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- PDB-4umm: The Cryo-EM structure of the palindromic DNA-bound USP-EcR nuclea... -

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Basic information

Entry
Database: PDB / ID: 4umm
TitleThe Cryo-EM structure of the palindromic DNA-bound USP-EcR nuclear receptor reveals an asymmetric organization with allosteric domain positioning
Components
  • (ECDYSONE RECEPTOR) x 2
  • 5'-D(*CP*AP*AP*GP*GP*GP*TP*TP*CP*AP*AP*TP*GP*CP *AP*CP*TP*TP*GP*TP)-3'
  • 5'-D(*DGP*AP*CP*AP*AP*GP*TP*GP*CP*AP*TP*TP*GP*DAP *AP*CP*CP*CP*TP*T)-3'
  • ECR-USP
  • GENE REGULATION PROTEINRegulation of gene expression
KeywordsNUCLEAR RECEPTOR / TRANSCRIPTION / ECDYSONE / USP-ECR / DNA RESPONSE ELEMENT / ALLOSTERY / CRYO ELECTRON MICROSCOPY
Function / homology
Function and homology information


ecdysone binding / ecdysone receptor-mediated signaling pathway / nuclear steroid receptor activity / nuclear receptor activity / sequence-specific DNA binding / regulation of DNA-templated transcription / DNA binding / zinc ion binding / nucleus
Similarity search - Function
Ecdysteroid receptor / Ecdysone receptor, ligand-binding domain / Retinoid X receptor/HNF4 / Nuclear hormone receptor / Nuclear hormones receptors DNA-binding region signature. / Zinc finger, nuclear hormone receptor-type / Zinc finger, C4 type (two domains) / Nuclear hormone receptors DNA-binding domain profile. / c4 zinc finger in nuclear hormone receptors / Nuclear hormone receptor, ligand-binding domain ...Ecdysteroid receptor / Ecdysone receptor, ligand-binding domain / Retinoid X receptor/HNF4 / Nuclear hormone receptor / Nuclear hormones receptors DNA-binding region signature. / Zinc finger, nuclear hormone receptor-type / Zinc finger, C4 type (two domains) / Nuclear hormone receptors DNA-binding domain profile. / c4 zinc finger in nuclear hormone receptors / Nuclear hormone receptor, ligand-binding domain / Nuclear hormone receptor-like domain superfamily / Ligand-binding domain of nuclear hormone receptor / Nuclear receptor (NR) ligand-binding (LBD) domain profile. / Ligand binding domain of hormone receptors / Zinc finger, NHR/GATA-type
Similarity search - Domain/homology
Chem-EPH / 2,3,14,20,22-PENTAHYDROXYCHOLEST-7-EN-6-ONE / DNA / DNA (> 10) / Ecdysone receptor / Gene regulation protein
Similarity search - Component
Biological speciesHELIOTHIS VIRESCENS (tobacco budworm)
SYNTHETIC CONSTRUCT (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 11.6 Å
AuthorsMaletta, M. / Orlov, I. / Moras, D. / Billas, I.M.L. / Klaholz, B.P.
CitationJournal: Nat Commun / Year: 2014
Title: The palindromic DNA-bound USP/EcR nuclear receptor adopts an asymmetric organization with allosteric domain positioning.
Authors: Massimiliano Maletta / Igor Orlov / Pierre Roblin / Yannick Beck / Dino Moras / Isabelle M L Billas / Bruno P Klaholz /
Abstract: Nuclear receptors (NRs) regulate gene expression through DNA- and ligand-binding and thus represent crucial therapeutic targets. The ultraspiracle protein/ecdysone receptor (USP/EcR) complex binds to ...Nuclear receptors (NRs) regulate gene expression through DNA- and ligand-binding and thus represent crucial therapeutic targets. The ultraspiracle protein/ecdysone receptor (USP/EcR) complex binds to half-sites with a one base pair spaced inverted repeat (IR1), a palindromic DNA response element (RE) reminiscent of IRs observed for vertebrate steroid hormone receptors. Here we present the cryo electron microscopy structure of the USP/EcR complex bound to an IR1 RE which provides the first description of a full IR-bound NR complex. The structure reveals that even though the DNA is almost symmetric, the complex adopts a highly asymmetric architecture in which the ligand-binding domains (LBDs) are positioned 5' off-centred. Additional interactions of the USP LBD with the 5'-flanking sequence trigger transcription activity as monitored by transfection assays. The comparison with DR-bound NR complexes suggests that DNA is the major allosteric driver in inversely positioning the LBDs, which serve as the main binding-site for transcriptional regulators.
History
DepositionMay 19, 2014Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 25, 2014Provider: repository / Type: Initial release
Revision 1.1Jul 2, 2014Group: Database references / Other
Revision 1.2Oct 3, 2018Group: Data collection / Derived calculations
Category: diffrn_radiation / diffrn_radiation_wavelength ...diffrn_radiation / diffrn_radiation_wavelength / em_software / ndb_struct_na_base_pair / ndb_struct_na_base_pair_step
Item: _em_software.image_processing_id / _ndb_struct_na_base_pair.propeller ..._em_software.image_processing_id / _ndb_struct_na_base_pair.propeller / _ndb_struct_na_base_pair_step.helical_rise / _ndb_struct_na_base_pair_step.helical_twist / _ndb_struct_na_base_pair_step.roll / _ndb_struct_na_base_pair_step.slide / _ndb_struct_na_base_pair_step.tip / _ndb_struct_na_base_pair_step.twist

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Structure visualization

Movie
  • Deposited structure unit
  • Imaged by Jmol
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  • Superimposition on EM map
  • EMDB-2631
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Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: ECR-USP
C: 5'-D(*CP*AP*AP*GP*GP*GP*TP*TP*CP*AP*AP*TP*GP*CP *AP*CP*TP*TP*GP*TP)-3'
D: 5'-D(*DGP*AP*CP*AP*AP*GP*TP*GP*CP*AP*TP*TP*GP*DAP *AP*CP*CP*CP*TP*T)-3'
E: ECDYSONE RECEPTOR
F: GENE REGULATION PROTEIN
G: ECDYSONE RECEPTOR
hetero molecules


Theoretical massNumber of molelcules
Total (without water)93,1308
Polymers91,9556
Non-polymers1,1752
Water1448
1


  • Idetical with deposited unit
  • defined by software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA

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Components

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Protein , 4 types, 4 molecules AEFG

#1: Protein ECR-USP


Mass: 9256.789 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) HELIOTHIS VIRESCENS (tobacco budworm) / Organ: NUCLEOUS / Production host: ESCHERICHIA COLI BL21(DE3) (bacteria)
#4: Protein ECDYSONE RECEPTOR / / 20-HYDROXY-ECDYSONE RECEPTOR / 20E RECEPTOR / ECRH / ECDYSTEROID RECEPTOR / HVECR / NUCLEAR ...20-HYDROXY-ECDYSONE RECEPTOR / 20E RECEPTOR / ECRH / ECDYSTEROID RECEPTOR / HVECR / NUCLEAR RECEPTOR SUBFAMILY 1 GROUP H MEMBER 1


Mass: 10099.977 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) HELIOTHIS VIRESCENS (tobacco budworm) / Organ: NUCLEOUS / Production host: ESCHERICHIA COLI BL21(DE3) (bacteria) / References: UniProt: O18473
#5: Protein GENE REGULATION PROTEIN / Regulation of gene expression / ECR-USP


Mass: 29962.717 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) HELIOTHIS VIRESCENS (tobacco budworm) / Organ: NUCLEOUS / Production host: ESCHERICHIA COLI BL21(DE3) (bacteria) / References: UniProt: Q7SIF6
#6: Protein ECDYSONE RECEPTOR / / 20-HYDROXY-ECDYSONE RECEPTOR / 20E RECEPTOR / ECRH / ECDYSTEROID RECEPTOR / HVECR / NUCLEAR ...20-HYDROXY-ECDYSONE RECEPTOR / 20E RECEPTOR / ECRH / ECDYSTEROID RECEPTOR / HVECR / NUCLEAR RECEPTOR SUBFAMILY 1 GROUP H MEMBER 1


Mass: 30368.961 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) HELIOTHIS VIRESCENS (tobacco budworm) / Organ: NUCLEOUS / Production host: ESCHERICHIA COLI BL21(DE3) (bacteria) / References: UniProt: O18473

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DNA chain , 2 types, 2 molecules CD

#2: DNA chain 5'-D(*CP*AP*AP*GP*GP*GP*TP*TP*CP*AP*AP*TP*GP*CP *AP*CP*TP*TP*GP*TP)-3'


Mass: 6148.989 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) SYNTHETIC CONSTRUCT (others)
#3: DNA chain 5'-D(*DGP*AP*CP*AP*AP*GP*TP*GP*CP*AP*TP*TP*GP*DAP *AP*CP*CP*CP*TP*T)-3'


Mass: 6117.979 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) SYNTHETIC CONSTRUCT (others)

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Non-polymers , 3 types, 10 molecules

#7: Chemical ChemComp-EPH / L-ALPHA-PHOSPHATIDYL-BETA-OLEOYL-GAMMA-PALMITOYL-PHOSPHATIDYLETHANOLAMINE / Phosphatidylethanolamine


Mass: 709.933 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C39H68NO8P / Comment: phospholipid*YM
#8: Chemical ChemComp-P1A / 2,3,14,20,22-PENTAHYDROXYCHOLEST-7-EN-6-ONE / PONASTERONE A, 25-DEOXYECDYSTERONE, 25-DEOXY-20-HYDROXYECDYSONE,


Mass: 464.635 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C27H44O6
#9: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 8 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: BRUNO P. KLAHOLZ / Type: COMPLEX
Buffer solutionName: 10 MM TRIS PH 7.5, 100 MM NACL, 10 MM MGCL2, 10 MM TCEP
pH: 7.5
Details: 10 MM TRIS PH 7.5, 100 MM NACL, 10 MM MGCL2, 10 MM TCEP
SpecimenConc.: 0.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: CARBON
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE
Details: VITRIFICATION 1 -- CRYOGEN- ETHANE, HUMIDITY- 90, TEMPERATURE- 120, INSTRUMENT- FEI VITROBOT MARK IV, METHOD- 2 SECONDS, FORCE 4,

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F30 / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI F30 / Date: Nov 20, 2009
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 100 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 59000 X / Calibrated magnification: 64244 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1500 nm / Cs: 2 mm
Image recordingElectron dose: 20 e/Å2 / Film or detector model: FEI EAGLE (4k x 4k)
Image scansNum. digital images: 600

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Processing

EM softwareName: IMAGIC / Version: 5 / Category: 3D reconstruction
CTF correctionDetails: CCD IMAGES 4096X4096
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionMethod: CROSS-COMMON LINES / Resolution: 11.6 Å / Num. of particles: 50000
Details: SUBMISSION BASED ON EXPERIMENTAL DATA FROM EMDB EMD -2631. (DEPOSITION ID: 12447).
Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL / Details: METHOD--RIGID BODY
Atomic model buildingPDB-ID: 1R1K

1r1k

RefinementHighest resolution: 11.6 Å
Refinement stepCycle: LAST / Highest resolution: 11.6 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5128 812 82 8 6030

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