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- EMDB-6485: Mechanisms of Ribosome Stalling by SecM at Multiple Elongation Steps -

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Entry
Database: EMDB / ID: 6485
TitleMechanisms of Ribosome Stalling by SecM at Multiple Elongation Steps
Map dataCryo EM reconstruction of SecM-Pro-RNC ( ribosomal-nascent-peptide-complex) without 50S-tRNA mask
SampleSecM-Pro-RNC(ribosome-nascent-peptide-complex) complex without mask:
ribosome-prokaryote
Keywordselectron microscopy / single particle analysis / ribosome stalling
Function / homologyRibosomal protein L35 / Ribosomal protein L21-like / Ribosomal protein L36 superfamily / Ribosomal protein L20, C-terminal / L28p-like / Ribosomal protein L5, N-terminal / Ribosomal protein L5, C-terminal / Ribosomal protein L15 / Ribosomal protein L25 / Ribosomal protein S10 domain ...Ribosomal protein L35 / Ribosomal protein L21-like / Ribosomal protein L36 superfamily / Ribosomal protein L20, C-terminal / L28p-like / Ribosomal protein L5, N-terminal / Ribosomal protein L5, C-terminal / Ribosomal protein L15 / Ribosomal protein L25 / Ribosomal protein S10 domain / Ribosomal protein S8 superfamily / 30s ribosomal protein S13, C-terminal / Ribosomal protein L28/L24 / Ribosomal protein S16 domain superfamily / Ribosomal protein S7 domain / Ribosomal protein S2, flavodoxin-like domain superfamily / Ribosomal protein S19, superfamily / Ribosomal protein L4 domain superfamily / Ribosomal protein L13, conserved site / Ribosomal protein S6 superfamily / Ribosomal protein L29/L35 superfamily / Ribosomal protein S9, bacterial/plastid / Ribosomal protein S10 domain superfamily / Ribosomal protein S11 superfamily / Ribosomal protein L9, N-terminal domain superfamily / Ribosomal protein L10e/L16 superfamily / Ribosomal protein L30, ferredoxin-like fold domain superfamily / Ribosomal protein L13 superfamily / Ribosomal protein S18 superfamily / Ribosomal protein L14 superfamily / Ribosomal protein S7 domain superfamily / L21-like superfamily / Ribosomal protein L9, C-terminal domain superfamily / Ribosomal protein L6, alpha-beta domain superfamily / OmpA-like domain superfamily / Ribosomal protein S20 superfamily / Ribosomal protein S3, C-terminal domain superfamily / Ribosomal protein L22/L17 superfamily / Ribosomal protein L17 superfamily / Ribosomal L18e/L15P superfamily / Ribosomal protein S14, bacterial/plastid / Ribosomal protein L5 domain superfamily / Ribosomal protein L28/L24 superfamily / Ribosomal protein S3, conserved site / Ribosomal protein S11, bacterial-type / Ribosomal protein S13, bacterial-type / Ribosomal protein S17, conserved site / Ribosomal protein L14P, conserved site / Ribosomal protein L3, bacterial/organelle-type / Ribosomal protein L3, conserved site / Ribosomal protein L6, bacterial-type / Ribosomal protein S21, conserved site / Ribosomal protein L6, alpha-beta domain / Ribosomal protein S18, conserved site / Ribosomal protein S14, conserved site / Ribosomal protein S13, conserved site / Ribosomal protein S10, conserved site / Ribosomal protein L35, conserved site / Ribosomal protein L33, conserved site / Ribosomal protein L27, conserved site / Ribosomal protein L22/L17, conserved site / 30S ribosomal protein S17 / Ribosomal protein L25, short-form / Ribosomal protein S4/S9 / Ribosomal protein S6, conserved site / Ribosomal protein L2, conserved site / Ribosomal protein L2, C-terminal / Ribosomal Proteins L2, RNA binding domain / Ribosomal protein L18e/L15P / Ribosomal protein L34, conserved site / Ribosomal protein S19 conserved site / Ribosomal protein L5, bacterial-type / Ribosomal protein L5, conserved site / Ribosomal protein S6, plastid/chloroplast / Ribosomal protein L25/Gln-tRNA synthetase, N-terminal / Ribosomal protein L16, conserved site / Ribosomal protein S7, conserved site / Ribosomal protein L9, bacteria/chloroplast / Ribosomal protein S16, conserved site / Ribosomal protein S9, conserved site / Ribosomal protein S5 domain 2-type fold / Ribosomal protein L9, N-terminal / Ribosomal protein L9, C-terminal / RNA-binding S4 domain superfamily / Ribosomal protein L35 superfamily / Ribosomal protein L16 signature 1. / Ribosomal protein S20 / Ribosomal protein L14 signature. / Ribosomal proteins 50S L24/mitochondrial 39S L24 / KH domain / Ribosomal protein L9, C-terminal domain / Ribosomal Proteins L2, C-terminal domain / Ribosomal protein S5, C-terminal domain / Ribosomal L32p protein family / Ribosomal protein L35 / Ribosomal protein S7 signature. / S4 domain / OmpA-like transmembrane domain / Ribosomal L25p family / Ribosomal protein L9, N-terminal domain
Function and homology information
SourceEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / 3.32 Å resolution
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
DateDeposition: Oct 15, 2015 / Header (metadata) release: Feb 24, 2016 / Map release: Feb 24, 2016 / Last update: Feb 24, 2016

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.03
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.03
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_6485.map.gz (map file in CCP4 format, 128001 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
320 pix
1.32 Å/pix.
= 422.4 Å
320 pix
1.32 Å/pix.
= 422.4 Å
320 pix
1.32 Å/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
Contour Level:0.03 (by author), 0.03 (movie #1):
Minimum - Maximum-0.08421799 - 0.16016082
Average (Standard dev.)0.00010774 (0.00801697)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions320320320
Origin000
Limit319319319
Spacing320320320
CellA=B=C: 422.40002 Å
α=β=γ: 90.0 deg.

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
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.0840.1600.000

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Supplemental data

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Sample components

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Entire SecM-Pro-RNC(ribosome-nascent-peptide-complex) complex without mask

EntireName: SecM-Pro-RNC(ribosome-nascent-peptide-complex) complex without mask
Number of components: 1
MassTheoretical: 2.3 MDa

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Component #1: ribosome-prokaryote, SecM-Pro-RNC without mask

Ribosome-prokaryoteName: SecM-Pro-RNC without mask / a.k.a: SecM-Pro-stalled RNC without mask / Prokaryote: ALL / Recombinant expression: Yes
MassTheoretical: 2.3 MDa
SourceSpecies: Escherichia coli (E. coli)
Source (engineered)Expression System: Escherichia coli (E. coli) / Vector: pET21 / Strain: Escherichia coli MG1655

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionBuffer solution: 20mM HEPES, 50mM KOAc, 6mM Mg[OAc]2, 1mM DTT, 500 ug/ml chloramphenicol,0.05% Nikkol,0.5% pill/ml Complete EDTA-free Protease inhibitor cocktail,0.1 U/ml RNasin and 125mM sucrose
pH: 7
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 277.15 K / Humidity: 100 % / Method: Blot for 1.5 seconds before plunging

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Electron microscopy imaging #1

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS / Date: May 8, 2014
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 16 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 22500.0 X (nominal), 37878 X (calibrated) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 1000 - 3500 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 (4k x 4k)

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Electron microscopy imaging #2

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS / Date: Jun 16, 2014
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 16 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 22500.0 X (nominal), 37878 X (calibrated) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 1000 - 3500 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 (4k x 4k)

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Electron microscopy imaging #3

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS / Date: Aug 30, 2014
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 16 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 22500.0 X (nominal), 37878 X (calibrated) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 1000 - 3500 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 3908 / Sampling size: 4 microns
Details: Every image is the average of 14 frames recorded by the direct electron detector

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 60354
3D reconstructionAlgorithm: reference-based reconstruction / Software: RELION / CTF correction: CTFFIND / Resolution: 3.32 Å / Resolution method: FSC 0.143, gold-standard
FSC plot
(resolution estimation)

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Atomic model buiding

Modeling #1Software: Chimera, EMFit, Coot / Refinement protocol: flexible / Refinement space: RECIPROCAL
Input PDB model: 4V7T
Chain ID: A, B

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