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- EMDB-6311: Cryo-EM structure of tetracycline resistance protein TetM bound t... -

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Basic information

Entry
Database: EMDB / ID: EMD-6311
TitleCryo-EM structure of tetracycline resistance protein TetM bound to a translating E. coli ribosome
Map dataReconstruction of TetM bound to translating E. coli ribosomes.
Sample
  • Sample: TetM bound to ErmCL-RNCs
  • Complex: ErmCL-stalled ribosome
  • Protein or peptide: Tetracycline resistance protein TetMTetracycline
Keywordsantibiotics / protein synthesis / resistance / ribosome / TetM / tetracycline / tigecycline / translation
Function / homology
Function and homology information


negative regulation of cytoplasmic translational initiation / stringent response / mRNA base-pairing translational repressor activity / ornithine decarboxylase inhibitor activity / misfolded RNA binding / transcription antitermination factor activity, RNA binding / Group I intron splicing / RNA folding / transcriptional attenuation / endoribonuclease inhibitor activity ...negative regulation of cytoplasmic translational initiation / stringent response / mRNA base-pairing translational repressor activity / ornithine decarboxylase inhibitor activity / misfolded RNA binding / transcription antitermination factor activity, RNA binding / Group I intron splicing / RNA folding / transcriptional attenuation / endoribonuclease inhibitor activity / RNA-binding transcription regulator activity / positive regulation of ribosome biogenesis / negative regulation of cytoplasmic translation / translational termination / DnaA-L2 complex / four-way junction DNA binding / translation repressor activity / negative regulation of translational initiation / translational initiation / negative regulation of DNA-templated DNA replication initiation / regulation of mRNA stability / ribosome assembly / mRNA regulatory element binding translation repressor activity / response to reactive oxygen species / assembly of large subunit precursor of preribosome / transcription elongation factor complex / positive regulation of RNA splicing / DNA endonuclease activity / : / cytosolic ribosome assembly / regulation of DNA-templated transcription elongation / transcription antitermination / regulation of cell growth / maintenance of translational fidelity / DNA-templated transcription termination / response to radiation / mRNA 5'-UTR binding / ribosomal small subunit biogenesis / small ribosomal subunit rRNA binding / ribosomal small subunit assembly / ribosomal large subunit assembly / cytosolic small ribosomal subunit / large ribosomal subunit rRNA binding / ribosome binding / large ribosomal subunit / ribosome biogenesis / regulation of translation / small ribosomal subunit / 5S rRNA binding / cytosolic large ribosomal subunit / cytoplasmic translation / transferase activity / tRNA binding / negative regulation of translation / rRNA binding / molecular adaptor activity / ribosome / structural constituent of ribosome / translation / response to antibiotic / mRNA binding / GTPase activity / negative regulation of DNA-templated transcription / GTP binding / DNA binding / RNA binding / zinc ion binding / membrane / cytosol / cytoplasm
Similarity search - Function
Tetracycline resistance protein, C-terminal / Elongation Factor G, domain II / Elongation Factor G, domain III / Translation elongation factor EFG/EF2, domain IV / Elongation factor G, domain IV / Elongation factor G, domain IV / Ribosomal protein L10, eubacterial, conserved site / Ribosomal protein L10 signature. / Elongation factor EFG, domain V-like / Elongation factor G C-terminus ...Tetracycline resistance protein, C-terminal / Elongation Factor G, domain II / Elongation Factor G, domain III / Translation elongation factor EFG/EF2, domain IV / Elongation factor G, domain IV / Elongation factor G, domain IV / Ribosomal protein L10, eubacterial, conserved site / Ribosomal protein L10 signature. / Elongation factor EFG, domain V-like / Elongation factor G C-terminus / Ribosomal protein L10 / EF-G domain III/V-like / : / Tr-type G domain, conserved site / Translational (tr)-type guanine nucleotide-binding (G) domain signature. / Ribosomal protein S21, conserved site / Ribosomal protein S21 signature. / Ribosomal protein L25, short-form / Ribosomal protein S14, bacterial/plastid / Ribosomal protein L11, bacterial-type / Ribosomal protein L31 type A / Ribosomal protein S21 superfamily / Ribosomal protein S21 / Ribosomal protein S16, conserved site / Ribosomal protein S16 signature. / Translational (tr)-type GTP-binding domain / Elongation factor Tu GTP binding domain / Translational (tr)-type guanine nucleotide-binding (G) domain profile. / Ribosomal protein L31 signature. / Ribosomal protein S21 / 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 L9 signature. / Ribosomal protein L9, bacteria/chloroplast / Ribosomal protein L9, C-terminal / Ribosomal protein L9, C-terminal domain / Ribosomal protein L9, C-terminal domain superfamily / 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 L25p family / Ribosomal protein L25 / Ribosomal protein L28/L24 superfamily / Ribosomal protein L36 signature. / Ribosomal protein L25/Gln-tRNA synthetase, N-terminal / Ribosomal protein L32p, bacterial type / Ribosomal protein L25/Gln-tRNA synthetase, anti-codon-binding domain superfamily / Ribosomal protein L9, N-terminal domain superfamily / Ribosomal protein L9 / Ribosomal protein L9, N-terminal / Ribosomal protein L9, N-terminal domain / 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 L9/RNase H1, N-terminal / 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
Similarity search - Domain/homology
: / Small ribosomal subunit protein bS6 / Small ribosomal subunit protein uS7 / Large ribosomal subunit protein uL15 / Large ribosomal subunit protein uL10 / Large ribosomal subunit protein uL11 / Large ribosomal subunit protein bL19 / Large ribosomal subunit protein bL20 / Large ribosomal subunit protein bL27 / Large ribosomal subunit protein bL28 ...: / Small ribosomal subunit protein bS6 / Small ribosomal subunit protein uS7 / Large ribosomal subunit protein uL15 / Large ribosomal subunit protein uL10 / Large ribosomal subunit protein uL11 / Large ribosomal subunit protein bL19 / Large ribosomal subunit protein bL20 / Large ribosomal subunit protein bL27 / Large ribosomal subunit protein bL28 / Large ribosomal subunit protein uL29 / Large ribosomal subunit protein bL31 / Large ribosomal subunit protein bL32 / Large ribosomal subunit protein bL33 / Large ribosomal subunit protein bL34 / Large ribosomal subunit protein bL35 / Large ribosomal subunit protein bL36A / Large ribosomal subunit protein bL9 / Small ribosomal subunit protein uS10 / Small ribosomal subunit protein uS11 / Small ribosomal subunit protein uS12 / Small ribosomal subunit protein uS13 / Small ribosomal subunit protein bS16 / Small ribosomal subunit protein bS18 / Small ribosomal subunit protein uS19 / Small ribosomal subunit protein bS20 / Small ribosomal subunit protein uS2 / Small ribosomal subunit protein uS3 / Small ribosomal subunit protein uS4 / Small ribosomal subunit protein uS5 / Small ribosomal subunit protein uS8 / Small ribosomal subunit protein uS9 / Large ribosomal subunit protein uL13 / Large ribosomal subunit protein uL14 / Large ribosomal subunit protein uL16 / Large ribosomal subunit protein uL23 / Small ribosomal subunit protein uS15 / Large ribosomal subunit protein bL17 / Large ribosomal subunit protein bL21 / Large ribosomal subunit protein uL30 / Large ribosomal subunit protein uL6 / Small ribosomal subunit protein uS14 / Small ribosomal subunit protein uS17 / Large ribosomal subunit protein uL18 / Tetracycline resistance protein TetM from transposon Tn916 / Large ribosomal subunit protein uL2 / Large ribosomal subunit protein uL3 / Large ribosomal subunit protein uL24 / Large ribosomal subunit protein uL4 / Large ribosomal subunit protein uL22 / Large ribosomal subunit protein uL5 / Small ribosomal subunit protein bS21 / Large ribosomal subunit protein bL25
Similarity search - Component
Biological speciesEscherichia coli (E. coli) / Enterococcus faecalis (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsArenz S / Nguyen F / Beckmann R / Wilson DN
CitationJournal: Proc Natl Acad Sci U S A / Year: 2015
Title: Cryo-EM structure of the tetracycline resistance protein TetM in complex with a translating ribosome at 3.9-Å resolution.
Authors: Stefan Arenz / Fabian Nguyen / Roland Beckmann / Daniel N Wilson /
Abstract: Ribosome protection proteins (RPPs) confer resistance to tetracycline by binding to the ribosome and chasing the drug from its binding site. Current models for RPP action are derived from 7.2- to 16- ...Ribosome protection proteins (RPPs) confer resistance to tetracycline by binding to the ribosome and chasing the drug from its binding site. Current models for RPP action are derived from 7.2- to 16-Å resolution structures of RPPs bound to vacant or nontranslating ribosomes. Here we present a cryo-electron microscopy reconstruction of the RPP TetM in complex with a translating ribosome at 3.9-Å resolution. The structure reveals the contacts of TetM with the ribosome, including interaction between the conserved and functionally critical C-terminal extension of TetM with a unique splayed conformation of nucleotides A1492 and A1493 at the decoding center of the small subunit. The resolution enables us to unambiguously model the side chains of the amino acid residues comprising loop III in domain IV of TetM, revealing that the tyrosine residues Y506 and Y507 are not responsible for drug-release as suggested previously but rather for intrafactor contacts that appear to stabilize the conformation of loop III. Instead, Pro509 at the tip of loop III is located directly within the tetracycline binding site where it interacts with nucleotide C1054 of the 16S rRNA, such that RPP action uses Pro509, rather than Y506/Y507, to directly dislodge and release tetracycline from the ribosome.
History
DepositionMar 23, 2015-
Header (metadata) releaseApr 15, 2015-
Map releaseApr 15, 2015-
UpdateMay 27, 2015-
Current statusMay 27, 2015Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0025
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.0025
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3j9y
  • Surface level: 0.0025
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_6311.png

Downloads & links

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Map

FileDownload / File: emd_6311.map.gz / Format: CCP4 / Size: 185.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of TetM bound to translating E. coli ribosomes.
Voxel sizeX=Y=Z: 1.108 Å
Density
Contour LevelBy AUTHOR: 0.0025 / Movie #1: 0.0025
Minimum - Maximum-0.00462703 - 0.01004424
Average (Standard dev.)0.00000486 (±0.00067502)
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
start NX/NY/NZ-72-72-72
NX/NY/NZ145145145
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS368368368
D min/max/mean-0.0050.0100.000

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Supplemental data

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Sample components

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Entire : TetM bound to ErmCL-RNCs

EntireName: TetM bound to ErmCL-RNCs
Components
  • Sample: TetM bound to ErmCL-RNCs
  • Complex: ErmCL-stalled ribosome
  • Protein or peptide: Tetracycline resistance protein TetMTetracycline

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Supramolecule #1000: TetM bound to ErmCL-RNCs

SupramoleculeName: TetM bound to ErmCL-RNCs / type: sample / ID: 1000 / Number unique components: 2

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Supramolecule #1: ErmCL-stalled ribosome

SupramoleculeName: ErmCL-stalled ribosome / type: complex / ID: 1 / Recombinant expression: No / Database: NCBI / Ribosome-details: ribosome-prokaryote: ALL
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #1: Tetracycline resistance protein TetM

MacromoleculeName: Tetracycline resistance protein TetM / type: protein_or_peptide / ID: 1 / Name.synonym: TetM / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Enterococcus faecalis (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)

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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: 7.4
Details: 50 mM HEPES-KOH, 100 mM KOAc, 25 mM Mg(OAc)2, 6 mM b-mercaptoethanol
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: 3.5 µm / Nominal defocus min: 0.7 µm / Nominal magnification: 125085 / Cs: mm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Cs0
DateMar 14, 2014
Image recordingCategory: CCD / Film or detector model: FEI FALCON II (4k x 4k) / Number real images: 2753 / 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, an FSC cut-off value of 0.143 was used for average resolution determination of 3.9 A ...Details: Since images from microscopy were processed in the absence of spatial frequencies higher than 8 A, an FSC cut-off value of 0.143 was used for average resolution determination of 3.9 A (Scheres and Chen, 2012). The final map was sharpened by applying an automatically determined negative B-factor using the program EMBFACTOR (Fernandez et al, 2008).
Number images used: 78186

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Atomic model buiding 1

Initial modelPDB ID:

5afi

SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-3j9y:
Cryo-EM structure of tetracycline resistance protein TetM bound to a translating E.coli ribosome

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