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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-alpha
  • 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 / regulation of glucocorticoid biosynthetic process / nuclear glucocorticoid receptor activity / cellular heat acclimation / steroid hormone binding / negative regulation of inclusion body assembly / C3HC4-type RING finger domain binding / glucocorticoid metabolic process ...: / : / Regulation of NPAS4 gene transcription / regulation of glucocorticoid biosynthetic process / nuclear glucocorticoid receptor activity / cellular heat acclimation / steroid hormone binding / negative regulation of inclusion body assembly / C3HC4-type RING finger domain binding / glucocorticoid metabolic process / response to cortisol / positive regulation of nucleotide-binding oligomerization domain containing 2 signaling pathway / PTK6 Expression / neuroinflammatory response / dynein axonemal particle / positive regulation of microtubule nucleation / mammary gland duct morphogenesis / ATP-dependent protein disaggregase activity / microglia differentiation / misfolded protein binding / maternal behavior / astrocyte differentiation / positive regulation of tumor necrosis factor-mediated signaling pathway / regulation of mitotic spindle assembly / cellular response to interleukin-7 / protein folding chaperone complex / RND1 GTPase cycle / aggresome / adrenal gland development / regulation of gluconeogenesis / cellular response to glucocorticoid stimulus / sulfonylurea receptor binding / sperm mitochondrial sheath / dATP binding / CTP binding / positive regulation of protein polymerization / Scavenging by Class F Receptors / cellular response to steroid hormone stimulus / vRNP Assembly / UTP binding / sperm plasma membrane / chaperone-mediated autophagy / Rho GDP-dissociation inhibitor binding / Respiratory syncytial virus genome replication / telomerase holoenzyme complex assembly / mitochondrial transport / Uptake and function of diphtheria toxin / protein insertion into mitochondrial outer membrane / 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 / TPR domain binding / dendritic growth cone / mRNA catabolic process / motor behavior / Assembly and release of respiratory syncytial virus (RSV) virions / PIWI-interacting RNA (piRNA) biogenesis / non-chaperonin molecular chaperone ATPase / : / Sema3A PAK dependent Axon repulsion / protein unfolding / regulation of protein ubiquitination / positive regulation of cell size / Regulation of HSF1-mediated heat shock response / estrogen response element binding / HSF1-dependent transactivation / cellular response to unfolded protein / response to unfolded protein / negative regulation of extrinsic apoptotic signaling pathway in absence of ligand / enzyme-substrate adaptor activity / skeletal muscle contraction / HSF1 activation / nuclear receptor-mediated steroid hormone signaling pathway / regulation of protein-containing complex assembly / FOXO-mediated transcription of oxidative stress, metabolic and neuronal genes / telomere maintenance via telomerase / Attenuation phase / chaperone-mediated protein complex assembly / cellular response to transforming growth factor beta stimulus / regulation of postsynaptic membrane neurotransmitter receptor levels / neurofibrillary tangle assembly / axonal growth cone / RHOBTB2 GTPase cycle / core promoter sequence-specific DNA binding / ATP metabolic process / positive regulation of lamellipodium assembly / eNOS activation / vesicle-mediated transport / nitric oxide metabolic process / Tetrahydrobiopterin (BH4) synthesis, recycling, salvage and regulation / inclusion body / DNA polymerase binding / heat shock protein binding
Similarity search - Function
STI1/HOP, DP domain / STI1/HOP, DP domain / Tetratricopeptide repeat 2 / Tetratricopeptide repeat / Glucocorticoid receptor / Glucocorticoid receptor / Glucocorticoid receptor / Heat shock chaperonin-binding / Heat shock chaperonin-binding / Heat shock chaperonin-binding motif. ...STI1/HOP, DP domain / STI1/HOP, DP domain / Tetratricopeptide repeat 2 / Tetratricopeptide repeat / Glucocorticoid receptor / Glucocorticoid receptor / Glucocorticoid receptor / Heat shock chaperonin-binding / Heat shock chaperonin-binding / Heat shock chaperonin-binding motif. / 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 / Histidine kinase/HSP90-like ATPase / 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 superfamily / Nuclear hormone receptor / Nuclear hormones receptors DNA-binding region signature. / Zinc finger, nuclear hormone receptor-type / Double treble clef zinc finger, C4 type / 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 / ATPase, nucleotide binding domain / 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
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.4 Å/pix.
x 120 pix.
= 288. Å		2.4 Å/pix.
x 120 pix.
= 288. Å		2.4 Å/pix.
x 120 pix.
= 288. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.4 Å
Density

Histogram

Histogram (log scale)
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-alpha
  • 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
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
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.2 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 2.2 µm / Nominal magnification: 60000
Sample stageSpecimen holder model: OTHER
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

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

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