel: Quantifoil R3.5/1 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR / Details: The grid was ultrathin carbon coated by flotation.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Number grids imaged: 1 / Number real images: 6710 / Average exposure time: 1.74 sec. / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 1.67 mm / Nominal defocus max: 2.4 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: cryoSPARC (ver. 4.3.0) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionNumber classes used: 1 / Resolution.type: BY AUTHOR / Resolution: 2.29 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.3.0) / Number images used: 373741
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.3.0)
Final 3D classificationNumber classes: 4 / Software - Name: cryoSPARC (ver. 4.3.0)
FSC plot (resolution estimation)

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