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- EMDB-8506: Structural Basis of Co-translational Quality Control by ArfA and ... -

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Basic information

Entry
Database: EMDB / ID: EMD-8506
TitleStructural Basis of Co-translational Quality Control by ArfA and RF2 Binding to Ribosome
Map data
Sample
  • Complex: Non-stop ribosomal complex with ArfA and RF2
    • Complex: 30S subunitProkaryotic small ribosomal subunit
      • Organelle or cellular component: 16S rRNA
        • RNA: 16S rRNA
      • Organelle or cellular component: 30S ribosomal protein S3
        • Protein or peptide: 30S ribosomal protein S3
      • Organelle or cellular component: 30S ribosomal protein S4
        • Protein or peptide: 30S ribosomal protein S4
      • Organelle or cellular component: 30S ribosomal protein S5
      • Organelle or cellular component: 30S ribosomal protein S12
      • Protein or peptide: 30S ribosomal protein S5
      • Protein or peptide: 30S ribosomal protein S12
    • Complex: 50S subunitProkaryotic large ribosomal subunit
      • Organelle or cellular component: 23S rRNA23S ribosomal RNA
        • RNA: 23S rRNA23S ribosomal RNA
    • Organelle or cellular component: Alternative ribosome-rescue factor A
      • Protein or peptide: Alternative ribosome-rescue factor A
    • Organelle or cellular component: Peptide chain release factor 2
      • Protein or peptide: Peptide chain release factor 2
    • Organelle or cellular component: P-site tRNA fMet
      • RNA: P-site tRNA fMet
    • Organelle or cellular component: mRNAMessenger RNA
      • RNA: mRNAMessenger RNA
KeywordsRibosome / ArfA / RF2 / nonstop translation
Function / homology
Function and homology information


translation release factor activity, codon specific / ribosomal large subunit binding / misfolded RNA binding / Group I intron splicing / RNA folding / rescue of stalled ribosome / translational termination / negative regulation of translational initiation / mRNA regulatory element binding translation repressor activity / positive regulation of RNA splicing ...translation release factor activity, codon specific / ribosomal large subunit binding / misfolded RNA binding / Group I intron splicing / RNA folding / rescue of stalled ribosome / translational termination / negative regulation of translational initiation / mRNA regulatory element binding translation repressor activity / positive regulation of RNA splicing / transcription antitermination / maintenance of translational fidelity / DNA-templated transcription termination / mRNA 5'-UTR binding / ribosomal small subunit biogenesis / ribosomal small subunit assembly / cytosolic small ribosomal subunit / regulation of translation / small ribosomal subunit / cytoplasmic translation / tRNA binding / rRNA binding / ribosome / structural constituent of ribosome / translation / response to antibiotic / mRNA binding / cytosol / cytoplasm
Similarity search - Function
Alternative ribosome-rescue factor A / Alternative ribosome-rescue factor A / Peptide chain release factor 2 / Peptide chain release factor / PCRF domain / PCRF / Peptide chain release factor class I superfamily / Prokaryotic-type class I peptide chain release factors signature. / Peptide chain release factor class I / RF-1 domain ...Alternative ribosome-rescue factor A / Alternative ribosome-rescue factor A / Peptide chain release factor 2 / Peptide chain release factor / PCRF domain / PCRF / Peptide chain release factor class I superfamily / Prokaryotic-type class I peptide chain release factors signature. / Peptide chain release factor class I / RF-1 domain / Ribosomal protein S3, bacterial-type / Ribosomal protein S4, bacterial-type / Ribosomal protein S5, bacterial-type / Ribosomal protein S12, bacterial-type / K Homology domain / K homology RNA-binding domain / Ribosomal protein S3, conserved site / K Homology domain, type 2 / Ribosomal protein S3, C-terminal / Ribosomal protein S3, C-terminal domain superfamily / KH domain / Ribosomal protein S3, C-terminal domain / Ribosomal protein S3 signature. / K homology domain superfamily, prokaryotic type / Ribosomal protein S4/S9 N-terminal domain / Ribosomal protein S4/S9, N-terminal / Ribosomal protein S4, conserved site / Type-2 KH domain profile. / Ribosomal protein S4/S9 N-terminal domain / Ribosomal protein S4/S9 / K homology domain-like, alpha/beta / Ribosomal protein S5, N-terminal, conserved site / Ribosomal protein S5 signature. / Ribosomal protein S5 / Ribosomal protein S5, N-terminal / S5 double stranded RNA-binding domain profile. / Ribosomal protein S5, C-terminal / Ribosomal protein S5, N-terminal domain / Ribosomal protein S5, C-terminal domain / S4 RNA-binding domain / RNA-binding S4 domain / RNA-binding S4 domain superfamily / S4 domain / Ribosomal protein S4 signature. / Ribosomal protein S12/S23 / S4 RNA-binding domain profile. / Ribosomal protein S12/S23 / Ribosomal protein S12 signature. / Ribosomal protein S5 domain 2-type fold, subgroup / Ribosomal protein S5 domain 2-type fold / Nucleic acid-binding, OB-fold
Similarity search - Domain/homology
Peptide chain release factor RF2 / Small ribosomal subunit protein uS12 / Small ribosomal subunit protein uS3 / Small ribosomal subunit protein uS4 / Small ribosomal subunit protein uS5 / Alternative ribosome-rescue factor A
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsZeng F / Chen Y
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM120552 United States
CitationJournal: Nature / Year: 2017
Title: Structural basis of co-translational quality control by ArfA and RF2 bound to ribosome.
Authors: Fuxing Zeng / Yanbo Chen / Jonathan Remis / Mrinal Shekhar / James C Phillips / Emad Tajkhorshid / Hong Jin /
Abstract: Quality control mechanisms intervene appropriately when defective translation events occur, in order to preserve the integrity of protein synthesis. Rescue of ribosomes translating on messenger RNAs ...Quality control mechanisms intervene appropriately when defective translation events occur, in order to preserve the integrity of protein synthesis. Rescue of ribosomes translating on messenger RNAs that lack stop codons is one of the co-translational quality control pathways. In many bacteria, ArfA recognizes stalled ribosomes and recruits the release factor RF2, which catalyses the termination of protein synthesis. Although an induced-fit mechanism of nonstop mRNA surveillance mediated by ArfA and RF2 has been reported, the molecular interaction between ArfA and RF2 in the ribosome that is responsible for the mechanism is unknown. Here we report an electron cryo-microscopy structure of ArfA and RF2 in complex with the 70S ribosome bound to a nonstop mRNA. The structure, which is consistent with our kinetic and biochemical data, reveals the molecular interactions that enable ArfA to specifically recruit RF2, not RF1, into the ribosome and to enable RF2 to release the truncated protein product in this co-translational quality control pathway. The positively charged C-terminal domain of ArfA anchors in the mRNA entry channel of the ribosome. Furthermore, binding of ArfA and RF2 induces conformational changes in the ribosomal decoding centre that are similar to those seen in other protein-involved decoding processes. Specific interactions between residues in the N-terminal domain of ArfA and RF2 help RF2 to adopt a catalytically competent conformation for peptide release. Our findings provide a framework for understanding recognition of the translational state of the ribosome by new proteins, and expand our knowledge of the decoding potential of the ribosome.
History
DepositionDec 4, 2016-
Header (metadata) releaseJan 4, 2017-
Map releaseJan 11, 2017-
UpdateMar 13, 2024-
Current statusMar 13, 2024Processing site: RCSB / 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-5u4j
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-5u4j
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8506.png

Downloads & links

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Map

FileDownload / File: emd_8506.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.19 Å/pix.
x 384 pix.
= 458.112 Å		1.19 Å/pix.
x 384 pix.
= 458.112 Å		1.19 Å/pix.
x 384 pix.
= 458.112 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.193 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.03 / Movie #1: 0.03
Minimum - Maximum-0.19296288 - 0.23596789
Average (Standard dev.)0.0000067479664 (±0.005431493)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 458.112 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.1931.1931.193
M x/y/z384384384
origin x/y/z0.0000.0000.000
length x/y/z458.112458.112458.112
α/β/γ90.00090.00090.000
start NX/NY/NZ-200-200-200
NX/NY/NZ400400400
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS384384384
D min/max/mean-0.1930.2360.000

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Supplemental data

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Half map: half map 1

Fileemd_8506_half_map_1.map
Annotationhalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map 2

Fileemd_8506_half_map_2.map
Annotationhalf map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Non-stop ribosomal complex with ArfA and RF2

EntireName: Non-stop ribosomal complex with ArfA and RF2
Components
  • Complex: Non-stop ribosomal complex with ArfA and RF2
    • Complex: 30S subunitProkaryotic small ribosomal subunit
      • Organelle or cellular component: 16S rRNA
        • RNA: 16S rRNA
      • Organelle or cellular component: 30S ribosomal protein S3
        • Protein or peptide: 30S ribosomal protein S3
      • Organelle or cellular component: 30S ribosomal protein S4
        • Protein or peptide: 30S ribosomal protein S4
      • Organelle or cellular component: 30S ribosomal protein S5
      • Organelle or cellular component: 30S ribosomal protein S12
      • Protein or peptide: 30S ribosomal protein S5
      • Protein or peptide: 30S ribosomal protein S12
    • Complex: 50S subunitProkaryotic large ribosomal subunit
      • Organelle or cellular component: 23S rRNA23S ribosomal RNA
        • RNA: 23S rRNA23S ribosomal RNA
    • Organelle or cellular component: Alternative ribosome-rescue factor A
      • Protein or peptide: Alternative ribosome-rescue factor A
    • Organelle or cellular component: Peptide chain release factor 2
      • Protein or peptide: Peptide chain release factor 2
    • Organelle or cellular component: P-site tRNA fMet
      • RNA: P-site tRNA fMet
    • Organelle or cellular component: mRNAMessenger RNA
      • RNA: mRNAMessenger RNA

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Supramolecule #1: Non-stop ribosomal complex with ArfA and RF2

SupramoleculeName: Non-stop ribosomal complex with ArfA and RF2 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#10
Source (natural)Organism: Escherichia coli (E. coli)

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Supramolecule #2: 30S subunit

SupramoleculeName: 30S subunit / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1, #3-#5, #8
Source (natural)Organism: Escherichia coli (E. coli)

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Supramolecule #3: 50S subunit

SupramoleculeName: 50S subunit / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Escherichia coli (E. coli)

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Supramolecule #4: Alternative ribosome-rescue factor A

SupramoleculeName: Alternative ribosome-rescue factor A / type: organelle_or_cellular_component / ID: 4 / Parent: 1 / Macromolecule list: #10
Source (natural)Organism: Escherichia coli (E. coli)

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Supramolecule #5: Peptide chain release factor 2

SupramoleculeName: Peptide chain release factor 2 / type: organelle_or_cellular_component / ID: 5 / Parent: 1 / Macromolecule list: #9
Source (natural)Organism: Escherichia coli (E. coli)

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Supramolecule #6: P-site tRNA fMet

SupramoleculeName: P-site tRNA fMet / type: organelle_or_cellular_component / ID: 6 / Parent: 1 / Macromolecule list: #7 / Details: P-site tRNA (fMet) was in vitro transcribed.
Source (natural)Organism: Escherichia coli (E. coli)

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Supramolecule #7: mRNA

SupramoleculeName: mRNA / type: organelle_or_cellular_component / ID: 7 / Parent: 1 / Macromolecule list: #6

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Supramolecule #8: 23S rRNA

SupramoleculeName: 23S rRNA / type: organelle_or_cellular_component / ID: 8 / Parent: 3 / Macromolecule list: #2
Source (natural)Organism: Escherichia coli (E. coli)

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Supramolecule #9: 16S rRNA

SupramoleculeName: 16S rRNA / type: organelle_or_cellular_component / ID: 9 / Parent: 2 / Macromolecule list: #1
Source (natural)Organism: Escherichia coli (E. coli)

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Supramolecule #10: 30S ribosomal protein S3

SupramoleculeName: 30S ribosomal protein S3 / type: organelle_or_cellular_component / ID: 10 / Parent: 2 / Macromolecule list: #3
Source (natural)Organism: Escherichia coli (E. coli)

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Supramolecule #11: 30S ribosomal protein S4

SupramoleculeName: 30S ribosomal protein S4 / type: organelle_or_cellular_component / ID: 11 / Parent: 2 / Macromolecule list: #4
Source (natural)Organism: Escherichia coli (E. coli)

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Supramolecule #12: 30S ribosomal protein S5

SupramoleculeName: 30S ribosomal protein S5 / type: organelle_or_cellular_component / ID: 12 / Parent: 2
Source (natural)Organism: Escherichia coli (E. coli)

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Supramolecule #13: 30S ribosomal protein S12

SupramoleculeName: 30S ribosomal protein S12 / type: organelle_or_cellular_component / ID: 13 / Parent: 2
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #1: 16S rRNA

MacromoleculeName: 16S rRNA / type: rna / ID: 1 / Number of copies: 1
Source (natural)Organism: Escherichia coli (E. coli) / Strain: MRE600
Molecular weightTheoretical: 497.075812 KDa
SequenceString: AAUUGAAGAG UUUGAUCAUG GCUCAGAUUG AACGCUGGCG GCAGGCCUAA CACAUGCAAG UCGAACGGUA ACAGGAAGAA GCUUGCUUC UUUGCUGACG AGUGGCGGAC GGGUGAGUAA UGUCUGGGAA ACUGCCUGAU GGAGGGGGAU AACUACUGGA A ACGGUAGC ...String:
AAUUGAAGAG UUUGAUCAUG GCUCAGAUUG AACGCUGGCG GCAGGCCUAA CACAUGCAAG UCGAACGGUA ACAGGAAGAA GCUUGCUUC UUUGCUGACG AGUGGCGGAC GGGUGAGUAA UGUCUGGGAA ACUGCCUGAU GGAGGGGGAU AACUACUGGA A ACGGUAGC UAAUACCGCA UAACGUCGCA AGACCAAAGA GGGGGACCUU CGGGCCUCUU GCCAUCGGAU GUGCCCAGAU GG GAUUAGC UAGUAGGUGG GGUAACGGCU CACCUAGGCG ACGAUCCCUA GCUGGUCUGA GAGGAUGACC AGCCACACUG GAA CUGAGA CACGGUCCAG ACUCCUACGG GAGGCAGCAG UGGGGAAUAU UGCACAAUGG GCGCAAGCCU GAUGCAGCCA UGCC GCGUG UAUGAAGAAG GCCUUCGGGU UGUAAAGUAC UUUCAGCGGG GAGGAAGGGA GUAAAGUUAA UACCUUUGCU CAUUG ACGU UACCCGCAGA AGAAGCACCG GCUAACUCCG (PSU)GCCAGCAGC C(G7M)CGGUAAUA CGGAGGGUGC AAGCGUUA A UCGGAAUUAC UGGGCGUAAA GCGCACGCAG GCGGUUUGUU AAGUCAGAUG UGAAAUCCCC GGGCUCAACC UGGGAACUG CAUCUGAUAC UGGCAAGCUU GAGUCUCGUA GAGGGGGGUA GAAUUCCAGG UGUAGCGGUG AAAUGCGUAG AGAUCUGGAG GAAUACCGG UGGCGAAGGC GGCCCCCUGG ACGAAGACUG ACGCUCAGGU GCGAAAGCGU GGGGAGCAAA CAGGAUUAGA U ACCCUGGU AGUCCACGCC GUAAACGAUG UCGACUUGGA GGUUGUGCCC UUGAGGCGUG GCUUCCGGAG CUAACGCGUU AA GUCGACC GCCUGGGGAG UACGGCCGCA AGGUUAAAAC UCAAAUGAAU UGACGGGGGC CCGCACAAGC GGUGGAGCAU GUG GUUUAA UUCGAU(2MG)(5MC)AA CGCGAAGAAC CUUACCUGGU CUUGACAUCC ACGGAAGUUU UCAGAGAUGA GAAUGU GCC UUCGGGAACC GUGAGACAGG UGCUGCAUGG CUGUCGUCAG CUCGUGUUGU GAAAUGUUGG GUUAAGUCCC GCAACGA GC GCAACCCUUA UCCUUUGUUG CCAGCGGUCC GGCCGGGAAC UCAAAGGAGA CUGCCAGUGA UAAACUGGAG GAAGGUGG G GAUGACGUCA AGUCAUCAUG (2MG)CCCUUACGA CCAGGGCUAC ACACGUGCUA CAAUGGCGCA UACAAAGAGA AGCGA CCUC GCGAGAGCAA GCGGACCUCA UAAAGUGCGU CGUAGUCCGG AUUGGAGUCU GCAACUCGAC UCCAUGAAGU CGGAAU CGC UAGUAAUCGU GGAUCAGAAU GCCACGGUGA AUACGUUCCC GGGCCUUGUA CACACCG(4OC)CC GU(5MC)ACACCA UGGGAGUGGG UUGCAAAAGA AGUAGGUAGC UUAACCUUCG GGAGGGCGCU UACCACUUUG UGAUUCAUGA CUGGGGUGAA GUCG(UR3)AACA AGGUAACCGU AGG(2MG)G(MA6)(MA6)CCU GCGGUUGGAU CA

GENBANK: GENBANK: CP011320.1

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Macromolecule #2: 23S rRNA

MacromoleculeName: 23S rRNA / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Escherichia coli (E. coli) / Strain: MRE600
Molecular weightTheoretical: 941.790625 KDa
SequenceString: GGUUAAGCGA CUAAGCGUAC ACGGUGGAUG CCCUGGCAGU CAGAGGCGAU GAAGGACGUG CUAAUCUGCG AUAAGCGUCG GUAAGGUGA UAUGAACCGU UAUAACCGGC GAUUUCCGAA UGGGGAAACC CAGUGUGUUU CGACACACUA UCAUUAACUG A AUCCAUAG ...String:
GGUUAAGCGA CUAAGCGUAC ACGGUGGAUG CCCUGGCAGU CAGAGGCGAU GAAGGACGUG CUAAUCUGCG AUAAGCGUCG GUAAGGUGA UAUGAACCGU UAUAACCGGC GAUUUCCGAA UGGGGAAACC CAGUGUGUUU CGACACACUA UCAUUAACUG A AUCCAUAG GUUAAUGAGG CGAACCGGGG GAACUGAAAC AUCUAAGUAC CCCGAGGAAA AGAAAUCAAC CGAGAUUCCC CC AGUAGCG GCGAGCGAAC GGGGAGCAGC CCAGAGCCUG AAUCAGUGUG UGUGUUAGUG GAAGCGUCUG GAAAGGCGCG CGA UACAGG GUGACAGCCC CGUACACAAA AAUGCACAUG CUGUGAGCUC GAUGAGUAGG GCGGGACACG UGGUAUCCUG UCUG AAUAU GGGGGGACCA UCCUCCAAGG CUAAAUACUC CUGACUGACC GAUAGUGAAC CAGUACCGUG AGGGAAAGGC GAAAA GAAC CCCGGCGAGG GGAGUGAAAA AGAACCUGAA ACCGUGUACG UACAAGCAGU GGGAGCACGC UUAGGCGUGU GACUGC GUA CCUUUUGUAU AAUGGGUCAG CGACUUAUAU UCUGUAGCAA GGUUAACCGA AUAGGGGAGC CGAAGGGAAA CCGAGUC UU AACUGGGCGU UAAGUUGCAG GGUAUAGACC CGAAACCCGG UGAUCUAGCC AUGGGCAGGU UGAAGGUUGG GUAACACU A ACUGGAGGAC CGAACCGACU AAU(1MG)(PSU)(5MU)GAAA AAUUAGCGGA UGACUUGUGG CUGGGGGUGA AAGGCCA AU CAAACCGGGA GAUAGCUGGU UCUCCCCGAA AGCUAUUUAG GUAGCGCCUC GUGAAUUCAU CUCCGGGGGU AGAGCACU G UUUCGGCAAG GGGGUCAACC CGACUUACCA ACCCGAUGCA AACUGCGAAU ACCGGAGAAU GUUAUCACGG GAGACACAC GGCGGG(PSU)GCU AACGUCCGUC GUGAAGAGGG AAACAACCCA GACCGCCAGC UAAGGUCCCA AAGUCAUGGU UAAGUG GGA AACGAUGUGG GAAGGCCCAG ACAGCCAGGA UGUUGGCUUA GAAGCAGCCA UCAUUUAAAG AAAGCGUAAU AGCUCAC UG GUCGAGUCGG CCUGCGCGGA AGAUGUAACG GGGCUAAACC AUGCACCGAA GCUGCGGCAG CGACGCUUAU GCGUUGUU G GGUAGGGGAG CGUUCUGUAA GCCUGCGAAG GUGUGCUGUG AGGCAUGCUG GAGGUAUCAG AAGUGCGAAU GCUGACAUA AGUAACGAUA AAGCGGGUGA AAAGCCCGCU CGCCGGAAGA CCAAGGGUUC CUGUCCAACG UUAAUCGGGG CAGGGUGAGU CGACCCCUA AGGCGAGGCC GAAAGGCGUA GUCGAUGGGA AACAGGUUAA UAUUCCUGUA CUUGGUGUUA CUGCGAAGGG G GGACGGAG AAGGCUAUGU UGGCCGGGCG ACGGUUGUCC CGGUUUAAGC GUGUAGGCUG GUUUUCCAGG CAAAUCCGGA AA AUCAAGG CUGAGGCGUG AUGACGAGGC ACUACGGUGC UGAAGCAACA AAUGCCCUGC UUCCAGGAAA AGCCUCUAAG CAU CAGGUA ACAUCAAAUC GUACCCCAAA CCGACACAGG UGGUCAGGUA GAGAAUACCA AGGCGCUUGA GAGAACUCGG GUGA AGGAA CUAGGCAAAA UGGUGCCGUA ACUUCGGGAG AAGGCACGCU GAUAUGUAGG UGAGGUCCCU CGCGGAUGGA GCUGA AAUC AGUCGAAGAU ACCAGCUGGC UGCAACUGUU UAUUAAAAAC ACAGCACUGU GCAAACACGA AAGUGGACGU AUACGG UGU GACGCCU(2MG)CC CGGUGCCGGA AGGUUAAUUG AUGGGGUUAG CGCAAGCGAA GCUCUUGAUC GAAGCCCCGG UAA ACGGCG GCCG(PSU)AACUA (PSU)AACGGUCCU AAGGUAGCGA AA(5MU)UCCUUGU CGGGUAAGUU CCGAC(5MC)UG C ACGAAUGGCG UAAUGAUGGC CAGGCUGUCU CCACCCGAGA CUCAGUGAAA UUGAACUCGC UGUGAAGAUG CAGUGUACC CGCGGCAAGA CGGAAAGACC CCGU(G7M)AACCU UUACUAUAGC UUGACACUGA ACAUUGAGCC UUGAUGUGUA GGAUAG GUG GGAGGCUUUG AAGUGUGGAC GCCAGUCUGC AUGGAGCCGA CCUUGAAAUA CCACCCUUUA AUGUUUGAUG UUCUAAC GU UGACCCGUAA UCCGGGUUGC GGACAGUGUC UGGUGGGUAG UUUGACUG(OMG)G GCGGUCUCCU CCUAAAGAGU AACG GAGGA GCACGAAGGU UGGCUAAUCC UGGUCGGACA UCAGGAGGUU AGUGCAAUGG CAUAAGCCAG CUUGACUGCG AGCGU GACG GCGCGAGCAG GUGCGAAAGC AGGUCAUAGU GAUCCGGUGG UUCUGAAUGG AAGGGCCAUC GCUCAACGGA UAAAAG GUA CUCCG(2MG)GGAU AACAGGC(PSU)GA UACCGCCCAA GAGUUCAUAU CGACGGCGGU GUUUGGCA(OMC)C UCG (2MA)(PSU)GUCG GCUCAUCACA UCCUGGGGCU GAAGUAGGUC CCAAGGGUAU GGC(OMU)GUUCGC CAUUUAAAGU GG UACGCGA GC(PSU)GGGUUUA GAACGUCGUG AGACAGU(PSU)CG GUCCCUAUCU GCCGUGGGCG CUGGAGAACU GAGGG GGGC UGCUCCUAGU ACGAGAGGAC CGGAGUGGAC GCAUCACUGG UGUUCGGGUU GUCAUGCCAA UGGCACUGCC CGGUAG CUA AAUGCGGAAG AGAUAAGUGC UGAAAGCAUC UAAGCACGAA ACUUGCCCCG AGAUGAGUUC UCCCUGACCC UUUAAGG GU CCUGAAGGAA CGUUGAAGAC GACGACGUUG AUAGGCCGGG UGUGUAAGCG CAGCGAUGCG UUGAGCUAAC CGGUACUA A UGAACCGUGA GGCUUAACCU U

GENBANK: GENBANK: V00331.1

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Macromolecule #6: mRNA

MacromoleculeName: mRNA / type: rna / ID: 6 / Number of copies: 1
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 5.922644 KDa
SequenceString:
GGCAAGGAGG UAAAAAUG

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Macromolecule #7: P-site tRNA fMet

MacromoleculeName: P-site tRNA fMet / type: rna / ID: 7 / Number of copies: 1
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 24.786785 KDa
SequenceString:
CGCGGGGUGG AGCAGCCUGG UAGCUCGUCG GGCUCAUAAC CCGAAGAUCG UCGGUUCAAA UCCGGCCCCC GCAACCA

GENBANK: GENBANK: LM993812.1

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Macromolecule #3: 30S ribosomal protein S3

MacromoleculeName: 30S ribosomal protein S3 / type: protein_or_peptide / ID: 3 / Details: Gene Name(s): rpsC b3314 JW3276 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli) / Strain: MRE600
Molecular weightTheoretical: 26.031316 KDa
SequenceString: MGQKVHPNGI RLGIVKPWNS TWFANTKEFA DNLDSDFKVR QYLTKELAKA SVSRIVIERP AKSIRVTIHT ARPGIVIGKK GEDVEKLRK VVADIAGVPA QINIAEVRKP ELDAKLVADS ITSQLERRVM FRRAMKRAVQ NAMRLGAKGI KVEVSGRLGG A EIARTEWY ...String:
MGQKVHPNGI RLGIVKPWNS TWFANTKEFA DNLDSDFKVR QYLTKELAKA SVSRIVIERP AKSIRVTIHT ARPGIVIGKK GEDVEKLRK VVADIAGVPA QINIAEVRKP ELDAKLVADS ITSQLERRVM FRRAMKRAVQ NAMRLGAKGI KVEVSGRLGG A EIARTEWY REGRVPLHTL RADIDYNTSE AHTTYGVIGV KVWIFKGEIL GGMAAVEQPE KPAAQPKKQQ RKGRK

UniProtKB: Small ribosomal subunit protein uS3

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Macromolecule #4: 30S ribosomal protein S4

MacromoleculeName: 30S ribosomal protein S4 / type: protein_or_peptide / ID: 4 / Details: Gene Name(s): rpsD ramA b3296 JW3258 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli) / Strain: MRE600
Molecular weightTheoretical: 23.514199 KDa
SequenceString: MARYLGPKLK LSRREGTDLF LKSGVRAIDT KCKIEQAPGQ HGARKPRLSD YGVQLREKQK VRRIYGVLER QFRNYYKEAA RLKGNTGEN LLALLEGRLD NVVYRMGFGA TRAEARQLVS HKAIMVNGRV VNIASYQVSP NDVVSIREKA KKQSRVKAAL E LAEQREKP ...String:
MARYLGPKLK LSRREGTDLF LKSGVRAIDT KCKIEQAPGQ HGARKPRLSD YGVQLREKQK VRRIYGVLER QFRNYYKEAA RLKGNTGEN LLALLEGRLD NVVYRMGFGA TRAEARQLVS HKAIMVNGRV VNIASYQVSP NDVVSIREKA KKQSRVKAAL E LAEQREKP TWLEVDAGKM EGTFKRKPER SDLSADINEH LIVELYSK

UniProtKB: Small ribosomal subunit protein uS4

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Macromolecule #5: 30S ribosomal protein S5

MacromoleculeName: 30S ribosomal protein S5 / type: protein_or_peptide / ID: 5 / Details: Gene Name(s): rpsE spc b3303 JW3265 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli) / Strain: MRE600
Molecular weightTheoretical: 17.629398 KDa
SequenceString:
MAHIEKQAGE LQEKLIAVNR VSKTVKGGRI FSFTALTVVG DGNGRVGFGY GKAREVPAAI QKAMEKARRN MINVALNNGT LQHPVKGVH TGSRVFMQPA SEGTGIIAGG AMRAVLEVAG VHNVLAKAYG STNPINVVRA TIDGLENMNS PEMVAAKRGK S VEEILGK

UniProtKB: Small ribosomal subunit protein uS5

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Macromolecule #8: 30S ribosomal protein S12

MacromoleculeName: 30S ribosomal protein S12 / type: protein_or_peptide / ID: 8 / Details: Gene Name(s): rpsL strA b3342 JW3304 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli) / Strain: MRE600
Molecular weightTheoretical: 13.768157 KDa
SequenceString:
MATVNQLVRK PRARKVAKSN VPALEACPQK RGVCTRVYTT TPKKPNSALR KVCRVRLTNG FEVTSYIGGE GHNLQEHSVI LIRGGRVKD LPGVRYHTVR GALDCSGVKD RKQARSKYGV KRPKA

UniProtKB: Small ribosomal subunit protein uS12

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Macromolecule #9: Peptide chain release factor 2

MacromoleculeName: Peptide chain release factor 2 / type: protein_or_peptide / ID: 9 / Details: Gene Name(s): prfB ECK2886 JW5847 supK / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli) / Strain: MRE600
Molecular weightTheoretical: 43.299844 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: HHHHHHSAAL EVLFQGPGMF EINPVNNRIQ DLTERSDVLR GYLDYDAKKE RLEEVNAELE QPDVWNEPER AQALGKERSS LEAVVDTLD QMKQGLEDVS GLLELAVEAD DEETFNEAVA ELDALEEKLA QLEFRRMFSG EYDSADCYLD IQAGSGGTEA Q DWASMLER ...String:
HHHHHHSAAL EVLFQGPGMF EINPVNNRIQ DLTERSDVLR GYLDYDAKKE RLEEVNAELE QPDVWNEPER AQALGKERSS LEAVVDTLD QMKQGLEDVS GLLELAVEAD DEETFNEAVA ELDALEEKLA QLEFRRMFSG EYDSADCYLD IQAGSGGTEA Q DWASMLER MYLRWAESRG FKTEIIEESE GEVAGIKSVT IKISGDYAYG WLRTETGVHR LVRKSPFDSG GRRHTSFSSA FV YPEVDDD IDIEINPADL RIDVYRTSGA GGQHVNRTES AVRITHIPTG IVTQCQNDRS QHKNKDQAMK QMKAKLYELE MQK KNAEKQ AMEDNKSDIG WGSQIRSYVL DDSRIKDLRT GVETRNTQAV LDGSLDQFIE ASLKAGL

UniProtKB: Peptide chain release factor RF2

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Macromolecule #10: Alternative ribosome-rescue factor A

MacromoleculeName: Alternative ribosome-rescue factor A / type: protein_or_peptide / ID: 10 / Details: Gene Name(s): arfA ECK3278 JW3253 yhdL / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli) / Strain: MRE600
Molecular weightTheoretical: 6.513396 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
GSMSRYQHTK GQIKDNAIEA LLHDPLFRQR VEKNKKGKGS YMRKGKHGNR GNWEASG

UniProtKB: Alternative ribosome-rescue factor A

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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

Concentration0.2 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4S4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
15.0 mMMg(CH3COO)2magnesium acetate
150.0 mMCH3CO2Kpotassium acetate
4.0 mMHOCH2CH2SH2-mercapthoethanol
2.0 mMC7H19N3spermidine
0.05 mMC10H26N4spermin
GridModel: C-flat-2/0.5 4C / Material: COPPER / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: Grids were blotted for 3.5 s.

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Electron microscopy

MicroscopeJEOL 3200FSC
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 83822 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.7000000000000001 µm
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Frames/image: 4-25 / Average exposure time: 6.0 sec. / Average electron dose: 20.0 e/Å2

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Image processing

Startup modelType of model: EMDB MAP
EMDB ID:

1005

Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 2.0)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 2.0)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.0) / Number images used: 155440
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: RECIPROCAL / Protocol: FLEXIBLE FIT / Overall B value: 108.501 / Target criteria: Average FSC
Output model

PDB-5u4j:
Structural Basis of Co-translational Quality Control by ArfA and RF2 Bound to Ribosome

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