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- EMDB-6149: Electron cryo-microscopy structure of EngA bound with the 50S rib... -

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Basic information

Entry
Database: EMDB / ID: EMD-6149
TitleElectron cryo-microscopy structure of EngA bound with the 50S ribosomal subunit
Map dataReconstruction of E. coli 50S subunit and EngA
Sample
  • Sample: EngA bound with E. coli 50S subunit
  • Complex: 50S ribosomeProkaryotic large ribosomal subunit
  • Protein or peptide: EngA
KeywordsRibosome assembly / EngA / Der / YphC / GTPase / RNA folding
Function / homology
Function and homology information


GTPase activating protein binding / guanosine tetraphosphate binding / stringent response / ribosomal large subunit binding / transcriptional attenuation / endoribonuclease inhibitor activity / RNA-binding transcription regulator activity / positive regulation of ribosome biogenesis / negative regulation of cytoplasmic translation / translational termination ...GTPase activating protein binding / guanosine tetraphosphate binding / stringent response / ribosomal large subunit binding / 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 / negative regulation of translational initiation / translation repressor activity / negative regulation of DNA-templated DNA replication initiation / ribosome assembly / mRNA regulatory element binding translation repressor activity / response to reactive oxygen species / assembly of large subunit precursor of preribosome / cytosolic ribosome assembly / regulation of cell growth / DNA-templated transcription termination / : / response to radiation / mRNA 5'-UTR binding / large ribosomal subunit rRNA binding / ribosome binding / ribosomal large subunit assembly / large ribosomal subunit / 5S rRNA binding / cytosolic large ribosomal subunit / cytoplasmic translation / transferase activity / tRNA binding / negative regulation of translation / ribosome / rRNA binding / structural constituent of ribosome / translation / response to antibiotic / GTPase activity / mRNA binding / negative regulation of DNA-templated transcription / GTP binding / DNA binding / RNA binding / zinc ion binding / cytosol / cytoplasm
Similarity search - Function
GTP-binding protein EngA / EngA-type guanine nucleotide-binding (G) domain / EngA-type guanine nucleotide-binding (G) domain profile. / GTPase Der, C-terminal KH-domain-like / KH-domain-like of EngA bacterial GTPase enzymes, C-terminal / 50S ribosome-binding GTPase / Ribosomal protein L1, bacterial-type / GTP binding domain / Ribosomal protein L1, conserved site / Ribosomal protein L1 ...GTP-binding protein EngA / EngA-type guanine nucleotide-binding (G) domain / EngA-type guanine nucleotide-binding (G) domain profile. / GTPase Der, C-terminal KH-domain-like / KH-domain-like of EngA bacterial GTPase enzymes, C-terminal / 50S ribosome-binding GTPase / Ribosomal protein L1, bacterial-type / GTP binding domain / Ribosomal protein L1, conserved site / Ribosomal protein L1 / Ribosomal protein L1 signature. / Ribosomal protein L1, 3-layer alpha/beta-sandwich / Ribosomal protein L25, short-form / Ribosomal protein L1-like / Ribosomal protein L1/ribosomal biogenesis protein / Ribosomal protein L1p/L10e family / Ribosomal protein L11, bacterial-type / Ribosomal protein L21, conserved site / Ribosomal protein L21 signature. / Ribosomal protein L11, conserved site / 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 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 L25p family / Ribosomal protein L25 / Ribosomal protein L11, N-terminal / Ribosomal protein L11, N-terminal domain / 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, RNA binding domain / Ribosomal protein L11/L12 / 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 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 L27, conserved site / Ribosomal protein L27 signature. / Ribosomal protein L14P, bacterial-type / Ribosomal protein L34, conserved site / Ribosomal protein L34 signature. / Ribosomal protein L22, bacterial/chloroplast-type / Ribosomal protein L35 / Ribosomal protein L35 superfamily / Ribosomal protein L2, bacterial/organellar-type / Ribosomal protein L35 / Ribosomal L28 family / Ribosomal protein L28/L24 / : / Ribosomal protein L30, bacterial-type / Ribosomal protein L16 / Ribosomal protein L18 / Ribosomal L18 of archaea, bacteria, mitoch. and chloroplast / L28p-like / Ribosomal protein L20 / Ribosomal protein L20 / Ribosomal protein L20, C-terminal / Ribosomal protein L21 / Ribosomal protein L27 / Ribosomal L27 protein / Ribosomal protein L19 / Ribosomal protein L19 superfamily / Ribosomal protein L19 / Ribosomal proteins 50S L24/mitochondrial 39S L24 / Ribosomal protein L17 / Ribosomal protein L17 superfamily / Ribosomal protein L17 / Ribosomal protein L21-like / L21-like superfamily / Ribosomal prokaryotic L21 protein
Similarity search - Domain/homology
: / : / : / : / : / : / : / : / : / : ...: / : / : / : / : / : / : / : / : / : / : / : / : / : / : / : / : / : / : / : / : / : / : / : / : / : / : / : / : / Large ribosomal subunit protein uL15 / GTPase Der / Large ribosomal subunit protein uL11 / Large ribosomal subunit protein bL19 / Large ribosomal subunit protein uL1 / Large ribosomal subunit protein bL20 / Large ribosomal subunit protein bL27 / Large ribosomal subunit protein bL28 / Large ribosomal subunit protein uL29 / Large ribosomal subunit protein bL32 / Large ribosomal subunit protein bL34 / Large ribosomal subunit protein bL35 / Large ribosomal subunit protein bL36A / Large ribosomal subunit protein bL9 / Large ribosomal subunit protein uL13 / Large ribosomal subunit protein uL14 / Large ribosomal subunit protein uL16 / Large ribosomal subunit protein uL23 / Large ribosomal subunit protein bL17 / Large ribosomal subunit protein bL21 / Large ribosomal subunit protein uL30 / Large ribosomal subunit protein uL6 / Large ribosomal subunit protein uL18 / 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 / Large ribosomal subunit protein bL25
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.0 Å
AuthorsZhang X / Yan K / Lei J / Gao N
CitationJournal: Nucleic Acids Res / Year: 2014
Title: Structural insights into the function of a unique tandem GTPase EngA in bacterial ribosome assembly.
Authors: Xiaoxiao Zhang / Kaige Yan / Yixiao Zhang / Ningning Li / Chengying Ma / Zhifei Li / Yanqing Zhang / Boya Feng / Jing Liu / Yadong Sun / Yanji Xu / Jianlin Lei / Ning Gao /
Abstract: Many ribosome-interacting GTPases, with proposed functions in ribosome biogenesis, are also implicated in the cellular regulatory coupling between ribosome assembly process and various growth control ...Many ribosome-interacting GTPases, with proposed functions in ribosome biogenesis, are also implicated in the cellular regulatory coupling between ribosome assembly process and various growth control pathways. EngA is an essential GTPase in bacteria, and intriguingly, it contains two consecutive GTPase domains (GD), being one-of-a-kind among all known GTPases. EngA is required for the 50S subunit maturation. However, its molecular role remains elusive. Here, we present the structure of EngA bound to the 50S subunit. Our data show that EngA binds to the peptidyl transferase center (PTC) and induces dramatic conformational changes on the 50S subunit, which virtually returns the 50S subunit to a state similar to that of the late-stage 50S assembly intermediates. Very interestingly, our data show that the two GDs exhibit a pseudo-two-fold symmetry in the 50S-bound conformation. Our results indicate that EngA recognizes certain forms of the 50S assembly intermediates, and likely facilitates the conformational maturation of the PTC of the 23S rRNA in a direct manner. Furthermore, in a broad context, our data also suggest that EngA might be a sensor of the cellular GTP/GDP ratio, endowed with multiple conformational states, in response to fluctuations in cellular nucleotide pool, to facilitate and regulate ribosome assembly.
History
DepositionOct 23, 2014-
Header (metadata) releaseNov 26, 2014-
Map releaseNov 26, 2014-
UpdateJun 3, 2015-
Current statusJun 3, 2015Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.045
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.045
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3j8g
  • Surface level: 0.045
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_6149.map.gz / Format: CCP4 / Size: 122.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of E. coli 50S subunit and EngA
Voxel sizeX=Y=Z: 1.1659 Å
Density
Contour LevelBy AUTHOR: 0.045 / Movie #1: 0.045
Minimum - Maximum-0.09316271 - 0.23179024
Average (Standard dev.)0.00017538 (±0.01283344)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-159-159-159
Dimensions320320320
Spacing320320320
CellA=B=C: 373.088 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.16591.16591.1659
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z373.088373.088373.088
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-159-159-159
NC/NR/NS320320320
D min/max/mean-0.0930.2320.000

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Supplemental data

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Sample components

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Entire : EngA bound with E. coli 50S subunit

EntireName: EngA bound with E. coli 50S subunit
Components
  • Sample: EngA bound with E. coli 50S subunit
  • Complex: 50S ribosomeProkaryotic large ribosomal subunit
  • Protein or peptide: EngA

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Supramolecule #1000: EngA bound with E. coli 50S subunit

SupramoleculeName: EngA bound with E. coli 50S subunit / type: sample / ID: 1000 / Number unique components: 2

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Supramolecule #1: 50S ribosome

SupramoleculeName: 50S ribosome / type: complex / ID: 1 / Recombinant expression: No / Database: NCBI
Ribosome-details: ribosome-prokaryote: LSU 50S, LSU RNA 23S, LSU RNA 5S
Source (natural)Organism: Escherichia coli K-12 (bacteria)

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Macromolecule #1: EngA

MacromoleculeName: EngA / type: protein_or_peptide / ID: 1 / Name.synonym: Der / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: BL21(DE3) / Recombinant plasmid: pET28a

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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: 7.5
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: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
DateApr 1, 2012
Image recordingCategory: CCD / Film or detector model: FEI EAGLE (4k x 4k)
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionResolution.type: BY AUTHOR / Resolution: 5.0 Å / Resolution method: OTHER / Software - Name: Relion / Number images used: 189614
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

2wwq
PDB Unreleased entry

RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-3j8g:
Electron cryo-microscopy structure of EngA bound with the 50S ribosomal subunit

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Atomic model buiding 2

Initial modelPDB ID:
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-3j8g:
Electron cryo-microscopy structure of EngA bound with the 50S ribosomal subunit

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