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- EMDB-3341: Atomic cryoEM structure of Hsp90/Cdc37/Cdk4 complex -

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Entry
Database: EMDB / ID: EMD-3341
TitleAtomic cryoEM structure of Hsp90/Cdc37/Cdk4 complex
Map dataReconstruction of Hsp90:Cdc37:Cdk4 complex. Part of series of maps, the highest resolution map being EMD-3337. This is a different subclass from the same particles as in EMD-3337, having well defined Cdk4 N-Lobe. Other relevant maps are EMD-3338, EMD-3339, EMD-3340.
Sample
  • Sample: Complex of Human Hsp90 beta, human Cdc37 and human Cdk4
  • Protein or peptide: Heat Shock Protein HSP 90 betaHeat shock response
  • 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 / regulation of transcription initiation by RNA polymerase II / 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 type II interferon-mediated signaling pathway / Drug-mediated inhibition of CDK4/CDK6 activity ...cyclin D3-CDK4 complex / cyclin D1-CDK4 complex / regulation of transcription initiation by RNA polymerase II / 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 type II interferon-mediated signaling pathway / 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 B pancreatic cell proliferation / : / hypoxia-inducible factor-asparagine dioxygenase / : / very long-chain fatty acid metabolic process / [protein]-asparagine 3-dioxygenase activity / HSP90-CDC37 chaperone complex / positive regulation of cyclin-dependent protein kinase activity / peptidyl-histidine dioxygenase activity / peptidyl-aspartic acid 3-dioxygenase activity / positive regulation of mitophagy in response to mitochondrial depolarization / Aryl hydrocarbon receptor signalling / negative regulation of proteasomal protein catabolic process / Cellular response to hypoxia / dynein axonemal particle / aryl hydrocarbon receptor complex / histone methyltransferase binding / carboxylic acid binding / positive regulation of vasculogenesis / Transcriptional regulation by RUNX2 / cellular response to phorbol 13-acetate 12-myristate / mitochondrial genome maintenance / protein kinase regulator activity / ankyrin repeat binding / positive regulation of protein localization to cell surface / ATP-dependent protein binding / oxygen sensor activity / protein folding chaperone complex / negative regulation of protein metabolic process / Notch binding / cyclin-dependent protein serine/threonine kinase regulator activity / positive regulation of tau-protein kinase activity / post-transcriptional regulation of gene expression / telomerase holoenzyme complex assembly / Uptake and function of diphtheria toxin / 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 / negative regulation of Notch signaling pathway / TPR domain binding / PTK6 Regulates Cell Cycle / regulation of cyclin-dependent protein serine/threonine kinase activity / positive regulation of transforming growth factor beta receptor signaling pathway / dendritic growth cone / Defective binding of RB1 mutants to E2F1,(E2F2, E2F3) / positive regulation of phosphoprotein phosphatase activity / regulation of type I interferon-mediated signaling pathway / The NLRP3 inflammasome / Sema3A PAK dependent Axon repulsion / regulation of protein ubiquitination / HSF1-dependent transactivation / telomere maintenance via telomerase / negative regulation of proteasomal ubiquitin-dependent protein catabolic process / response to unfolded protein / NF-kappaB binding / bicellular tight junction / cyclin-dependent protein kinase holoenzyme complex / chaperone-mediated protein complex assembly / HSF1 activation / protein targeting / positive regulation of myoblast differentiation / Attenuation phase / cyclin-dependent kinase / RHOBTB2 GTPase cycle / cyclin-dependent protein serine/threonine kinase activity / Purinergic signaling in leishmaniasis infection / DNA polymerase binding / supramolecular fiber organization / axonal growth cone / Signaling by ERBB2 / positive regulation of telomerase activity / heat shock protein binding / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / positive regulation of G2/M transition of mitotic cell cycle / regulation of G2/M transition of mitotic cell cycle / nitric-oxide synthase regulator activity / cellular response to interleukin-4 / Constitutive Signaling by Overexpressed ERBB2 / ESR-mediated signaling / response to organic substance / placenta development
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 / Hypoxia-inducible factor 1-alpha inhibitor, domain II / Cupin-like domain 8 / Cupin-like domain / 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 / A domain family that is part of the cupin metalloenzyme superfamily. / JmjC domain / JmjC domain profile. / RmlC-like jelly roll fold / 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 / Hypoxia-inducible factor 1-alpha inhibitor
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 9.0 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: 0.02
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5fwl
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_3341.map.gz / Format: CCP4 / Size: 62.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of Hsp90:Cdc37:Cdk4 complex. Part of series of maps, the highest resolution map being EMD-3337. This is a different subclass from the same particles as in EMD-3337, having well defined Cdk4 N-Lobe. Other relevant maps are EMD-3338, EMD-3339, EMD-3340.
Voxel sizeX=Y=Z: 1.315 Å
Density
Contour LevelBy AUTHOR: 0.02 / Movie #1: 0.02
Minimum - Maximum-0.02356639 - 0.08513857
Average (Standard dev.)0.00003483 (±0.00293237)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 336.64 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.3151.3151.315
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z336.640336.640336.640
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0240.0850.000

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Supplemental data

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Sample components

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

EntireName: Complex of Human Hsp90 beta, human Cdc37 and human Cdk4
Components
  • Sample: Complex of Human Hsp90 beta, human Cdc37 and human Cdk4
  • Protein or peptide: Heat Shock Protein HSP 90 betaHeat shock response
  • 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 Cdc37 and human Cdk4

SupramoleculeName: Complex of Human Hsp90 beta, human Cdc37 and human Cdk4
type: sample / ID: 1000 / Details: All three proteins were co-expressed in Sf9 cells.
Oligomeric state: One Hsp90 homodimer binds to one 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: Spodoptera frugiperda (fall armyworm) / Recombinant plasmid: pFastBacHT
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 / 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: Spodoptera frugiperda (fall armyworm) / Recombinant plasmid: pFastBacHT
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: Spodoptera frugiperda (fall armyworm) / Recombinant plasmid: pFastBacHT
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-HCl (pH 7.5), 150 mM NaCl, 10 mM KCl, 10 mM MgCl2, 20 mM Na2MoO4, 2mM 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 TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.8 µm / Nominal defocus min: 1.4 µm / Nominal magnification: 22500
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Alignment procedureLegacy - Astigmatism: At high mag via FT.
DateNov 25, 2014
Image recordingCategory: CCD / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number real images: 3718 / Average electron dose: 44 e/Å2 / Details: 38 frames, 7.6 seconds total exposure / Bits/pixel: 8
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final two d classificationNumber classes: 1
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 9.0 Å / Resolution method: OTHER / Software - Name: Relion / Number images used: 45974
DetailsImage stacks were corrected for motion and summed as described previously, resulting in binned sums (1.315A/pix). For particle picking the images were binned to 5.2A/pix and Gaussian bandpass filtered between 15A and 500A using EMAN2. SamViewer template based picking was then used to pick particles from all the micrographs, followed by manual review of all the picks. After such procedure 802877 particles were picked in total and extracted from images binned to 2.6A/pix. CTFFIND4 was used to estimate defocus parameters for all the images. Relion 1.4 was used for all the following steps unless noted otherwise. Reference free 2D classification into 300 classes for 75 iterations was performed followed by manual examination of the resulting class averages. Low resolution/signal to noise/feature class averages and contributing particles were discarded, resulting in 670000 particles left. The resulting particles were 3D classified into 4 classes resulting in two classes having high-resolution features (390000 particles). At this stage particles were extracted from 1.315A/pix micrographs and all the following processing was done with these particles. Using 3D Auto-refine in Relion 1.4, a reconstruction was obtained from 390000 particles resulting from 3D classification above (using highest resolution 3D class as initial model, low pass filtered to 20A). Using the resulting parameters, the particles were further drift corrected per particle and dose weighted using the Particle Polishing feature. The B-factor weighing curve was fit by a polynomial (with a rationale that such a curve should be smooth) and used to generate new weighting parameters for Particle Polishing, with which 390000 particles were then polished. All further data processing was done using the polished particles. Re-refinement of the 390000 particles after polishing yielded the map at about 4A resolution (determined using gold standard FSC in the PostProcessing tab). The 390000 particles were then 3D classified into four different classes without particle re-alignment, using the alignment parameters from 4A reconstruction and subtraction procedure described previously. Particles contributing to each of the four classes were grouped and a full 3D refinement with a spherical 200A mask was performed with each of the four groups of particles using the same initial model, low pass filtered to 20A. This is one of the resulting reconstructions.
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B / Chain - #2 - Chain ID: E / Chain - #3 - Chain ID: K
SoftwareName: Chimera, Rosetta
DetailsResidues 5-85 (N-lobe) of Cdk4 from 3G33 were fit in Chimera as rigid body into EMD-3341. Such fit N-lobe of Cdk4 was further tweaked in Coot in the context of 5FWK to join the Cdk4 chain and minimize clashes for each of the maps. To relieve atomic clashes or bond length/angle distortions at the linker regions, these models were subjected to "Cartesian space relax" protocol within corresponding density maps using Rosetta. Final models were selected using the combined score of Rosetta all-atom physically-realistic score and electron density score.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 200
Target criteria: cross correlation of fit into the density with Rosetta force field score
Output model

PDB-5fwl:
Atomic cryoEM structure of Hsp90-Cdc37-Cdk4 complex

PDB-5jwl:
Factor Inhibiting HIF D201E in Complex with Zn, and Alpha-Ketoglutarate

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Atomic model buiding 2

Initial modelPDB ID:
SoftwareName: Chimera, Rosetta
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 200
Target criteria: cross correlation of fit into the density with Rosetta force field score
Output model

PDB-5fwl:
Atomic cryoEM structure of Hsp90-Cdc37-Cdk4 complex

PDB-5jwl:
Factor Inhibiting HIF D201E in Complex with Zn, and Alpha-Ketoglutarate

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