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- EMDB-5981: Cryo-Electron Microscopy Reconstruction of Glucocorticoid Recepto... -

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Basic information

Entry
Database: EMDB / ID: EMD-5981
TitleCryo-Electron Microscopy Reconstruction of Glucocorticoid Receptor-bound Hsp90-Hsp70-Hop Chaperone Complex
Map dataReconstruction of client-bound Hsp90-Hsp70-Hop chaperone complex
Sample
  • Sample: Human chaperones Hsp90 and Hsp70 bound with the human co-chaperone Hop and the ligand binding domain of the human glucocorticoid receptor N-terminally fused with the maltose binding protein
  • Protein or peptide: Heat shock protein 90-alphaHeat shock response
  • Protein or peptide: Heat shock 70 kDa protein
  • Protein or peptide: Glucocorticoid Receptor Ligand Binding Domain
  • Protein or peptide: Stress-induced-phosphoprotein 1
KeywordsHsp90 / Hsp70 / Hop / Glucocorticoid Receptor
Function / homology
Function and homology information


: / Regulation of NPAS4 gene transcription / cellular heat acclimation / regulation of glucocorticoid biosynthetic process / nuclear glucocorticoid receptor activity / negative regulation of inclusion body assembly / positive regulation of nucleotide-binding oligomerization domain containing 2 signaling pathway / : / dynein axonemal particle / C3HC4-type RING finger domain binding ...: / Regulation of NPAS4 gene transcription / cellular heat acclimation / regulation of glucocorticoid biosynthetic process / nuclear glucocorticoid receptor activity / negative regulation of inclusion body assembly / positive regulation of nucleotide-binding oligomerization domain containing 2 signaling pathway / : / dynein axonemal particle / C3HC4-type RING finger domain binding / steroid hormone binding / PTK6 Expression / ATP-dependent protein disaggregase activity / glucocorticoid metabolic process / positive regulation of microtubule nucleation / neuroinflammatory response / microglia differentiation / maternal behavior / mammary gland duct morphogenesis / nucleus localization / misfolded protein binding / regulation of mitotic spindle assembly / positive regulation of tumor necrosis factor-mediated signaling pathway / astrocyte differentiation / protein folding chaperone complex / cellular response to interleukin-7 / cellular response to glucocorticoid stimulus / aggresome / RND1 GTPase cycle / motor behavior / sperm mitochondrial sheath / dATP binding / Scavenging by Class F Receptors / sulfonylurea receptor binding / CTP binding / positive regulation of protein polymerization / vRNP Assembly / UTP binding / sperm plasma membrane / positive regulation of tau-protein kinase activity / protein insertion into mitochondrial outer membrane / regulation of gluconeogenesis / adrenal gland development / telomerase holoenzyme complex assembly / chaperone-mediated autophagy / Rho GDP-dissociation inhibitor binding / cellular response to steroid hormone stimulus / Uptake and function of diphtheria toxin / mitochondrial transport / Drug-mediated inhibition of ERBB2 signaling / Resistance of ERBB2 KD mutants to trastuzumab / Resistance of ERBB2 KD mutants to sapitinib / Resistance of ERBB2 KD mutants to tesevatinib / Resistance of ERBB2 KD mutants to neratinib / Resistance of ERBB2 KD mutants to osimertinib / Resistance of ERBB2 KD mutants to afatinib / Resistance of ERBB2 KD mutants to AEE788 / Resistance of ERBB2 KD mutants to lapatinib / Drug resistance in ERBB2 TMD/JMD mutants / PIWI-interacting RNA (piRNA) biogenesis / TPR domain binding / mRNA catabolic process / non-chaperonin molecular chaperone ATPase / regulation of postsynaptic membrane neurotransmitter receptor levels / dendritic growth cone / chaperone cofactor-dependent protein refolding / Sema3A PAK dependent Axon repulsion / regulation of protein ubiquitination / positive regulation of cell size / skeletal muscle contraction / protein unfolding / regulation of protein-containing complex assembly / HSF1-dependent transactivation / telomere maintenance via telomerase / response to unfolded protein / negative regulation of extrinsic apoptotic signaling pathway in absence of ligand / Regulation of HSF1-mediated heat shock response / HSF1 activation / chaperone-mediated protein complex assembly / Attenuation phase / cellular response to unfolded protein / estrogen response element binding / FOXO-mediated transcription of oxidative stress, metabolic and neuronal genes / RHOBTB2 GTPase cycle / DNA polymerase binding / positive regulation of lamellipodium assembly / axonal growth cone / eNOS activation / intracellular steroid hormone receptor signaling pathway / ATP metabolic process / protein folding chaperone / inclusion body / core promoter sequence-specific DNA binding / endocytic vesicle lumen / Tetrahydrobiopterin (BH4) synthesis, recycling, salvage and regulation / negative regulation of protein ubiquitination / positive regulation of defense response to virus by host / Loss of Nlp from mitotic centrosomes / Loss of proteins required for interphase microtubule organization from the centrosome / positive regulation of cardiac muscle contraction
Similarity search - Function
STI1/HOP, DP domain / STI1/HOP, DP domain / Tetratricopeptide repeat 2 / Tetratricopeptide repeat / Heat shock chaperonin-binding / Heat shock chaperonin-binding / Heat shock chaperonin-binding motif. / Glucocorticoid receptor / Glucocorticoid receptor / Glucocorticoid receptor ...STI1/HOP, DP domain / STI1/HOP, DP domain / Tetratricopeptide repeat 2 / Tetratricopeptide repeat / Heat shock chaperonin-binding / Heat shock chaperonin-binding / Heat shock chaperonin-binding motif. / Glucocorticoid receptor / Glucocorticoid receptor / Glucocorticoid receptor / TPR repeat / Tetratricopeptide repeat / Heat shock hsp70 proteins family signature 2. / Heat shock hsp70 proteins family signature 1. / Heat shock hsp70 proteins family signature 3. / Heat shock protein 70, conserved site / Heat shock protein 70kD, peptide-binding domain superfamily / Heat shock protein 70 family / Heat shock protein 70 family / Hsp70 protein / Heat shock protein 70kD, C-terminal domain superfamily / Tetratricopeptide repeat / Heat shock protein Hsp90, conserved site / Heat shock hsp90 proteins family signature. / HSP90, C-terminal domain / Heat shock protein Hsp90, N-terminal / Heat shock protein Hsp90 family / Heat shock protein Hsp90 family / Hsp90 protein / Histidine kinase-, DNA gyrase B-, and HSP90-like ATPase / TPR repeat region circular profile. / TPR repeat profile. / Tetratricopeptide repeats / Tetratricopeptide repeat / Histidine kinase-like ATPases / Histidine kinase/HSP90-like ATPase / Histidine kinase/HSP90-like ATPase superfamily / ATPase, nucleotide binding domain / 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 / Tetratricopeptide-like helical domain superfamily / Ribosomal protein S5 domain 2-type fold
Similarity search - Domain/homology
Glucocorticoid receptor / Heat shock protein HSP 90-alpha / Heat shock 70 kDa protein 1B / Stress-induced-phosphoprotein 1
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 38.0 Å
AuthorsKirschke E / Goswami D / Southworth DR / Griffin PR / Agard DA
CitationJournal: Cell / Year: 2014
Title: Glucocorticoid receptor function regulated by coordinated action of the Hsp90 and Hsp70 chaperone cycles.
Authors: Elaine Kirschke / Devrishi Goswami / Daniel Southworth / Patrick R Griffin / David A Agard /
Abstract: The glucocorticoid receptor (GR), like many signaling proteins, depends on the Hsp90 molecular chaperone for in vivo function. Although Hsp90 is required for ligand binding in vivo, purified apo GR ...The glucocorticoid receptor (GR), like many signaling proteins, depends on the Hsp90 molecular chaperone for in vivo function. Although Hsp90 is required for ligand binding in vivo, purified apo GR is capable of binding ligand with no enhancement from Hsp90. We reveal that Hsp70, known to facilitate client delivery to Hsp90, inactivates GR through partial unfolding, whereas Hsp90 reverses this inactivation. Full recovery of ligand binding requires ATP hydrolysis on Hsp90 and the Hop and p23 cochaperones. Surprisingly, Hsp90 ATP hydrolysis appears to regulate client transfer from Hsp70, likely through a coupling of the two chaperone's ATP cycles. Such coupling is embodied in contacts between Hsp90 and Hsp70 in the GR:Hsp70:Hsp90:Hop complex imaged by cryoelectron microscopy. Whereas GR released from Hsp70 is aggregation prone, release from Hsp90 protects GR from aggregation and enhances its ligand affinity. Together, this illustrates how coordinated chaperone interactions can enhance stability, function, and regulation.
History
DepositionMay 30, 2014-
Header (metadata) releaseJun 18, 2014-
Map releaseJun 18, 2014-
UpdateJul 16, 2014-
Current statusJul 16, 2014Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3.12
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 3.12
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5981.png

Downloads & links

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Map

FileDownload / File: emd_5981.map.gz / Format: CCP4 / Size: 6.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of client-bound Hsp90-Hsp70-Hop chaperone complex
Voxel sizeX=Y=Z: 2.4 Å
Density
Contour LevelBy AUTHOR: 3.12 / Movie #1: 3.12
Minimum - Maximum-9.243871690000001 - 17.204744340000001
Average (Standard dev.)-0.10103421 (±1.29138064)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-25-25-25
Dimensions120120120
Spacing120120120
CellA=B=C: 288.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.42.42.4
M x/y/z120120120
origin x/y/z0.0000.0000.000
length x/y/z288.000288.000288.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ969680
MAP C/R/S123
start NC/NR/NS-25-25-25
NC/NR/NS120120120
D min/max/mean-9.24417.205-0.101

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Supplemental data

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Sample components

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Entire : Human chaperones Hsp90 and Hsp70 bound with the human co-chaperon...

EntireName: Human chaperones Hsp90 and Hsp70 bound with the human co-chaperone Hop and the ligand binding domain of the human glucocorticoid receptor N-terminally fused with the maltose binding protein
Components
  • Sample: Human chaperones Hsp90 and Hsp70 bound with the human co-chaperone Hop and the ligand binding domain of the human glucocorticoid receptor N-terminally fused with the maltose binding protein
  • Protein or peptide: Heat shock protein 90-alphaHeat shock response
  • Protein or peptide: Heat shock 70 kDa protein
  • Protein or peptide: Glucocorticoid Receptor Ligand Binding Domain
  • Protein or peptide: Stress-induced-phosphoprotein 1

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Supramolecule #1000: Human chaperones Hsp90 and Hsp70 bound with the human co-chaperon...

SupramoleculeName: Human chaperones Hsp90 and Hsp70 bound with the human co-chaperone Hop and the ligand binding domain of the human glucocorticoid receptor N-terminally fused with the maltose binding protein
type: sample / ID: 1000
Details: Complex was purified using size exclusion chromatography.
Oligomeric state: One homodimer of Hsp90 bound to one Hsp70, one Hop, and one MBP-GRLBD
Number unique components: 4
Molecular weightTheoretical: 380 KDa

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Macromolecule #1: Heat shock protein 90-alpha

MacromoleculeName: Heat shock protein 90-alpha / type: protein_or_peptide / ID: 1 / Name.synonym: Hsp90 / Number of copies: 1 / Oligomeric state: Dimer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 84.66 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21 / Recombinant plasmid: pET151-D
SequenceUniProtKB: Heat shock protein HSP 90-alpha / InterPro: Heat shock protein Hsp90 family

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Macromolecule #2: Heat shock 70 kDa protein

MacromoleculeName: Heat shock 70 kDa protein / type: protein_or_peptide / ID: 2 / Name.synonym: Hsp70 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 70.052 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm) / Recombinant cell: Sf9
SequenceUniProtKB: Heat shock 70 kDa protein 1B / InterPro: Heat shock protein 70 family

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Macromolecule #3: Glucocorticoid Receptor Ligand Binding Domain

MacromoleculeName: Glucocorticoid Receptor Ligand Binding Domain / type: protein_or_peptide / ID: 3 / Name.synonym: GRLBD
Details: Maltose Binding Protein (MBP) tag attached to N-terminus
Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 74.926 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21 / Recombinant plasmid: pMal
SequenceUniProtKB: Glucocorticoid receptor / InterPro: Glucocorticoid receptor

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Macromolecule #4: Stress-induced-phosphoprotein 1

MacromoleculeName: Stress-induced-phosphoprotein 1 / type: protein_or_peptide / ID: 4 / Name.synonym: Hop / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 62.639 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21 / Recombinant plasmid: pET151-D
SequenceUniProtKB: Stress-induced-phosphoprotein 1 / InterPro: Heat shock chaperonin-binding

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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: 7.5
Details: 50 mM HEPES, 50 mM KCl, 2 mM DTT, 5 mM MgCl2, 0.2 mM ADP
GridDetails: C-flat holey carbon grid, 2 micron hole, 0.5 micron space
VitrificationCryogen name: ETHANE / Chamber humidity: 70 % / Chamber temperature: 75 K / Instrument: FEI VITROBOT MARK I / Method: two one-second blots before plunging

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.2 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 2.2 µm / Nominal magnification: 60000
Sample stageSpecimen holder model: OTHER
Alignment procedureLegacy - Astigmatism: Astigmatism was corrected at 250,000 times magnification.
DateSep 21, 2011
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F816 (8k x 8k) / Number real images: 214 / Average electron dose: 30 e/Å2
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: CTF Find
Final angle assignmentDetails: EMAN: 15
Final reconstructionResolution.type: BY AUTHOR / Resolution: 38.0 Å / Resolution method: OTHER / Software - Name: EMAN / Number images used: 10149
DetailsThe particles were manually selected.

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