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- PDB-6o1m: Architectural principles for Hfq/Crc-mediated regulation of gene ... -

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Basic information

Entry
Database: PDB / ID: 6o1m
TitleArchitectural principles for Hfq/Crc-mediated regulation of gene expression. Hfq-Crc-amiE 2:4:2 complex
Components
  • Catabolite repression control protein
  • RNA (5'-R(*AP*AP*AP*AP*AP*UP*AP*AP*CP*AP*AP*CP*AP*AP*GP*AP*GP*G)-3')
  • RNA-binding protein Hfq
KeywordsRNA BINDING PROTEIN/RNA/HYDROLASE / Hfq / Crc / amiE / Carbon catabolite repression / RNA-protein interaction / RNA-mediated gene regulation / RNA BINDING PROTEIN / RNA BINDING PROTEIN-RNA-HYDROLASE complex
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
Specimen sourcePseudomonas aeruginosa (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 3.15 Å resolution
AuthorsPei, X.Y. / Dendooven, T. / Sonnleitner, E. / Chen, S. / Blasi, U. / Luisi, B.F.
Funding supportUnited Kingdom , 1 items
OrganizationGrant numberCountry
Wellcome Trust200873/Z/16/ZUnited 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.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Feb 20, 2019 / Release: Mar 13, 2019

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Structure visualization

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Assembly

Deposited unit
A: Catabolite repression control protein
B: Catabolite repression control protein
C: RNA-binding protein Hfq
D: RNA-binding protein Hfq
E: RNA-binding protein Hfq
F: RNA-binding protein Hfq
G: RNA-binding protein Hfq
H: RNA-binding protein Hfq
I: RNA-binding protein Hfq
J: RNA-binding protein Hfq
K: RNA-binding protein Hfq
L: RNA-binding protein Hfq
M: RNA-binding protein Hfq
N: RNA-binding protein Hfq
O: RNA (5'-R(*AP*AP*AP*AP*AP*UP*AP*AP*CP*AP*AP*CP*AP*AP*GP*AP*GP*G)-3')
P: RNA (5'-R(*AP*AP*AP*AP*AP*UP*AP*AP*CP*AP*AP*CP*AP*AP*GP*AP*GP*G)-3')
R: Catabolite repression control protein
S: Catabolite repression control protein


Theoretical massNumber of molelcules
Total (without water)224,35518
Polyers224,35518
Non-polymers00
Water0
1


  • idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, Gel filtration and Multi Angle Light Scattering
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TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein/peptide
Catabolite repression control protein / Exodeoxyribonuclease / Exodeoxyribonuclease III


Mass: 30101.869 Da / Num. of mol.: 4 / Source: (gene. exp.) Pseudomonas aeruginosa (bacteria)
Gene: crc, exoA, exoA_2, C0044_39460, C8257_32550, CAZ10_32350, CGU42_22755, DT376_09125, DZ940_17015, DZ962_18070, NCTC13719_05871, PAERUG_E15_London_28_01_14_10555, PAMH19_3299, RW109_RW109_06940
Plasmid name: pETM14lic-6His-Crc / Production host: Escherichia coli (E. coli) / Strain (production host): BL21DE3 / References: UniProt: Q51380, exodeoxyribonuclease III
#2: Protein/peptide
RNA-binding protein Hfq


Mass: 7685.984 Da / Num. of mol.: 12
Source: (gene. exp.) 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
Gene: hfq, PA4944 / Plasmid name: pKEHfqPae / Production host: Escherichia coli (E. coli) / Strain (production host): JW413F' / References: UniProt: Q9HUM0
#3: RNA chain RNA (5'-R(*AP*AP*AP*AP*AP*UP*AP*AP*CP*AP*AP*CP*AP*AP*GP*AP*GP*G)-3')


Mass: 5857.660 Da / Num. of mol.: 2 / Source: (synth.) Pseudomonas aeruginosa (bacteria)

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / Reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Assembly of Hfq-Crc-amiE with a 2:4:2 stoichiometry / Type: COMPLEX / Entity ID: 1, 2, 3 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.252 MDa / Experimental value: YES
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.9
Buffer component
IDConc.NameBuffer ID
120 mMHEPES pH 7.91
240 mMNaCl1
310 mMKCl1
41 mMMgCl21
52 mMTCEP1
SpecimenDetails: Monodisperse / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: unspecified
VitrificationCryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 kelvins

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 125000 / Calibrated magnification: 130841 / Nominal defocus max: 3 nm / Nominal defocus min: 1.25 nm / Cs: 2.7 mm / C2 aperture diameter: 5 microns / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 100 kelvins / Temperature (min): 70 kelvins
Image recordingAverage exposure time: 60 sec. / Electron dose: 28 e/Å2
Details: Images were collected in movie-counting mode, 75 frames per movie stack, 60s exposure per movie
Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Number of grids imaged: 1 / Number of real images: 917
Image scansSampling size: 5 microns

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Processing

EM software
IDNameVersionCategoryDetails
2EPU1.10.1.1938RELimage acquisition
4Gctf1.06CTF correction
7UCSF Chimeramodel fitting
8MOLREPmodel fitting
10RELION3binitial Euler assignmentbeta version
11RELION3bfinal Euler assignmentbeta version
12RELION3bclassificationbeta version
13RELION3b3D reconstruction
14PHENIXmodel refinementreal-space refine
15REFMACmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNumber of particles selected: 435000
SymmetryPoint symmetry: C2
3D reconstructionResolution: 3.15 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 35376 / Symmetry type: POINT
Atomic model buildingRef protocol: RIGID BODY FIT / Ref space: REAL / Target criteria: Cross-correlation
Atomic model building
IDPDB-ID 3D fitting ID
11U1T1
24JG31

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