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- EMDB-6306: Cryo-EM structure of the Bacillus subtilis MifM-stalled ribosome ... -

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Basic information

Entry
Database: EMDB / ID: EMD-6306
TitleCryo-EM structure of the Bacillus subtilis MifM-stalled ribosome complex
Map dataCryo-EM structure of the B. subtilis MifM-stalled ribosome complex
Sample
  • Sample: B. subtilis MifM-stalled ribosome complex
  • Complex: 70S ribosomeRibosome
  • Protein or peptide: Membrane protein insertion and folding monitorBiological membrane
  • RNA: tRNA-Asp
KeywordsBacillus subtilis / ribosome / cryo-EM / atomic model / stalling / translation arrest / MifM / L22
Function / homology
Function and homology information


positive regulation of rRNA processing / nucleoid / ribosomal small subunit biogenesis / small ribosomal subunit rRNA binding / rRNA processing / ribosomal small subunit assembly / cytosolic small ribosomal subunit / large ribosomal subunit rRNA binding / large ribosomal subunit / small ribosomal subunit ...positive regulation of rRNA processing / nucleoid / ribosomal small subunit biogenesis / small ribosomal subunit rRNA binding / rRNA processing / ribosomal small subunit assembly / cytosolic small ribosomal subunit / large ribosomal subunit rRNA binding / large ribosomal subunit / small ribosomal subunit / 5S rRNA binding / cytoplasmic translation / cytosolic large ribosomal subunit / transferase activity / negative regulation of translation / tRNA binding / membrane => GO:0016020 / rRNA binding / ribosome / structural constituent of ribosome / translation / ribonucleoprotein complex / response to antibiotic / mRNA binding / DNA binding / RNA binding / zinc ion binding / metal ion binding / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Ribosomal protein L10, eubacterial, conserved site / Ribosomal protein L10 signature. / Ribosomal protein L10 / : / Ribosomal protein S14, type Z / Ribosomal protein L11, bacterial-type / Ribosomal protein L31 type A / Ribosomal protein S16, conserved site / Ribosomal protein S16 signature. / Ribosomal protein L31 signature. ...Ribosomal protein L10, eubacterial, conserved site / Ribosomal protein L10 signature. / Ribosomal protein L10 / : / Ribosomal protein S14, type Z / Ribosomal protein L11, bacterial-type / Ribosomal protein L31 type A / Ribosomal protein S16, conserved site / Ribosomal protein S16 signature. / Ribosomal protein L31 signature. / Ribosomal protein L31 / Ribosomal protein L31 superfamily / Ribosomal protein L31 / Ribosomal protein L11, conserved site / Ribosomal protein L10-like domain superfamily / Ribosomal protein L21, conserved site / Ribosomal protein L21 signature. / Ribosomal protein L10P / Ribosomal protein L10 / Ribosomal protein L11 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 L11, N-terminal / Ribosomal protein L17 signature. / Ribosomal protein L11/L12 / Ribosomal protein L11, C-terminal / Ribosomal protein L11, C-terminal domain superfamily / Ribosomal protein L11/L12, N-terminal domain superfamily / Ribosomal protein L11/L12 / Ribosomal protein L11, N-terminal domain / Ribosomal protein L11, RNA binding domain / Ribosomal protein S14/S29 / Ribosomal protein L28/L24 superfamily / Ribosomal protein L36 signature. / Ribosomal protein L32p, bacterial type / 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 L6, bacterial-type / Ribosomal protein L18, bacterial-type / Ribosomal protein L19, conserved site / Ribosomal protein L19 signature. / Ribosomal protein L36 / Ribosomal protein L36 superfamily / Ribosomal protein L36 / Ribosomal protein L20 signature. / Ribosomal protein S3, bacterial-type / Ribosomal protein L27, conserved site / Ribosomal protein S6, conserved site / Ribosomal protein L27 signature. / Ribosomal protein S6 signature. / Ribosomal protein S19, bacterial-type / Ribosomal protein S7, bacterial/organellar-type / Ribosomal protein S11, bacterial-type / Ribosomal protein S13, bacterial-type / Ribosomal protein S20 / Ribosomal protein S20 superfamily / Ribosomal protein S20 / Ribosomal protein S9, bacterial/plastid / Ribosomal protein L14P, bacterial-type / Ribosomal protein S4, bacterial-type / Ribosomal protein L34, conserved site / Ribosomal protein L34 signature. / 30S ribosomal protein S17 / Ribosomal protein S5, bacterial-type / Ribosomal protein L22, bacterial/chloroplast-type / Ribosomal protein S6, plastid/chloroplast / Ribosomal protein L35 / Ribosomal protein L35 superfamily / Ribosomal protein L2, bacterial/organellar-type / Ribosomal protein L35 / Ribosomal protein S2, bacteria/mitochondria/plastid / Ribosomal L28 family / Ribosomal protein L33 / Ribosomal protein L33 / Ribosomal protein L28/L24 / Ribosomal protein L33 superfamily / : / Ribosomal protein L30, bacterial-type / Ribosomal protein L16 / Ribosomal protein L18 / Ribosomal L18 of archaea, bacteria, mitoch. and chloroplast / Ribosomal protein S18, conserved site / Ribosomal protein S18 signature. / L28p-like / Ribosomal protein L20 / Ribosomal protein S16 / Ribosomal protein S16 / Ribosomal protein L20 / Ribosomal protein L20, C-terminal / Ribosomal protein S16 domain superfamily / Ribosomal protein L21
Similarity search - Domain/homology
Large ribosomal subunit protein bL19 / Large ribosomal subunit protein bL32 / Small ribosomal subunit protein uS11 / Large ribosomal subunit protein bL34 / Large ribosomal subunit protein bL27 / Large ribosomal subunit protein uL24 / Large ribosomal subunit protein uL29 / Small ribosomal subunit protein uS17 / Large ribosomal subunit protein uL14 / Large ribosomal subunit protein uL5 ...Large ribosomal subunit protein bL19 / Large ribosomal subunit protein bL32 / Small ribosomal subunit protein uS11 / Large ribosomal subunit protein bL34 / Large ribosomal subunit protein bL27 / Large ribosomal subunit protein uL24 / Large ribosomal subunit protein uL29 / Small ribosomal subunit protein uS17 / Large ribosomal subunit protein uL14 / Large ribosomal subunit protein uL5 / Small ribosomal subunit protein uS14B / Small ribosomal subunit protein uS8 / Large ribosomal subunit protein uL16 / Large ribosomal subunit protein uL15 / Large ribosomal subunit protein uL30 / Large ribosomal subunit protein bL17 / Large ribosomal subunit protein bL36 / Small ribosomal subunit protein uS13 / Small ribosomal subunit protein uS2 / Small ribosomal subunit protein uS3 / Small ribosomal subunit protein uS4 / Small ribosomal subunit protein uS5 / Small ribosomal subunit protein bS6 / Small ribosomal subunit protein uS7 / Small ribosomal subunit protein uS9 / Small ribosomal subunit protein uS10 / Small ribosomal subunit protein uS12 / Small ribosomal subunit protein uS15 / Small ribosomal subunit protein bS16 / Small ribosomal subunit protein bS18 / Small ribosomal subunit protein uS19 / Small ribosomal subunit protein bS20 / Large ribosomal subunit protein bL21 / Large ribosomal subunit protein bL28 / Large ribosomal subunit protein uL22 / Large ribosomal subunit protein uL2 / Large ribosomal subunit protein uL3 / Large ribosomal subunit protein uL4 / Large ribosomal subunit protein uL10 / Large ribosomal subunit protein uL23 / Large ribosomal subunit protein uL6 / Large ribosomal subunit protein uL18 / Large ribosomal subunit protein bL20 / Large ribosomal subunit protein bL35 / Large ribosomal subunit protein bL33A / Large ribosomal subunit protein uL13 / Large ribosomal subunit protein bL31 / Large ribosomal subunit protein uL11 / Membrane protein insertion and folding monitor
Similarity search - Component
Biological speciesBacillus subtilis (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsSohmen D / Chiba S / Shimokawa-Chiba N / Innis A / Berninghausen O / Beckmann R / Wilson DN
CitationJournal: Nat Commun / Year: 2015
Title: Structure of the Bacillus subtilis 70S ribosome reveals the basis for species-specific stalling.
Authors: Daniel Sohmen / Shinobu Chiba / Naomi Shimokawa-Chiba / C Axel Innis / Otto Berninghausen / Roland Beckmann / Koreaki Ito / Daniel N Wilson /
Abstract: Ribosomal stalling is used to regulate gene expression and can occur in a species-specific manner. Stalling during translation of the MifM leader peptide regulates expression of the downstream ...Ribosomal stalling is used to regulate gene expression and can occur in a species-specific manner. Stalling during translation of the MifM leader peptide regulates expression of the downstream membrane protein biogenesis factor YidC2 (YqjG) in Bacillus subtilis, but not in Escherichia coli. In the absence of structures of Gram-positive bacterial ribosomes, a molecular basis for species-specific stalling has remained unclear. Here we present the structure of a Gram-positive B. subtilis MifM-stalled 70S ribosome at 3.5-3.9 Å, revealing a network of interactions between MifM and the ribosomal tunnel, which stabilize a non-productive conformation of the PTC that prevents aminoacyl-tRNA accommodation and thereby induces translational arrest. Complementary genetic analyses identify a single amino acid within ribosomal protein L22 that dictates the species specificity of the stalling event. Such insights expand our understanding of how the synergism between the ribosome and the nascent chain is utilized to modulate the translatome in a species-specific manner.
History
DepositionMar 16, 2015-
Header (metadata) releaseApr 29, 2015-
Map releaseApr 29, 2015-
UpdateFeb 17, 2016-
Current statusFeb 17, 2016Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.00155
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.00155
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3j9w
  • Surface level: 0.00155
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-3j9w
  • Surface level: 0.0013
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_6306.jpg

Downloads & links

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Map

FileDownload / File: emd_6306.map.gz / Format: CCP4 / Size: 185.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM structure of the B. subtilis MifM-stalled ribosome complex
Voxel sizeX=Y=Z: 1.108 Å
Density
Contour LevelBy AUTHOR: 0.00155 / Movie #1: 0.00155
Minimum - Maximum-0.00492832 - 0.00883723
Average (Standard dev.)0.00000473 (±0.0005257)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions368368368
Spacing368368368
CellA=B=C: 407.74402 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.1081.1081.108
M x/y/z368368368
origin x/y/z0.0000.0000.000
length x/y/z407.744407.744407.744
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS368368368
D min/max/mean-0.0050.0090.000

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Supplemental data

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Sample components

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Entire : B. subtilis MifM-stalled ribosome complex

EntireName: B. subtilis MifM-stalled ribosome complex
Components
  • Sample: B. subtilis MifM-stalled ribosome complex
  • Complex: 70S ribosomeRibosome
  • Protein or peptide: Membrane protein insertion and folding monitorBiological membrane
  • RNA: tRNA-Asp

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Supramolecule #1000: B. subtilis MifM-stalled ribosome complex

SupramoleculeName: B. subtilis MifM-stalled ribosome complex / type: sample / ID: 1000 / Number unique components: 3

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Supramolecule #1: 70S ribosome

SupramoleculeName: 70S ribosome / type: complex / ID: 1 / Details: stalled / Recombinant expression: No / Database: NCBI
Ribosome-details: ribosome-prokaryote: LSU 50S, LSU RNA 23S, LSU RNA 5S, SSU 30S, PSR16s, ALL
Source (natural)Organism: Bacillus subtilis (bacteria) / Strain: 168

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Macromolecule #1: Membrane protein insertion and folding monitor

MacromoleculeName: Membrane protein insertion and folding monitor / type: protein_or_peptide / ID: 1 / Name.synonym: MifM / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Bacillus subtilis (bacteria) / Strain: 168

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Macromolecule #2: tRNA-Asp

MacromoleculeName: tRNA-Asp / type: rna / ID: 2 / Classification: TRANSFER / Structure: DOUBLE HELIX / Synthetic?: No
Source (natural)Organism: Bacillus subtilis (bacteria) / Strain: 168

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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: 7.2
GridDetails: 2 nm pre-coated Quantifoil R3/3 holey carbon support grids
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 125085 / Cs: mm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Cs0
DateApr 12, 2014
Image recordingCategory: CCD / Film or detector model: FEI FALCON II (4k x 4k) / Average electron dose: 28 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: defocus groups
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: OTHER / Software - Name: SPIDER
Details: Since images from microscopy were processed in the absence of spatial frequencies higher than 8 A, a FSC cut-off value of 0.143 was used for average resolution determination of 3.9 A (Scheres and Chen, 2012).
Number images used: 305045

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