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- PDB-6pcq: E. coli 50S ribosome bound to VM2 -

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Basic information

Entry
Database: PDB / ID: 6pcq
TitleE. coli 50S ribosome bound to VM2
Components
  • (50S ribosomal protein ...) x 5
  • 23S ribosomal RNA
  • 5S ribosomal RNA
KeywordsRIBOSOME / E. coli ribosome / streptogramin A analog / antibiotics
Function / homology
Function and homology information


transcriptional attenuation / endoribonuclease inhibitor activity / RNA-binding transcription regulator activity / negative regulation of cytoplasmic translation / DnaA-L2 complex / translation repressor activity / negative regulation of DNA-templated DNA replication initiation / ribosome assembly / DNA-templated transcription termination / mRNA 5'-UTR binding ...transcriptional attenuation / endoribonuclease inhibitor activity / RNA-binding transcription regulator activity / negative regulation of cytoplasmic translation / DnaA-L2 complex / translation repressor activity / negative regulation of DNA-templated DNA replication initiation / ribosome assembly / DNA-templated transcription termination / mRNA 5'-UTR binding / large ribosomal subunit / large ribosomal subunit rRNA binding / transferase activity / ribosomal large subunit assembly / cytoplasmic translation / cytosolic large ribosomal subunit / negative regulation of translation / rRNA binding / ribosome / structural constituent of ribosome / translation / response to antibiotic / negative regulation of DNA-templated transcription / mRNA binding / DNA binding / RNA binding / zinc ion binding / cytosol / cytoplasm
Similarity search - Function
Ribosomal protein L2, bacterial/organellar-type / Ribosomal protein L13, bacterial-type / Ribosomal protein L3, bacterial/organelle-type / Ribosomal protein L15, bacterial-type / 50S ribosomal protein uL4 / Ribosomal protein L2 signature. / Ribosomal protein L15, conserved site / Ribosomal protein L15 signature. / Ribosomal protein L2, conserved site / Ribosomal protein L13 signature. ...Ribosomal protein L2, bacterial/organellar-type / Ribosomal protein L13, bacterial-type / Ribosomal protein L3, bacterial/organelle-type / Ribosomal protein L15, bacterial-type / 50S ribosomal protein uL4 / Ribosomal protein L2 signature. / Ribosomal protein L15, conserved site / Ribosomal protein L15 signature. / Ribosomal protein L2, conserved site / Ribosomal protein L13 signature. / Ribosomal protein L2, domain 3 / Ribosomal protein L13, conserved site / Ribosomal Proteins L2, C-terminal domain / Ribosomal protein L2, C-terminal / Ribosomal Proteins L2, C-terminal domain / Ribosomal Proteins L2, RNA binding domain / Ribosomal Proteins L2, RNA binding domain / Ribosomal Proteins L2, RNA binding domain / Ribosomal protein L2 / Ribosomal protein L13 / Ribosomal protein L13 / Ribosomal protein L13 superfamily / Ribosomal protein L15 / Ribosomal proteins 50S-L15, 50S-L18e, 60S-L27A / Ribosomal protein L3, conserved site / Ribosomal protein L18e/L15P / Ribosomal L18e/L15P superfamily / Ribosomal protein L3 signature. / Ribosomal protein L3 / Ribosomal protein L3 / Ribosomal protein L4/L1e / Ribosomal protein L4 domain superfamily / Ribosomal protein L4/L1 family / Translation protein SH3-like domain superfamily / Translation protein, beta-barrel domain superfamily / Ribosomal protein L2, domain 2 / Nucleic acid-binding, OB-fold
Similarity search - Domain/homology
Chem-O8J / : / RNA / RNA (> 10) / RNA (> 100) / RNA (> 1000) / Large ribosomal subunit protein uL15 / 50S ribosomal protein L4 / 50S ribosomal protein L13 / Large ribosomal subunit protein uL15 ...Chem-O8J / : / RNA / RNA (> 10) / RNA (> 100) / RNA (> 1000) / Large ribosomal subunit protein uL15 / 50S ribosomal protein L4 / 50S ribosomal protein L13 / Large ribosomal subunit protein uL15 / Large ribosomal subunit protein uL13 / Large ribosomal subunit protein uL2 / Large ribosomal subunit protein uL3 / Large ribosomal subunit protein uL4
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.6 Å
AuthorsPellegrino, J. / Lee, D.J. / Fraser, J.S. / Seiple, I.B.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM123159 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM124149 United States
CitationJournal: Nature / Year: 2020
Title: Synthetic group A streptogramin antibiotics that overcome Vat resistance.
Authors: Qi Li / Jenna Pellegrino / D John Lee / Arthur A Tran / Hector A Chaires / Ruoxi Wang / Jesslyn E Park / Kaijie Ji / David Chow / Na Zhang / Axel F Brilot / Justin T Biel / Gydo van Zundert ...Authors: Qi Li / Jenna Pellegrino / D John Lee / Arthur A Tran / Hector A Chaires / Ruoxi Wang / Jesslyn E Park / Kaijie Ji / David Chow / Na Zhang / Axel F Brilot / Justin T Biel / Gydo van Zundert / Kenneth Borrelli / Dean Shinabarger / Cindy Wolfe / Beverly Murray / Matthew P Jacobson / Estelle Mühle / Olivier Chesneau / James S Fraser / Ian B Seiple /
Abstract: Natural products serve as chemical blueprints for most antibiotics in clinical use. The evolutionary process by which these molecules arise is inherently accompanied by the co-evolution of resistance ...Natural products serve as chemical blueprints for most antibiotics in clinical use. The evolutionary process by which these molecules arise is inherently accompanied by the co-evolution of resistance mechanisms that shorten the clinical lifetime of any given class of antibiotics. Virginiamycin acetyltransferase (Vat) enzymes are resistance proteins that provide protection against streptogramins, potent antibiotics against Gram-positive bacteria that inhibit the bacterial ribosome. Owing to the challenge of selectively modifying the chemically complex, 23-membered macrocyclic scaffold of group A streptogramins, analogues that overcome the resistance conferred by Vat enzymes have not been previously developed. Here we report the design, synthesis, and antibacterial evaluation of group A streptogramin antibiotics with extensive structural variability. Using cryo-electron microscopy and forcefield-based refinement, we characterize the binding of eight analogues to the bacterial ribosome at high resolution, revealing binding interactions that extend into the peptidyl tRNA-binding site and towards synergistic binders that occupy the nascent peptide exit tunnel. One of these analogues has excellent activity against several streptogramin-resistant strains of Staphylococcus aureus, exhibits decreased rates of acetylation in vitro, and is effective at lowering bacterial load in a mouse model of infection. Our results demonstrate that the combination of rational design and modular chemical synthesis can revitalize classes of antibiotics that are limited by naturally arising resistance mechanisms.
History
DepositionJun 18, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 17, 2020Provider: repository / Type: Initial release
Revision 1.1Sep 23, 2020Group: Database references / Derived calculations / Category: citation / citation_author / struct_conn
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name / _struct_conn.pdbx_dist_value / _struct_conn.pdbx_leaving_atom_flag / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn.ptnr2_label_seq_id
Revision 1.2Oct 7, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title

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Structure visualization

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Assembly

Deposited unit
I: 23S ribosomal RNA
J: 5S ribosomal RNA
K: 50S ribosomal protein L2
L: 50S ribosomal protein L15
M: 50S ribosomal protein L4
N: 50S ribosomal protein L3
O: 50S ribosomal protein L13
hetero molecules


Theoretical massNumber of molelcules
Total (without water)1,085,6228
Polymers1,085,0957
Non-polymers5281
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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RNA chain , 2 types, 2 molecules IJ

#1: RNA chain 23S ribosomal RNA


Mass: 941795.562 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli)
#2: RNA chain 5S ribosomal RNA


Mass: 38177.762 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: GenBank: 1266940032

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50S ribosomal protein ... , 5 types, 5 molecules KLMNO

#3: Protein 50S ribosomal protein L2 / Large ribosomal subunit protein uL2


Mass: 29663.244 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P60422
#4: Protein 50S ribosomal protein L15


Mass: 15008.471 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: A0A037Y8L6, UniProt: P02413*PLUS
#5: Protein 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) / References: UniProt: D7Z9F6, UniProt: P60723*PLUS
#6: Protein 50S ribosomal protein L3 / Large ribosomal subunit protein uL3


Mass: 22277.535 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P60438
#7: Protein 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) / References: UniProt: D7ZET0, UniProt: P0AA10*PLUS

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Non-polymers , 1 types, 1 molecules

#8: Chemical ChemComp-O8J / (3R,4R,5E,10E,12E,14S,26aR)-14-hydroxy-4,12-dimethyl-3-(propan-2-yl)-8,9,14,15,24,25,26,26a-octahydro-1H,3H,22H-21,18-(azeno)pyrrolo[2,1-c][1,8,4,19]dioxadiazacyclotetracosine-1,7,16,22(4H,17H)-tetrone


Mass: 527.609 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C28H37N3O7 / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY

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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: 50S E. coli ribosome / Type: RIBOSOME / Entity ID: #1-#7 / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 71.2 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 31630 / Symmetry type: POINT

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