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- PDB-9ha7: Pooled 50S subunit C-CP_(L22)-~H61 precursor states supplemented ... -

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Basic information

Entry
Database: PDB / ID: 9ha7
TitlePooled 50S subunit C-CP_(L22)-~H61 precursor states supplemented with Api137
Components
  • (Large ribosomal subunit protein ...) x 15
  • 23S ribosomal RNA
  • 50S ribosomal protein L3
  • 5S ribosomal RNA
  • Apidaecins type 22
KeywordsRIBOSOME / antimicrobial peptide / RNA / ribosomal protein / PrAMP / proline-rich peptide / antibiotics / 50S / Api137
Function / homology
Function and homology information


transcriptional attenuation / positive regulation of ribosome biogenesis / endoribonuclease inhibitor activity / RNA-binding transcription regulator activity / negative regulation of cytoplasmic translation / translation repressor activity / mRNA regulatory element binding translation repressor activity / assembly of large subunit precursor of preribosome / ribosome assembly / cytosolic ribosome assembly ...transcriptional attenuation / positive regulation of ribosome biogenesis / endoribonuclease inhibitor activity / RNA-binding transcription regulator activity / negative regulation of cytoplasmic translation / translation repressor activity / mRNA regulatory element binding translation repressor activity / assembly of large subunit precursor of preribosome / ribosome assembly / cytosolic ribosome assembly / regulation of cell growth / DNA-templated transcription termination / response to radiation / mRNA 5'-UTR binding / large ribosomal subunit / ribosome binding / 5S rRNA binding / ribosomal large subunit assembly / large ribosomal subunit rRNA binding / cytosolic large ribosomal subunit / cytoplasmic translation / tRNA binding / negative regulation of translation / rRNA binding / defense response to bacterium / ribosome / structural constituent of ribosome / translation / innate immune response / response to antibiotic / negative regulation of DNA-templated transcription / mRNA binding / DNA binding / RNA binding / extracellular region / zinc ion binding / cytosol / cytoplasm
Similarity search - Function
Apidaecin / Apidaecin / Ribosomal protein L25, short-form / : / Ribosomal protein L21, conserved site / Ribosomal protein L21 signature. / : / Ribosomal protein L17 signature. / Ribosomal L25p family / Ribosomal protein L25 ...Apidaecin / Apidaecin / Ribosomal protein L25, short-form / : / Ribosomal protein L21, conserved site / Ribosomal protein L21 signature. / : / Ribosomal protein L17 signature. / Ribosomal L25p family / Ribosomal protein L25 / Ribosomal protein L25/Gln-tRNA synthetase, N-terminal / Ribosomal protein L25/Gln-tRNA synthetase, anti-codon-binding domain superfamily / : / Ribosomal protein L5, bacterial-type / Ribosomal protein L18, bacterial-type / : / Ribosomal protein L27, conserved site / Ribosomal protein L27 signature. / Ribosomal protein L20 signature. / Ribosomal protein L34, conserved site / Ribosomal protein L34 signature. / Ribosomal protein L18 / Ribosomal L18 of archaea, bacteria, mitoch. and chloroplast / Ribosomal protein L30, bacterial-type / Ribosomal protein L27 / Ribosomal L27 protein / Ribosomal protein L20 / Ribosomal protein L20 / Ribosomal protein L20, C-terminal / Ribosomal protein L21 / Ribosomal protein L17 / Ribosomal protein L17 superfamily / Ribosomal protein L17 / Ribosomal proteins 50S L24/mitochondrial 39S L24 / Ribosomal protein L21-like / L21-like superfamily / Ribosomal prokaryotic L21 protein / Ribosomal protein L34 / Ribosomal protein L34 / Ribosomal protein L24 / Ribosomal protein L13, bacterial-type / Ribosomal protein L3, bacterial/organelle-type / Ribosomal protein L15, bacterial-type / 50S ribosomal protein uL4 / Ribosomal protein L23/L25, conserved site / Ribosomal protein L23 signature. / Ribosomal protein L30, conserved site / Ribosomal protein L30 signature. / Ribosomal protein L5, conserved site / Ribosomal protein L5 signature. / Ribosomal protein L29, conserved site / Ribosomal protein L29 signature. / Ribosomal protein L5, N-terminal / Ribosomal protein L5 / Ribosomal protein L15, conserved site / Ribosomal protein L15 signature. / Ribosomal protein L5, C-terminal / ribosomal L5P family C-terminus / Ribosomal protein L5 / Ribosomal protein L5 domain superfamily / Ribosomal protein L13 signature. / Ribosomal protein L13, conserved site / Ribosomal L29 protein / Ribosomal protein L29/L35 / Ribosomal protein L29/L35 superfamily / Ribosomal protein L24 signature. / Ribosomal protein L24/L26, conserved site / KOW (Kyprides, Ouzounis, Woese) motif. / Ribosomal protein L30, ferredoxin-like fold domain / Ribosomal protein L30p/L7e / Ribosomal protein L13 / Ribosomal protein L13 / Ribosomal protein L13 superfamily / Ribosomal protein L23 / Ribosomal protein L30, ferredoxin-like fold domain superfamily / Ribosomal protein L15 / Ribosomal protein L25/L23 / Ribosomal proteins 50S-L15, 50S-L18e, 60S-L27A / Ribosomal protein L26/L24, KOW domain / Ribosomal protein L3, conserved site / Ribosomal protein L3 signature. / Ribosomal protein L3 / Ribosomal protein L3 / Ribosomal protein L18e/L15P / Ribosomal L18e/L15P superfamily / Ribosomal protein L4/L1e / Ribosomal protein L4 domain superfamily / Ribosomal protein L4/L1 family / Translation protein SH3-like domain superfamily / Ribosomal protein L23/L15e core domain superfamily / KOW motif / KOW / Translation protein, beta-barrel domain superfamily / Ribosomal protein L2, domain 2 / Nucleotide-binding alpha-beta plait domain superfamily
Similarity search - Domain/homology
RNA / RNA (> 10) / RNA (> 100) / RNA (> 1000) / Large ribosomal subunit protein uL15 / Large ribosomal subunit protein bL20 / Large ribosomal subunit protein bL27 / Large ribosomal subunit protein uL29 / Large ribosomal subunit protein bL34 / Large ribosomal subunit protein uL13 ...RNA / RNA (> 10) / RNA (> 100) / RNA (> 1000) / Large ribosomal subunit protein uL15 / Large ribosomal subunit protein bL20 / Large ribosomal subunit protein bL27 / Large ribosomal subunit protein uL29 / Large ribosomal subunit protein bL34 / Large ribosomal subunit protein uL13 / Large ribosomal subunit protein uL23 / Large ribosomal subunit protein bL17 / Large ribosomal subunit protein bL21 / Large ribosomal subunit protein uL30 / Large ribosomal subunit protein uL18 / Apidaecins type 22 / Large ribosomal subunit protein uL3 / Large ribosomal subunit protein uL24 / Large ribosomal subunit protein uL4 / Large ribosomal subunit protein uL5 / Large ribosomal subunit protein bL25
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Apis mellifera (honey bee)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.37 Å
AuthorsLauer, S. / Nikolay, R. / Spahn, C.M.T.
Funding support Germany, 4items
OrganizationGrant numberCountry
German Federal Ministry for Education and Research16GW0300 Germany
German Federal Ministry for Education and Research16GW0299K Germany
German Research Foundation (DFG)EXC2008/1-390540038 Germany
German Research Foundation (DFG)INST 335/588-1 FUGG Germany
CitationJournal: Nat Commun / Year: 2025
Title: The proline-rich antimicrobial peptide Api137 disrupts large ribosomal subunit assembly and induces misfolding.
Authors: Simon Malte Lauer / Jakob Gasse / Andor Krizsan / Maren Reepmeyer / Thiemo Sprink / Rainer Nikolay / Christian M T Spahn / Ralf Hoffmann /
Abstract: The proline-rich antimicrobial designer peptide Api137 inhibits protein expression in bacteria by binding simultaneously to the ribosomal polypeptide exit tunnel and the release factor (RF), ...The proline-rich antimicrobial designer peptide Api137 inhibits protein expression in bacteria by binding simultaneously to the ribosomal polypeptide exit tunnel and the release factor (RF), depleting the cellular RF pool and leading to ribosomal arrest at stop codons. This study investigates the additional effect of Api137 on the assembly of ribosomes using an Escherichia coli reporter strain expressing one ribosomal protein per 30S and 50S subunit tagged with mCherry and EGFP, respectively. Separation of cellular extracts derived from cells exposed to Api137 in a sucrose gradient reveals elevated levels of partially assembled and not fully matured precursors of the 50S subunit (pre-50S). High-resolution structures obtained by cryogenic electron microscopy demonstrate that a large proportion of pre-50S states are missing up to five proteins (uL22, bL32, uL29, bL23, and uL16) and have misfolded helices in 23S rRNA domain IV. These data suggest a second mechanism for Api137, wherein it disrupts 50S subunit assembly by inducing the formation of misfolded precursor particles potentially incapable of evolving into active ribosomes, suggesting a bactericidal mechanism.
History
DepositionNov 1, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 22, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
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Assembly

Deposited unit
2: Large ribosomal subunit protein bL34
B: 5S ribosomal RNA
F: Large ribosomal subunit protein uL5
J: Large ribosomal subunit protein uL13
L: Large ribosomal subunit protein uL15
N: Large ribosomal subunit protein bL17
O: Large ribosomal subunit protein uL18
Q: Large ribosomal subunit protein bL20
R: Large ribosomal subunit protein bL21
T: Large ribosomal subunit protein uL23
U: Large ribosomal subunit protein uL24
V: Large ribosomal subunit protein bL25
W: Large ribosomal subunit protein bL27
Y: Large ribosomal subunit protein uL29
Z: Large ribosomal subunit protein uL30
A: 23S ribosomal RNA
D: 50S ribosomal protein L3
E: Large ribosomal subunit protein uL4
y: Apidaecins type 22


Theoretical massNumber of molelcules
Total (without water)1,188,60919
Polymers1,188,60919
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Large ribosomal subunit protein ... , 15 types, 15 molecules 2FJLNOQRTUVWYZE

#1: Protein/peptide Large ribosomal subunit protein bL34 / 50S ribosomal protein L34


Mass: 5397.463 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MC4100 / References: UniProt: P0A7P5
#3: Protein Large ribosomal subunit protein uL5 / 50S ribosomal protein L5


Mass: 20073.234 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MC4100 / References: UniProt: P62399
#4: Protein Large ribosomal subunit protein uL13 / 50S ribosomal protein L13


Mass: 16050.606 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MC4100 / References: UniProt: P0AA10
#5: Protein Large ribosomal subunit protein uL15 / 50S ribosomal protein L15


Mass: 14877.273 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MC4100 / References: UniProt: P02413
#6: Protein Large ribosomal subunit protein bL17 / 50S ribosomal protein L17


Mass: 13721.938 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MC4100 / References: UniProt: P0AG44
#7: Protein Large ribosomal subunit protein uL18 / 50S ribosomal protein L18


Mass: 12663.471 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MC4100 / References: UniProt: P0C018
#8: Protein Large ribosomal subunit protein bL20 / 50S ribosomal protein L20


Mass: 13396.828 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MC4100 / References: UniProt: P0A7L3
#9: Protein Large ribosomal subunit protein bL21 / 50S ribosomal protein L21


Mass: 11586.374 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MC4100 / References: UniProt: P0AG48
#10: Protein Large ribosomal subunit protein uL23 / 50S ribosomal protein L23


Mass: 10546.472 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MC4100 / References: UniProt: P0ADZ0
#11: Protein Large ribosomal subunit protein uL24 / 50S ribosomal protein L24


Mass: 11078.874 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MC4100 / References: UniProt: P60624
#12: Protein Large ribosomal subunit protein bL25 / 50S ribosomal protein L25


Mass: 10713.465 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MC4100 / References: UniProt: P68919
#13: Protein Large ribosomal subunit protein bL27 / 50S ribosomal protein L27


Mass: 8174.394 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MC4100 / References: UniProt: P0A7L8
#14: Protein Large ribosomal subunit protein uL29 / 50S ribosomal protein L29


Mass: 7286.464 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MC4100 / References: UniProt: P0A7M6
#15: Protein Large ribosomal subunit protein uL30 / 50S ribosomal protein L30


Mass: 6423.625 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MC4100 / References: UniProt: P0AG51
#18: Protein Large ribosomal subunit protein uL4 / 50S ribosomal protein L4


Mass: 22121.566 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MC4100 / References: UniProt: P60723

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RNA chain , 2 types, 2 molecules BA

#2: RNA chain 5S ribosomal RNA


Mass: 38813.133 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MC4100
#16: RNA chain 23S ribosomal RNA


Mass: 941322.188 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MC4100

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Protein / Protein/peptide , 2 types, 2 molecules Dy

#17: Protein 50S ribosomal protein L3 / ribosomal protein uL3


Mass: 22277.535 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MC4100 / References: UniProt: P60438
#19: Protein/peptide Apidaecins type 22


Mass: 2084.452 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Apis mellifera (honey bee) / References: UniProt: P35581

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Details

Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: pre50S precursor derived from Api137-treated cells supplemented with Api137
Type: RIBOSOME / Entity ID: all / Source: NATURAL
Source (natural)Organism: Escherichia coli (E. coli) / Strain: MC4100
Buffer solutionpH: 7.6
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Image recordingAverage exposure time: 1.2 sec. / Electron dose: 46.2 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of real images: 2794
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM softwareName: cryoSPARC / Version: 4.21 / Category: final Euler assignment
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4.37 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 28649 / Symmetry type: POINT

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