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- EMDB-0591: Architectural principles for Hfq/Crc-mediated regulation of gene ... -

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Basic information

Entry
Database: EMDB / ID: EMD-0591
TitleArchitectural principles for Hfq/Crc-mediated regulation of gene expression Hfq-Crc-amiE 2:3:2 complex
Map dataLocally sharpened Cryo-EM map of a Hfq-Crc-amiE assembly with stoichiometry 2:3:2, respectively.
Sample
  • Complex: Assembly of Hfq-Crc-amiE with a 2:3:2 stoichiometry
    • Protein or peptide: Catabolite repression control protein
    • Protein or peptide: RNA-binding protein Hfq
    • RNA: RNA (5'-R(*AP*AP*AP*AP*AP*UP*AP*AP*CP*AP*AP*CP*AP*AP*GP*AP*GP*G)-3')
KeywordsHfq / Crc / amiE / Carbon catabolite repression / RNA-protein interaction / RNA-mediated gene regulation / RNA BINDING PROTEIN / RNA BINDING PROTEIN-RNA-HYDROLASE complex
Function / homology
Function and homology information


regulation of carbon utilization / regulation of translation, ncRNA-mediated / regulation of RNA stability / quorum sensing / exodeoxyribonuclease III / double-stranded DNA 3'-5' DNA exonuclease activity / regulation of translation / DNA repair / regulation of DNA-templated transcription / RNA binding ...regulation of carbon utilization / regulation of translation, ncRNA-mediated / regulation of RNA stability / quorum sensing / exodeoxyribonuclease III / double-stranded DNA 3'-5' DNA exonuclease activity / regulation of translation / DNA repair / regulation of DNA-templated transcription / RNA binding / metal ion binding / cytosol
Similarity search - Function
Exodeoxyribonuclease III-like / RNA-binding protein Hfq / Hfq protein / AP endonuclease 1 / AP endonucleases family 1 profile. / Endonuclease/exonuclease/phosphatase / Endonuclease/Exonuclease/phosphatase family / : / Sm domain profile. / Endonuclease/exonuclease/phosphatase superfamily / LSM domain superfamily
Similarity search - Domain/homology
Catabolite repression control protein / RNA-binding protein Hfq
Similarity search - Component
Biological speciesPseudomonas aeruginosa (bacteria) / Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.37 Å
AuthorsPei XY / Dendooven T
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Wellcome Trust200873/Z/16/Z United Kingdom
CitationJournal: Elife / Year: 2019
Title: Architectural principles for Hfq/Crc-mediated regulation of gene expression.
Authors: Xue Yuan Pei / Tom Dendooven / Elisabeth Sonnleitner / Shaoxia Chen / Udo Bläsi / Ben F Luisi /
Abstract: In diverse bacterial species, the global regulator Hfq contributes to post-transcriptional networks that control expression of numerous genes. Hfq of the opportunistic pathogen inhibits translation ...In diverse bacterial species, the global regulator Hfq contributes to post-transcriptional networks that control expression of numerous genes. Hfq of the opportunistic pathogen inhibits translation of target transcripts by forming a regulatory complex with the catabolite repression protein Crc. This repressive complex acts as part of an intricate mechanism of preferred nutrient utilisation. We describe high-resolution cryo-EM structures of the assembly of Hfq and Crc bound to the translation initiation site of a target mRNA. The core of the assembly is formed through interactions of two cognate RNAs, two Hfq hexamers and a Crc pair. Additional Crc protomers are recruited to the core to generate higher-order assemblies with demonstrated regulatory activity in vivo. This study reveals how Hfq cooperates with a partner protein to regulate translation, and provides a structural basis for an RNA code that guides global regulators to interact cooperatively and regulate different RNA targets.
History
DepositionFeb 20, 2019-
Header (metadata) releaseMar 13, 2019-
Map releaseMar 13, 2019-
UpdateMar 20, 2024-
Current statusMar 20, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.27
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.27
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6o1l
  • Surface level: 0.27
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0591.png

Downloads & links

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Map

FileDownload / File: emd_0591.map.gz / Format: CCP4 / Size: 26.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLocally sharpened Cryo-EM map of a Hfq-Crc-amiE assembly with stoichiometry 2:3:2, respectively.
Voxel sizeX=Y=Z: 1.09 Å
Density
Contour LevelBy AUTHOR: 0.27 / Movie #1: 0.27
Minimum - Maximum-0.01850905 - 1.9583755
Average (Standard dev.)0.0037314594 (±0.054603253)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions190190190
Spacing190190190
CellA=B=C: 207.1 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.091.091.09
M x/y/z190190190
origin x/y/z0.0000.0000.000
length x/y/z207.100207.100207.100
α/β/γ90.00090.00090.000
start NX/NY/NZ-205-205-205
NX/NY/NZ411411411
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS190190190
D min/max/mean-0.0191.9580.004

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Supplemental data

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Mask #1

Fileemd_0591_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Globally sharpened Cryo-EM map of a Hfq-Crc-amiE assembly...

Fileemd_0591_additional.map
AnnotationGlobally sharpened Cryo-EM map of a Hfq-Crc-amiE assembly with stoichiometry 2:3:2, respectively.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1

Fileemd_0591_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_0591_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 : Assembly of Hfq-Crc-amiE with a 2:3:2 stoichiometry

EntireName: Assembly of Hfq-Crc-amiE with a 2:3:2 stoichiometry
Components
  • Complex: Assembly of Hfq-Crc-amiE with a 2:3:2 stoichiometry
    • Protein or peptide: Catabolite repression control protein
    • Protein or peptide: RNA-binding protein Hfq
    • RNA: RNA (5'-R(*AP*AP*AP*AP*AP*UP*AP*AP*CP*AP*AP*CP*AP*AP*GP*AP*GP*G)-3')

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Supramolecule #1: Assembly of Hfq-Crc-amiE with a 2:3:2 stoichiometry

SupramoleculeName: Assembly of Hfq-Crc-amiE with a 2:3:2 stoichiometry / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Molecular weightTheoretical: 222 KDa

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Macromolecule #1: Catabolite repression control protein

MacromoleculeName: Catabolite repression control protein / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO / EC number: exodeoxyribonuclease III
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Molecular weightTheoretical: 30.101869 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: GPAMRIISVN VNGIQAAAER GLLSWLQAQN ADVICLQDTR ASAFDLDDPS FQLDGYFLYA CDAELPEQGG VALYSRLQPK AVISGLGFE TADRYGRYLQ ADFDKVSIAT LLLPSGQSGD ESLNQKFKFM DDFTHYLSKQ RRKRREYIYC GSLYVAHQKM D VKNWRECQ ...String:
GPAMRIISVN VNGIQAAAER GLLSWLQAQN ADVICLQDTR ASAFDLDDPS FQLDGYFLYA CDAELPEQGG VALYSRLQPK AVISGLGFE TADRYGRYLQ ADFDKVSIAT LLLPSGQSGD ESLNQKFKFM DDFTHYLSKQ RRKRREYIYC GSLYVAHQKM D VKNWRECQ QMPGFLAPER AWLDEVFGNL GYADALREVS REGDQFSWWP DSEQAEMLNL GWRFDYQVLT PGLRRFVRNA KL PRQPRFS QHAPLIVDYD WQLSI

UniProtKB: Catabolite repression control protein

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Macromolecule #2: RNA-binding protein Hfq

MacromoleculeName: RNA-binding protein Hfq / type: protein_or_peptide / ID: 2 / Number of copies: 12 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1) (bacteria)
Strain: ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1
Molecular weightTheoretical: 7.685984 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
HSLQDPYLNT LRKERVPVSI YLVNGIKLQG QIESFDQFVI LLKNTVSQMV YKHAISTVVP SRPVRLP

UniProtKB: RNA-binding protein Hfq

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Macromolecule #3: RNA (5'-R(*AP*AP*AP*AP*AP*UP*AP*AP*CP*AP*AP*CP*AP*AP*GP*AP*GP*G)-3')

MacromoleculeName: RNA (5'-R(*AP*AP*AP*AP*AP*UP*AP*AP*CP*AP*AP*CP*AP*AP*GP*AP*GP*G)-3')
type: rna / ID: 3 / Number of copies: 2
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Molecular weightTheoretical: 5.85766 KDa
SequenceString:
AAAAAUAACA ACAAGAGG

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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: 7.9
Component:
ConcentrationName
20.0 mMHEPES pH 7.9
40.0 mMNaClSodium chloride
10.0 mMKCl
1.0 mMMgCl2
2.0 mMTCEP
GridModel: Quantifoil, UltrAuFoil, R1.2/1.3 / Material: GRAPHENE OXIDE / Mesh: 300 / Support film - Material: GRAPHENE OXIDE / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K
DetailsMonodisperse

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated magnification: 130841 / Illumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 0.003 µm / Nominal defocus min: 0.00125 µm / Nominal magnification: 125000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 70.0 K / Max: 100.0 K
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 917 / Average exposure time: 60.0 sec. / Average electron dose: 28.0 e/Å2
Details: Images were collected in movie-counting mode, 75 frames per movie stack, 60s exposure per movie
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 435000
Startup modelType of model: INSILICO MODEL / In silico model: ab-initio map generated in Relion3
Initial angle assignmentType: OTHER / Software - Name: RELION (ver. 3b) / Software - details: beta version / Details: Stochastic Gradient Descent
Final 3D classificationNumber classes: 6 / Avg.num./class: 35000 / Software - Name: RELION (ver. 3b) / Software - details: beta version
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3b) / Software - details: beta version
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.37 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3b) / Number images used: 45311
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

1u1t

source_name: PDB, initial_model_type: experimental model

4jg3

source_name: PDB, initial_model_type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: Cross-correlation
Output model

PDB-6o1l:
Architectural principles for Hfq/Crc-mediated regulation of gene expression Hfq-Crc-amiE 2:3:2 complex

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