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- EMDB-6483: Mechanisms of Ribosome Stalling by SecM at Multiple Elongation Steps -

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Entry
Database: EMDB / ID: EMD-6483
TitleMechanisms of Ribosome Stalling by SecM at Multiple Elongation Steps
Map dataCryo EM reconstruction of SecM-Gly-RNC (ribosomal-nascent-peptide-complex) with 50S-tRNA mask
Sample
  • Sample: SecM-Gly-RNC(ribosome-nascent-peptide-complex) complex with 50S mask
  • Complex: SecM-Gly-RNC with 50S mask
Keywordselectron microscopy / single particle analysis / ribosome stalling
Function / homology
Function and homology information


outer membrane protein complex / monoatomic ion transmembrane transporter activity / detection of virus / outer membrane / porin activity / pore complex / ornithine decarboxylase inhibitor activity / transcription antitermination factor activity, RNA binding / misfolded RNA binding / Group I intron splicing ...outer membrane protein complex / monoatomic ion transmembrane transporter activity / detection of virus / outer membrane / porin activity / pore complex / ornithine decarboxylase inhibitor activity / transcription antitermination factor activity, RNA binding / misfolded RNA binding / Group I intron splicing / RNA folding / transcriptional attenuation / endoribonuclease inhibitor activity / RNA-binding transcription regulator activity / positive regulation of ribosome biogenesis / negative regulation of cytoplasmic translation / four-way junction DNA binding / DnaA-L2 complex / monoatomic ion transport / translation repressor activity / negative regulation of DNA-templated DNA replication initiation / negative regulation of translational initiation / regulation of mRNA stability / mRNA regulatory element binding translation repressor activity / ribosome assembly / assembly of large subunit precursor of preribosome / positive regulation of RNA splicing / transcription elongation factor complex / cytosolic ribosome assembly / regulation of DNA-templated transcription elongation / DNA endonuclease activity / response to reactive oxygen species / transcription antitermination / regulation of cell growth / cell outer membrane / DNA-templated transcription termination / maintenance of translational fidelity / response to radiation / mRNA 5'-UTR binding / ribosomal small subunit biogenesis / small ribosomal subunit rRNA binding / large ribosomal subunit / ribosome biogenesis / ribosome binding / regulation of translation / ribosomal small subunit assembly / outer membrane-bounded periplasmic space / small ribosomal subunit / 5S rRNA binding / large ribosomal subunit rRNA binding / transferase activity / cytosolic small ribosomal subunit / ribosomal large subunit assembly / cytoplasmic translation / cytosolic large ribosomal subunit / tRNA binding / molecular adaptor activity / negative regulation of translation / rRNA binding / ribosome / structural constituent of ribosome / symbiont entry into host cell / ribonucleoprotein complex / translation / response to antibiotic / negative regulation of DNA-templated transcription / mRNA binding / DNA damage response / DNA binding / RNA binding / zinc ion binding / identical protein binding / membrane / cytosol / cytoplasm
Similarity search - Function
Outer membrane protein OmpA-like, transmembrane domain / Outer membrane protein, OmpA / OmpA-like transmembrane domain / Outer membrane protein, OmpA-like, conserved site / OmpA-like domain. / Outer membrane protein, bacterial / : / OmpA-like domain superfamily / OmpA family / OmpA-like domain ...Outer membrane protein OmpA-like, transmembrane domain / Outer membrane protein, OmpA / OmpA-like transmembrane domain / Outer membrane protein, OmpA-like, conserved site / OmpA-like domain. / Outer membrane protein, bacterial / : / OmpA-like domain superfamily / OmpA family / OmpA-like domain / OmpA-like domain profile. / Outer membrane protein/outer membrane enzyme PagP, beta-barrel / Ribosomal protein S21, conserved site / Ribosomal protein S21 signature. / Ribosomal protein L25, short-form / Ribosomal protein S14, bacterial/plastid / Ribosomal protein S21 superfamily / Ribosomal protein S21 / Ribosomal protein S16, conserved site / Ribosomal protein S16 signature. / Ribosomal protein S21 / : / Ribosomal protein L21, conserved site / Ribosomal protein L21 signature. / Ribosomal protein L16 signature 1. / : / Ribosomal protein L6, conserved site / Ribosomal protein L6 signature 1. / Ribosomal protein L16, conserved site / Ribosomal protein L16 signature 2. / Ribosomal protein L9 signature. / Ribosomal protein L9, bacteria/chloroplast / Ribosomal protein L9, C-terminal / Ribosomal protein L9, C-terminal domain / Ribosomal protein L9, C-terminal domain superfamily / Ribosomal protein L17 signature. / Ribosomal L25p family / Ribosomal protein L25 / Ribosomal protein L36 signature. / Ribosomal protein L28/L24 superfamily / Ribosomal protein L25/Gln-tRNA synthetase, N-terminal / Ribosomal protein L25/Gln-tRNA synthetase, anti-codon-binding domain superfamily / Ribosomal protein L32p, bacterial type / Ribosomal protein L9, N-terminal domain superfamily / Ribosomal protein L9 / Ribosomal protein L9, N-terminal / Ribosomal protein L9, N-terminal domain / : / : / Ribosomal protein L28 / Ribosomal protein L35, conserved site / Ribosomal protein L35 signature. / Ribosomal protein L33, conserved site / Ribosomal protein L33 signature. / Ribosomal protein L35, non-mitochondrial / Ribosomal protein L5, bacterial-type / Ribosomal protein L18, bacterial-type / Ribosomal protein L6, bacterial-type / Ribosomal protein L9/RNase H1, N-terminal / Ribosomal protein L19, conserved site / Ribosomal protein L19 signature. / Ribosomal protein S3, bacterial-type / Ribosomal protein S6, conserved site / Ribosomal protein S6 signature. / Ribosomal protein S19, bacterial-type / Ribosomal protein S13, bacterial-type / Ribosomal protein L36 / Ribosomal protein L36 superfamily / Ribosomal protein L36 / Ribosomal protein S7, bacterial/organellar-type / Ribosomal protein S11, bacterial-type / Ribosomal protein S20 / Ribosomal protein S20 superfamily / Ribosomal protein S20 / Ribosomal protein L20 signature. / Ribosomal protein S9, bacterial/plastid / Ribosomal protein L27, conserved site / Ribosomal protein L27 signature. / Ribosomal protein S4, bacterial-type / 30S ribosomal protein S17 / Ribosomal protein S5, bacterial-type / Ribosomal protein L14P, bacterial-type / Ribosomal protein S6, plastid/chloroplast / Ribosomal protein L22, bacterial/chloroplast-type / Ribosomal protein L2, bacterial/organellar-type / Ribosomal protein S2, bacteria/mitochondria/plastid / Ribosomal protein L35 / Ribosomal protein L35 superfamily / Ribosomal protein L35 / Ribosomal protein L34, conserved site / Ribosomal L28 family / Ribosomal protein L34 signature. / Ribosomal protein L33 / Ribosomal protein L33 / Ribosomal protein L28/L24 / Ribosomal protein L18 / Ribosomal L18 of archaea, bacteria, mitoch. and chloroplast / Ribosomal protein L33 superfamily / Ribosomal protein L30, bacterial-type / Ribosomal protein S18, conserved site
Similarity search - Domain/homology
Large ribosomal subunit protein bL25 / Small ribosomal subunit protein bS6 / Small ribosomal subunit protein uS7 / Large ribosomal subunit protein uL15 / Large ribosomal subunit protein bL19 / Large ribosomal subunit protein bL20 / Large ribosomal subunit protein bL27 / Large ribosomal subunit protein bL28 / Large ribosomal subunit protein uL29 / Large ribosomal subunit protein bL32 ...Large ribosomal subunit protein bL25 / Small ribosomal subunit protein bS6 / Small ribosomal subunit protein uS7 / Large ribosomal subunit protein uL15 / Large ribosomal subunit protein bL19 / Large ribosomal subunit protein bL20 / Large ribosomal subunit protein bL27 / Large ribosomal subunit protein bL28 / Large ribosomal subunit protein uL29 / Large ribosomal subunit protein bL32 / Large ribosomal subunit protein bL33 / Large ribosomal subunit protein bL34 / Large ribosomal subunit protein bL35 / Large ribosomal subunit protein bL36A / Large ribosomal subunit protein bL9 / Small ribosomal subunit protein uS10 / Small ribosomal subunit protein uS11 / Small ribosomal subunit protein uS12 / Small ribosomal subunit protein uS13 / Small ribosomal subunit protein bS16 / Small ribosomal subunit protein bS18 / Small ribosomal subunit protein uS19 / Small ribosomal subunit protein bS20 / Small ribosomal subunit protein uS2 / Small ribosomal subunit protein uS3 / Small ribosomal subunit protein uS4 / Small ribosomal subunit protein uS5 / Small ribosomal subunit protein uS8 / Small ribosomal subunit protein uS9 / Outer membrane protein A / Large ribosomal subunit protein uL13 / Large ribosomal subunit protein uL14 / Large ribosomal subunit protein uL16 / Large ribosomal subunit protein uL23 / Small ribosomal subunit protein uS15 / Large ribosomal subunit protein bL17 / Large ribosomal subunit protein bL21 / Large ribosomal subunit protein uL30 / Large ribosomal subunit protein uL6 / Small ribosomal subunit protein uS14 / Small ribosomal subunit protein uS17 / Large ribosomal subunit protein uL18 / Large ribosomal subunit protein uL2 / Large ribosomal subunit protein uL3 / Large ribosomal subunit protein uL24 / Large ribosomal subunit protein uL4 / Large ribosomal subunit protein uL22 / Large ribosomal subunit protein uL5 / Small ribosomal subunit protein bS21 / Large ribosomal subunit protein bL25
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.64 Å
AuthorsZhang J / Pan XJ / Yan KG / Sun S / Gao N / Sui SF
CitationJournal: Elife / Year: 2015
Title: Mechanisms of ribosome stalling by SecM at multiple elongation steps.
Authors: Jun Zhang / Xijiang Pan / Kaige Yan / Shan Sun / Ning Gao / Sen-Fang Sui /
Abstract: Regulation of translating ribosomes is a major component of gene expression control network. In Escherichia coli, ribosome stalling by the C-terminal arrest sequence of SecM regulates the SecA- ...Regulation of translating ribosomes is a major component of gene expression control network. In Escherichia coli, ribosome stalling by the C-terminal arrest sequence of SecM regulates the SecA-dependent secretion pathway. Previous studies reported many residues of SecM peptide and ribosome exit tunnel are critical for stalling. However, the underlying molecular mechanism is still not clear at the atomic level. Here, we present two cryo-EM structures of the SecM-stalled ribosomes at 3.3-3.7 Å resolution, which reveal two different stalling mechanisms at distinct elongation steps of the translation cycle: one is due to the inactivation of ribosomal peptidyl-transferase center which inhibits peptide bond formation with the incoming prolyl-tRNA; the other is the prolonged residence of the peptidyl-RNA at the hybrid A/P site which inhibits the full-scale tRNA translocation. These results demonstrate an elegant control of translation cycle by regulatory peptides through a continuous, dynamic reshaping of the functional center of the ribosome.
History
DepositionOct 15, 2015-
Header (metadata) releaseFeb 24, 2016-
Map releaseFeb 24, 2016-
UpdateFeb 24, 2016-
Current statusFeb 24, 2016Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3jbu
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_6483.tif

Downloads & links

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Map

FileDownload / File: emd_6483.map.gz / Format: CCP4 / Size: 122.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo EM reconstruction of SecM-Gly-RNC (ribosomal-nascent-peptide-complex) with 50S-tRNA mask
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.32 Å/pix.
x 320 pix.
= 422.4 Å		1.32 Å/pix.
x 320 pix.
= 422.4 Å		1.32 Å/pix.
x 320 pix.
= 422.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.32 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.03 / Movie #1: 0.03
Minimum - Maximum-0.09889516 - 0.18002944
Average (Standard dev.)0.00023492 (±0.00902666)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 422.40002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.321.321.32
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z422.400422.400422.400
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.0990.1800.000

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Supplemental data

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Segmentation: This mask represent the large subunit with P/P-tRNA

AnnotationThis mask represent the large subunit with P/P-tRNA
Fileemd_6483_msk_1.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 : SecM-Gly-RNC(ribosome-nascent-peptide-complex) complex with 50S mask

EntireName: SecM-Gly-RNC(ribosome-nascent-peptide-complex) complex with 50S mask
Components
  • Sample: SecM-Gly-RNC(ribosome-nascent-peptide-complex) complex with 50S mask
  • Complex: SecM-Gly-RNC with 50S mask

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Supramolecule #1000: SecM-Gly-RNC(ribosome-nascent-peptide-complex) complex with 50S mask

SupramoleculeName: SecM-Gly-RNC(ribosome-nascent-peptide-complex) complex with 50S mask
type: sample / ID: 1000 / Number unique components: 1
Molecular weightTheoretical: 2.3 MDa

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Supramolecule #1: SecM-Gly-RNC with 50S mask

SupramoleculeName: SecM-Gly-RNC with 50S mask / type: complex / ID: 1 / Name.synonym: SecM-Gly-stalled RNC with 50S mask / Recombinant expression: Yes / Ribosome-details: ribosome-prokaryote: ALL
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: Escherichia coli MG1655 / Recombinant plasmid: pET21
Molecular weightTheoretical: 2.3 MDa

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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
Details: 20mM HEPES, 50mM KOAc, 6mM Mg[OAc]2, 1mMDTT, 500ug/ml chloramphenicol, 0.05% Nikkol, 0.5% pill/ml Complete EDTA-free Protease inhibitor cocktail, 0.1U/ml RNasin and 125mM sucrose
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV / Method: Blot for 1.5 seconds before plunging

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Electron microscopy #1

Microscopy ID1
MicroscopeFEI TITAN KRIOS
DateMay 8, 2014
Image recordingCategory: CCD / Film or detector model: GATAN K2 (4k x 4k) / Digitization - Sampling interval: 4 µm / Number real images: 3908 / Average electron dose: 16 e/Å2
Details: Every image is the average of 14 frames recorded by the direct electron detector
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 37878 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 22500
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Electron microscopy #2

Microscopy ID2
MicroscopeFEI TITAN KRIOS
DateJun 16, 2014
Image recordingCategory: CCD / Film or detector model: GATAN K2 (4k x 4k) / Digitization - Sampling interval: 4 µm / Number real images: 3908 / Average electron dose: 16 e/Å2
Details: Every image is the average of 14 frames recorded by the direct electron detector
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 37878 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 22500
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Electron microscopy #3

Microscopy ID3
MicroscopeFEI TITAN KRIOS
DateAug 30, 2014
Image recordingCategory: CCD / Film or detector model: GATAN K2 (4k x 4k) / Digitization - Sampling interval: 4 µm / Number real images: 3908 / Average electron dose: 16 e/Å2
Details: Every image is the average of 14 frames recorded by the direct electron detector
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 37878 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 22500
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: CTFFIND
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 3.64 Å / Resolution method: OTHER / Software - Name: RELION
Details: The B-factor of final map was performed automated b-factor by using the postprocess step in relion
Number images used: 60354
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

4v7t


Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B
SoftwareName: Chimera, EMFit, Coot
RefinementSpace: RECIPROCAL / Protocol: FLEXIBLE FIT
Output model

PDB-3jbu:
Mechanisms of Ribosome Stalling by SecM at Multiple Elongation Steps

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