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- EMDB-2631: The Cryo-EM structure of the palindromic DNA-bound USP/EcR nuclea... -

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Basic information

Entry
Database: EMDB / ID: EMD-2631
TitleThe Cryo-EM structure of the palindromic DNA-bound USP/EcR nuclear receptor reveals an asymmetric organization with allosteric domain positioning
Map data-
Sample
  • Sample: USP/EcR
  • Protein or peptide: ECR
  • Protein or peptide: USP
  • DNA: DNA
KeywordsNuclear receptor / 100kDa complex / 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
Ecdysone receptor / Gene regulation protein
Similarity search - Component
Biological speciesHeliothis virescens (tobacco budworm) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 11.6 Å
AuthorsMaletta M / Orlov I / Moras D / Billas IML / Klaholz BP
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
DepositionApr 18, 2014-
Header (metadata) releaseMay 14, 2014-
Map releaseJul 2, 2014-
UpdateJul 2, 2014-
Current statusJul 2, 2014Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.5
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.5
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-4umm
  • Surface level: 0.5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2631-USP...

Downloads & links

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Map

FileDownload / File: emd_2631.map.gz / Format: CCP4 / Size: 3.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation-
Voxel sizeX=Y=Z: 1.7 Å
Density
Contour LevelBy AUTHOR: 0.5 / Movie #1: 0.5
Minimum - Maximum-2.77237272 - 13.81750774
Average (Standard dev.)0.12339457 (±0.8669368)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-47-48-48
Dimensions969696
Spacing969696
CellA=B=C: 163.20001 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.71.71.7
M x/y/z969696
origin x/y/z0.0000.0000.000
length x/y/z163.200163.200163.200
α/β/γ90.00090.00090.000
start NX/NY/NZ-184-184-183
NX/NY/NZ368368368
MAP C/R/S123
start NC/NR/NS-48-47-48
NC/NR/NS969696
D min/max/mean-2.77213.8180.123

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Supplemental data

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Sample components

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Entire : USP/EcR

EntireName: USP/EcR
Components
  • Sample: USP/EcR
  • Protein or peptide: ECR
  • Protein or peptide: USP
  • DNA: DNA

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Supramolecule #1000: USP/EcR

SupramoleculeName: USP/EcR / type: sample / ID: 1000 / Oligomeric state: a heterodimer of USP and EcR bound to DNA / Number unique components: 3
Molecular weightExperimental: 100 KDa / Theoretical: 100 KDa

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Macromolecule #1: ECR

MacromoleculeName: ECR / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Heliothis virescens (tobacco budworm) / Organelle: nucleus / Location in cell: nucleus
Molecular weightExperimental: 100 KDa / Theoretical: 100 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)

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Macromolecule #3: USP

MacromoleculeName: USP / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Heliothis virescens (tobacco budworm) / Organelle: nucleus / Location in cell: nucleus
Molecular weightExperimental: 100 KDa / Theoretical: 100 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)

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Macromolecule #2: DNA

MacromoleculeName: DNA / type: dna / ID: 2 / Classification: DNA / Structure: SINGLE STRANDED / Synthetic?: Yes
Source (natural)Organism: synthetic construct (others)
Molecular weightExperimental: 100 KDa / Theoretical: 100 KDa

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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

Concentration0.1 mg/mL
BufferDetails: 10 mM TRIS pH 7.5, 100 mM NaCl, 10 mM MgCl2, 10 mM TCEP
GridDetails: Quantifoil 2/2.2 300 mesh
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 120 K / Instrument: FEI VITROBOT MARK IV / Method: 2 seconds, force 4

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Electron microscopy

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 100 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 64244 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 59000
Sample stageSpecimen holder: FEI Polara cartrige / Specimen holder model: GATAN HELIUM
DateNov 20, 2009
Image recordingCategory: CCD / Film or detector model: FEI EAGLE (4k x 4k) / Number real images: 600 / Average electron dose: 20 e/Å2 / Bits/pixel: 16
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: ccd images 4096x4096
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 11.6 Å / Resolution method: OTHER / Software - Name: Imagic-V / Number images used: 50000
DetailsImagic-V, angular reconstitution angles determination

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Atomic model buiding 1

Initial modelPDB ID:

1r1k

DetailsThe domains (USP/EcR LBD and DBD heterodimers respectively, PDB IDs 1R1K and 2HAN) were separately fitted by manual docking using program Pymol.
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-4umm:
The Cryo-EM structure of the palindromic DNA-bound USP-EcR nuclear receptor reveals an asymmetric organization with allosteric domain positioning

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Atomic model buiding 2

Initial modelPDB ID:

2han

DetailsThe domains (USP/EcR LBD and DBD heterodimers respectively, PDB IDs 1R1K and 2HAN) were separately fitted by manual docking using program Pymol.
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-4umm:
The Cryo-EM structure of the palindromic DNA-bound USP-EcR nuclear receptor reveals an asymmetric organization with allosteric domain positioning

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