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

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Entry
Database: EMDB / ID: 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.
SampleAssembly of Hfq-Crc-amiE with a 2:3:2 stoichiometry:
Catabolite repression control protein / RNA-binding protein Hfq / nucleic-acidNucleic acid
Function / homologyAP endonucleases family 1 profile. / Hfq protein / Endonuclease/Exonuclease/phosphatase family / Endonuclease/exonuclease/phosphatase superfamily / LSM domain superfamily / Endonuclease/exonuclease/phosphatase / RNA-binding protein Hfq / AP endonuclease 1 / regulation of single-species biofilm formation / regulation of carbon utilization ...AP endonucleases family 1 profile. / Hfq protein / Endonuclease/Exonuclease/phosphatase family / Endonuclease/exonuclease/phosphatase superfamily / LSM domain superfamily / Endonuclease/exonuclease/phosphatase / RNA-binding protein Hfq / AP endonuclease 1 / regulation of single-species biofilm formation / regulation of carbon utilization / regulation of translation, ncRNA-mediated / exodeoxyribonuclease III / regulation of RNA stability / exodeoxyribonuclease III activity / quorum sensing / regulation of translation / pathogenesis / DNA repair / regulation of transcription, DNA-templated / RNA binding / cytosol / Catabolite repression control protein / RNA-binding protein Hfq
Function and homology information
SourcePseudomonas 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 / 3.37 Å resolution
AuthorsPei XY / Dendooven T / Sonnleitner E / Chen S / Blasi U / Luisi BF
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.
Validation ReportPDB-ID: 6o1l

SummaryFull reportAbout validation report
DateDeposition: Feb 20, 2019 / Header (metadata) release: Mar 13, 2019 / Map release: Mar 13, 2019 / Last update: Mar 13, 2019

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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
  • Download
  • 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

Downloads & links

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Map

Fileemd_0591.map.gz (map file in CCP4 format, 27437 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
190 pix
1.09 Å/pix.
= 207.1 Å
190 pix
1.09 Å/pix.
= 207.1 Å
190 pix
1.09 Å/pix.
= 207.1 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.09 Å
Density
Contour Level:0.27 (by author), 0.27 (movie #1):
Minimum - Maximum-0.01850905 - 1.9583755
Average (Standard dev.)0.0037314594 (0.054603253)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions190190190
Origin0.00.00.0
Limit189.0189.0189.0
Spacing190190190
CellA=B=C: 207.1 Å
α=β=γ: 90.0 deg.

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

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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 / Number of components: 4

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Component #1: protein, Assembly of Hfq-Crc-amiE with a 2:3:2 stoichiometry

ProteinName: Assembly of Hfq-Crc-amiE with a 2:3:2 stoichiometry / Recombinant expression: No
MassExperimental: 222 kDa
SourceSpecies: Pseudomonas aeruginosa (bacteria)

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Component #2: protein, Catabolite repression control protein

ProteinName: Catabolite repression control protein / Number of Copies: 3 / Recombinant expression: No
MassTheoretical: 30.101869 kDa
SourceSpecies: Pseudomonas aeruginosa (bacteria)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #3: protein, RNA-binding protein Hfq

ProteinName: RNA-binding protein Hfq / Number of Copies: 12 / Recombinant expression: No
MassTheoretical: 7.685984 kDa
SourceSpecies: 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
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #4: nucleic-acid, RNA (5'-R(*AP*AP*AP*AP*AP*UP*AP*AP*CP*AP*AP*CP*AP*A...

Nucleic-acidName: RNA (5'-R(*AP*AP*AP*AP*AP*UP*AP*AP*CP*AP*AP*CP*AP*AP*GP*AP*GP*G)-3')
Class: RNA / Structure: OTHER / Synthetic: No
Sequence:
AAAAAUAACA ACAAGAGG
MassTheoretical: 5.85766 kDa
SourceSpecies: Pseudomonas aeruginosa (bacteria)

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionpH: 7.9
VitrificationCryogen name: ETHANE / Temperature: 277 K / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 28 e/Å2 / Illumination mode: OTHER
LensMagnification: 125000.0 X (nominal), 130841.0 X (calibrated)
Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 1.25 - 3.0 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature: K ( 70.0 - 100.0 K)
CameraDetector: FEI FALCON III (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 917 / Sampling size: 5 microns
Details: Images were collected in movie-counting mode, 75 frames per movie stack, 60s exposure per movie

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 45311
3D reconstructionSoftware: RELION / Resolution: 3.37 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot
(resolution estimation)

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Atomic model buiding

Modeling #1Refinement protocol: rigid body / Target criteria: Cross-correlation / Refinement space: REAL
Input PDB model: 1U1T, 4JG3
Output model

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