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- EMDB-3343: Atomic cryoEM structure of Hsp90/Cdc37/Cdk4 complex -

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Entry
Database: EMDB / ID: EMD-3343
TitleAtomic cryoEM structure of Hsp90/Cdc37/Cdk4 complex
Map dataReconstruction of Hsp90:Cdc37:Cdk4 complex. Cdc37 is tagged at the N terminus with T4 lysozyme. Part of series of maps, the highest resolution map being EMD-3337, others being EMD-3338, EMD-3339, EMD-3340, EMD-3341, EMD-3342.
Sample
  • Sample: Complex of Human Hsp90 beta, human T4 lysozyme-Cdc37 and human Cdk4
  • Protein or peptide: Heat Shock Protein HSP 90 beta
  • Protein or peptide: Hsp90 co-chaperone Cdc37
  • Protein or peptide: Cyclin-dependent kinase 4
KeywordsHsp90 / Cdc37 / Cdk4 / chaperone / kinase / unfolding
Function / homology
Function and homology information


cyclin D3-CDK4 complex / cyclin D1-CDK4 complex / cyclin D2-CDK4 complex / Evasion of Oncogene Induced Senescence Due to Defective p16INK4A binding to CDK4 / Evasion of Oxidative Stress Induced Senescence Due to Defective p16INK4A binding to CDK4 / cellular response to ionomycin / citrulline metabolic process / regulation of transcription initiation by RNA polymerase II / Drug-mediated inhibition of CDK4/CDK6 activity / Evasion of Oncogene Induced Senescence Due to Defective p16INK4A binding to CDK4 and CDK6 ...cyclin D3-CDK4 complex / cyclin D1-CDK4 complex / cyclin D2-CDK4 complex / Evasion of Oncogene Induced Senescence Due to Defective p16INK4A binding to CDK4 / Evasion of Oxidative Stress Induced Senescence Due to Defective p16INK4A binding to CDK4 / cellular response to ionomycin / citrulline metabolic process / regulation of transcription initiation by RNA polymerase II / Drug-mediated inhibition of CDK4/CDK6 activity / Evasion of Oncogene Induced Senescence Due to Defective p16INK4A binding to CDK4 and CDK6 / Evasion of Oxidative Stress Induced Senescence Due to Defective p16INK4A binding to CDK4 and CDK6 / regulation of type II interferon-mediated signaling pathway / regulation of type B pancreatic cell proliferation / HSP90-CDC37 chaperone complex / receptor ligand inhibitor activity / very long-chain fatty acid metabolic process / negative regulation of proteasomal protein catabolic process / Aryl hydrocarbon receptor signalling / : / aryl hydrocarbon receptor complex / dynein axonemal particle / histone methyltransferase binding / Transcriptional regulation by RUNX2 / cellular response to phorbol 13-acetate 12-myristate / mitochondrial genome maintenance / ATP-dependent protein binding / positive regulation of protein localization to cell surface / protein kinase regulator activity / protein folding chaperone complex / cyclin-dependent protein serine/threonine kinase regulator activity / telomerase holoenzyme complex assembly / post-transcriptional regulation of gene expression / Respiratory syncytial virus genome replication / Uptake and function of diphtheria toxin / regulation of cyclin-dependent protein serine/threonine kinase activity / 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 / PTK6 Regulates Cell Cycle / Assembly and release of respiratory syncytial virus (RSV) virions / positive regulation of transforming growth factor beta receptor signaling pathway / dendritic growth cone / Defective binding of RB1 mutants to E2F1,(E2F2, E2F3) / regulation of type I interferon-mediated signaling pathway / The NLRP3 inflammasome / : / Sema3A PAK dependent Axon repulsion / regulation of protein ubiquitination / telomere maintenance via telomerase / HSF1-dependent transactivation / response to unfolded protein / chaperone-mediated protein complex assembly / HSF1 activation / bicellular tight junction / Attenuation phase / cyclin-dependent kinase / RHOBTB2 GTPase cycle / protein targeting / cyclin-dependent protein serine/threonine kinase activity / cellular response to interleukin-4 / Purinergic signaling in leishmaniasis infection / axonal growth cone / DNA polymerase binding / cyclin-dependent protein kinase holoenzyme complex / supramolecular fiber organization / chaperone-mediated protein folding / Signaling by ERBB2 / heat shock protein binding / negative regulation of proteasomal ubiquitin-dependent protein catabolic process / regulation of G2/M transition of mitotic cell cycle / protein folding chaperone / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / positive regulation of G2/M transition of mitotic cell cycle / nitric-oxide synthase regulator activity / cyclin binding / Constitutive Signaling by Overexpressed ERBB2 / ESR-mediated signaling / Ubiquitin-dependent degradation of Cyclin D / positive regulation of cell differentiation / ATP-dependent protein folding chaperone / Signaling by ERBB2 TMD/JMD mutants / peptide binding / Constitutive Signaling by EGFRvIII / Hsp90 protein binding / Signaling by ERBB2 ECD mutants / DDX58/IFIH1-mediated induction of interferon-alpha/beta / placenta development / Signaling by ERBB2 KD Mutants / tau protein binding / kinase binding / Oncogene Induced Senescence / Regulation of necroptotic cell death
Similarity search - Function
Cdc37, C-terminal / Cdc37, Hsp90 binding / Cdc37, Hsp90-binding domain superfamily / Cdc37 C terminal domain / Cdc37 Hsp90 binding domain / Cdc37 C terminal domain / Cdc37 Hsp90 binding domain / Cdc37 N terminal kinase binding / Cdc37 / Cdc37, N-terminal domain ...Cdc37, C-terminal / Cdc37, Hsp90 binding / Cdc37, Hsp90-binding domain superfamily / Cdc37 C terminal domain / Cdc37 Hsp90 binding domain / Cdc37 C terminal domain / Cdc37 Hsp90 binding domain / Cdc37 N terminal kinase binding / Cdc37 / Cdc37, N-terminal domain / Cdc37 N terminal kinase binding / 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 / : / Histidine kinase-like ATPases / Histidine kinase/HSP90-like ATPase / Histidine kinase/HSP90-like ATPase superfamily / Ribosomal protein S5 domain 2-type fold / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / Protein kinase domain / Serine/Threonine protein kinases, catalytic domain / Protein kinase, ATP binding site / Protein kinases ATP-binding region signature. / Protein kinase domain profile. / Protein kinase domain / Protein kinase domain / Protein kinase-like domain superfamily
Similarity search - Domain/homology
Heat shock protein HSP 90-beta / Cyclin-dependent kinase 4 / Hsp90 co-chaperone Cdc37
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 8.0 Å
AuthorsVerba KA / Wang RYR / Arakawa A / Liu Y / Shirouzu M / Yokoyama S / Agard DA
CitationJournal: Science / Year: 2016
Title: Atomic structure of Hsp90-Cdc37-Cdk4 reveals that Hsp90 traps and stabilizes an unfolded kinase.
Authors: Kliment A Verba / Ray Yu-Ruei Wang / Akihiko Arakawa / Yanxin Liu / Mikako Shirouzu / Shigeyuki Yokoyama / David A Agard /
Abstract: The Hsp90 molecular chaperone and its Cdc37 cochaperone help stabilize and activate more than half of the human kinome. However, both the mechanism by which these chaperones assist their "client" ...The Hsp90 molecular chaperone and its Cdc37 cochaperone help stabilize and activate more than half of the human kinome. However, both the mechanism by which these chaperones assist their "client" kinases and the reason why some kinases are addicted to Hsp90 while closely related family members are independent are unknown. Our structural understanding of these interactions is lacking, as no full-length structures of human Hsp90, Cdc37, or either of these proteins with a kinase have been elucidated. Here we report a 3.9 angstrom cryo-electron microscopy structure of the Hsp90-Cdc37-Cdk4 kinase complex. Surprisingly, the two lobes of Cdk4 are completely separated with the β4-β5 sheet unfolded. Cdc37 mimics part of the kinase N lobe, stabilizing an open kinase conformation by wedging itself between the two lobes. Finally, Hsp90 clamps around the unfolded kinase β5 strand and interacts with exposed N- and C-lobe interfaces, protecting the kinase in a trapped unfolded state. On the basis of this structure and an extensive amount of previously collected data, we propose unifying conceptual and mechanistic models of chaperone-kinase interactions.
History
DepositionFeb 18, 2016-
Header (metadata) releaseMar 9, 2016-
Map releaseJun 29, 2016-
UpdateJul 13, 2016-
Current statusJul 13, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.095
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.095
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3343.tif

Downloads & links

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Map

FileDownload / File: emd_3343.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of Hsp90:Cdc37:Cdk4 complex. Cdc37 is tagged at the N terminus with T4 lysozyme. Part of series of maps, the highest resolution map being EMD-3337, others being EMD-3338, EMD-3339, EMD-3340, EMD-3341, EMD-3342.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.44 Å/pix.
x 128 pix.
= 312.32 Å		2.44 Å/pix.
x 128 pix.
= 312.32 Å		2.44 Å/pix.
x 128 pix.
= 312.32 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.44 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.095 / Movie #1: 0.095
Minimum - Maximum-0.08927971 - 0.33076599
Average (Standard dev.)0.00093451 (±0.01407647)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 312.32 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.442.442.44
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z312.320312.320312.320
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-0.0890.3310.001

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Supplemental data

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Sample components

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Entire : Complex of Human Hsp90 beta, human T4 lysozyme-Cdc37 and human Cdk4

EntireName: Complex of Human Hsp90 beta, human T4 lysozyme-Cdc37 and human Cdk4
Components
  • Sample: Complex of Human Hsp90 beta, human T4 lysozyme-Cdc37 and human Cdk4
  • Protein or peptide: Heat Shock Protein HSP 90 beta
  • Protein or peptide: Hsp90 co-chaperone Cdc37
  • Protein or peptide: Cyclin-dependent kinase 4

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Supramolecule #1000: Complex of Human Hsp90 beta, human T4 lysozyme-Cdc37 and human Cdk4

SupramoleculeName: Complex of Human Hsp90 beta, human T4 lysozyme-Cdc37 and human Cdk4
type: sample / ID: 1000
Details: All three proteins were co-expressed in Saccharomyces cerevisiae cells.
Oligomeric state: One Hsp90 homodimer binds to one T4Lysozyme-Cdc37 and one Cdk4
Number unique components: 3
Molecular weightExperimental: 245 KDa / Theoretical: 245 KDa / Method: As cloned, verified by SDS-PAGE

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Macromolecule #1: Heat Shock Protein HSP 90 beta

MacromoleculeName: Heat Shock Protein HSP 90 beta / type: protein_or_peptide / ID: 1 / Name.synonym: Hsp90 / Number of copies: 2 / Oligomeric state: Dimer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Location in cell: cytoplasm
Molecular weightTheoretical: 83 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast) / Recombinant plasmid: 83nu
SequenceUniProtKB: Heat shock protein HSP 90-beta / GO: citrulline metabolic process / InterPro: Heat shock protein Hsp90 family

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Macromolecule #2: Hsp90 co-chaperone Cdc37

MacromoleculeName: Hsp90 co-chaperone Cdc37 / type: protein_or_peptide / ID: 2 / Name.synonym: Cdc37
Details: T4 lysozyme was fused recombinantly at the N terminus of Cdc37, with a GS linker in between. This was done to verify placement of proteins in the complex. Therefore, the total mass of T4lys- ...Details: T4 lysozyme was fused recombinantly at the N terminus of Cdc37, with a GS linker in between. This was done to verify placement of proteins in the complex. Therefore, the total mass of T4lys-Cdc37 is 63kDa rather that 44.5kDa for pure Cdc37.
Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Location in cell: throughout
Molecular weightTheoretical: 44.5 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast) / Recombinant plasmid: 83nu
SequenceUniProtKB: Hsp90 co-chaperone Cdc37 / GO: mitochondrial genome maintenance

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Macromolecule #3: Cyclin-dependent kinase 4

MacromoleculeName: Cyclin-dependent kinase 4 / type: protein_or_peptide / ID: 3 / Name.synonym: Cdk4 / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Location in cell: throughout
Molecular weightTheoretical: 33.7 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast) / Recombinant plasmid: 83nu
SequenceUniProtKB: Cyclin-dependent kinase 4 / GO: very long-chain fatty acid metabolic process / InterPro: Protein kinase domain

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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

Concentration0.27 mg/mL
BufferpH: 7.5
Details: 20mM Tris pH 7.5, 150mM NaCl, 10mM KCl, 20mM NaMoO4, 1mM DTT, 0.085mM DDM
GridDetails: Glow discharged for 30 sec, C-flat 400 mesh 1.2/1.3 thick carbon grids (Protochips)
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 95 K / Instrument: FEI VITROBOT MARK III / Method: Single blot from 4 to 6 seconds, at 20C

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Electron microscopy

MicroscopeFEI TECNAI F30
Alignment procedureLegacy - Astigmatism: At high mag via FT.
DateSep 25, 2015
Image recordingCategory: CCD / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number real images: 1634 / Average electron dose: 40 e/Å2 / Details: 30 frames, 6 seconds total exposure / Bits/pixel: 8
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 31000
Sample stageSpecimen holder: Polara 6 cartridge loader / Specimen holder model: OTHER
Experimental equipment
Model: Tecnai F30 / Image courtesy: FEI Company

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Image processing

DetailsImage stacks were dose weighted, drift corrected, binned to 1.22A/pix and summed using new UCSF DriftCorr program. The particles were picked and the CTF was estimated the same way as other maps in the series. Rounds of 2D classification (300 classes, 50 iterations) followed by 3D classification (2 classes, 50 iterations) in Relion 1.4 were used to eliminate low quality particles. Using the final set of particles, 3D Auto-refine feature in Relion 1.4 was used to generate the final maps.
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 8.0 Å / Resolution method: OTHER / Software - Name: Relion / Number images used: 29313
Final two d classificationNumber classes: 1
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

5fwk


Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B / Chain - #2 - Chain ID: E / Chain - #3 - Chain ID: K
SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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Atomic model buiding 2

Initial modelPDB ID:

2lzm


Chain - Chain ID: A
SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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