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- PDB-8gft: Hsp90 provides platform for CRaf dephosphorylation by PP5 -

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Entry
Database: PDB / ID: 8gft
TitleHsp90 provides platform for CRaf dephosphorylation by PP5
Components
  • Heat shock protein HSP 90-beta
  • Hsp90 co-chaperone Cdc37, N-terminally processed
  • RAF proto-oncogene serine/threonine-protein kinase
  • Serine/threonine-protein phosphatase 5
KeywordsCHAPERONE / kinase / phosphatase / complex
Function / homology
Function and homology information


regulation of type II interferon-mediated signaling pathway / response to arachidonate / HSP90-CDC37 chaperone complex / death-inducing signaling complex assembly / negative regulation of proteasomal protein catabolic process / Aryl hydrocarbon receptor signalling / aryl hydrocarbon receptor complex / : / peptidyl-serine dephosphorylation / peptidyl-threonine dephosphorylation ...regulation of type II interferon-mediated signaling pathway / response to arachidonate / HSP90-CDC37 chaperone complex / death-inducing signaling complex assembly / negative regulation of proteasomal protein catabolic process / Aryl hydrocarbon receptor signalling / aryl hydrocarbon receptor complex / : / peptidyl-serine dephosphorylation / peptidyl-threonine dephosphorylation / intermediate filament cytoskeleton organization / dynein axonemal particle / histone methyltransferase binding / receptor ligand inhibitor activity / type B pancreatic cell proliferation / regulation of Rho protein signal transduction / SHOC2 M1731 mutant abolishes MRAS complex function / Gain-of-function MRAS complexes activate RAF signaling / ATP-dependent protein binding / Rap1 signalling / positive regulation of protein localization to cell surface / regulation of cell motility / protein kinase regulator activity / insulin secretion involved in cellular response to glucose stimulus / protein folding chaperone complex / response to morphine / Negative feedback regulation of MAPK pathway / telomerase holoenzyme complex assembly / post-transcriptional regulation of gene expression / Respiratory syncytial virus genome replication / GP1b-IX-V activation signalling / Uptake and function of diphtheria toxin / IFNG signaling activates MAPKs / protein serine/threonine phosphatase 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 / regulation of cyclin-dependent protein serine/threonine kinase activity / myosin phosphatase activity / TPR domain binding / protein-serine/threonine phosphatase / ERBB2-ERBB3 signaling pathway / positive regulation of transforming growth factor beta receptor signaling pathway / Assembly and release of respiratory syncytial virus (RSV) virions / face development / dendritic growth cone / pseudopodium / phosphatase activity / regulation of type I interferon-mediated signaling pathway / regulation of cell differentiation / : / neurotrophin TRK receptor signaling pathway / somatic stem cell population maintenance / thyroid gland development / Sema3A PAK dependent Axon repulsion / The NLRP3 inflammasome / phosphoprotein phosphatase activity / regulation of protein ubiquitination / HSF1-dependent transactivation / response to unfolded protein / extrinsic apoptotic signaling pathway via death domain receptors / MAP kinase kinase kinase activity / HSF1 activation / telomere maintenance via telomerase / chaperone-mediated protein complex assembly / Attenuation phase / protein targeting / negative regulation of protein-containing complex assembly / RHOBTB2 GTPase cycle / cellular response to interleukin-4 / axonal growth cone / type II interferon-mediated signaling pathway / Purinergic signaling in leishmaniasis infection / Schwann cell development / DNA polymerase binding / activation of adenylate cyclase activity / negative regulation of extrinsic apoptotic signaling pathway via death domain receptors / supramolecular fiber organization / chaperone-mediated protein folding / Signaling by ERBB2 / negative regulation of proteasomal ubiquitin-dependent protein catabolic process / heat shock protein binding / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / nitric-oxide synthase regulator activity / response to muscle stretch / protein folding chaperone / CD209 (DC-SIGN) signaling / myelination / Constitutive Signaling by Overexpressed ERBB2 / ESR-mediated signaling / : / insulin-like growth factor receptor signaling pathway / thymus development
Similarity search - Function
PPP domain / PP5, C-terminal metallophosphatase domain / PPP5 TPR repeat region / : / Cdc37, C-terminal / Cdc37, Hsp90 binding / Cdc37, Hsp90-binding domain superfamily / Cdc37 C terminal domain / Cdc37 Hsp90 binding domain / Cdc37 C terminal domain ...PPP domain / PP5, C-terminal metallophosphatase domain / PPP5 TPR repeat region / : / 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 / Serine/threonine specific protein phosphatases signature. / Protein phosphatase 2A homologues, catalytic domain. / Raf-like Ras-binding domain / Raf-like Ras-binding / Ras-binding domain (RBD) profile. / Raf-like Ras-binding domain / Serine/threonine-specific protein phosphatase/bis(5-nucleosyl)-tetraphosphatase / Diacylglycerol/phorbol-ester binding / : / Tetratricopeptide repeat / Phorbol esters/diacylglycerol binding domain (C1 domain) / Zinc finger phorbol-ester/DAG-type signature. / Calcineurin-like phosphoesterase domain, ApaH type / Calcineurin-like phosphoesterase / Zinc finger phorbol-ester/DAG-type profile. / Protein kinase C conserved region 1 (C1) domains (Cysteine-rich domains) / Protein kinase C-like, phorbol ester/diacylglycerol-binding domain / Metallo-dependent phosphatase-like / 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 / Hsp90 protein / C1-like domain superfamily / 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 / Tetratricopeptide-like helical domain superfamily / Ubiquitin-like domain 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-like domain superfamily
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / ADENOSINE-5'-TRIPHOSPHATE / : / : / RAF proto-oncogene serine/threonine-protein kinase / Heat shock protein HSP 90-beta / Serine/threonine-protein phosphatase 5 / Hsp90 co-chaperone Cdc37
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsJaime-Garza, M. / Nowotny, C.A. / Coutandin, D. / Wang, F. / Tabios, M. / Agard, D.A.
Funding support United States, 4items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM118099 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1S10OD026881 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1S10OD020054 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1S10OD021741 United States
CitationJournal: Nat Commun / Year: 2023
Title: Hsp90 provides a platform for kinase dephosphorylation by PP5.
Authors: Maru Jaime-Garza / Carlos A Nowotny / Daniel Coutandin / Feng Wang / Mariano Tabios / David A Agard /
Abstract: The Hsp90 molecular chaperone collaborates with the phosphorylated Cdc37 cochaperone for the folding and activation of its many client kinases. As with many kinases, the Hsp90 client kinase CRaf is ...The Hsp90 molecular chaperone collaborates with the phosphorylated Cdc37 cochaperone for the folding and activation of its many client kinases. As with many kinases, the Hsp90 client kinase CRaf is activated by phosphorylation at specific regulatory sites. The cochaperone phosphatase PP5 dephosphorylates CRaf and Cdc37 in an Hsp90-dependent manner. Although dephosphorylating Cdc37 has been proposed as a mechanism for releasing Hsp90-bound kinases, here we show that Hsp90 bound kinases sterically inhibit Cdc37 dephosphorylation indicating kinase release must occur before Cdc37 dephosphorylation. Our cryo-EM structure of PP5 in complex with Hsp90:Cdc37:CRaf reveals how Hsp90 both activates PP5 and scaffolds its association with the bound CRaf to dephosphorylate phosphorylation sites neighboring the kinase domain. Thus, we directly show how Hsp90's role in maintaining protein homeostasis goes beyond folding and activation to include post translationally modifying its client kinases.
History
DepositionMar 8, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 3, 2023Provider: repository / Type: Initial release
Revision 1.1Nov 13, 2024Group: Data collection / Refinement description / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_3d_fitting_list.initial_refinement_model_id / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Heat shock protein HSP 90-beta
B: Heat shock protein HSP 90-beta
C: Hsp90 co-chaperone Cdc37, N-terminally processed
D: RAF proto-oncogene serine/threonine-protein kinase
E: Serine/threonine-protein phosphatase 5
hetero molecules


Theoretical massNumber of molelcules
Total (without water)305,82513
Polymers304,6545
Non-polymers1,1718
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: assay for oligomerization, FCS was used to test for PP5 binding of Hsp90:Cdc37:CRaf complex., cross-linking, Complex was crosslinked and ran through a sizing column. The complex was the ...Evidence: assay for oligomerization, FCS was used to test for PP5 binding of Hsp90:Cdc37:CRaf complex., cross-linking, Complex was crosslinked and ran through a sizing column. The complex was the expected size for the Hsp90:Cdc37:CRaf:PP5 complex., electron microscopy, All four components can be seen in the assembly. PP5 is heterogeneously bound to the complex., gel filtration, The Hsp90:Cdc37:CRaf complex was incubated with PP5, ran through a sizing column, and analyzed by running a gel. All four components co-eluted and were present.
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 4 types, 5 molecules ABCDE

#1: Protein Heat shock protein HSP 90-beta / HSP 90 / Heat shock 84 kDa / HSP 84 / HSP84


Mass: 83595.484 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: Hsp90 sequence with HRV 3C cleavage site and one residue glycine linker.
Source: (gene. exp.) Homo sapiens (human) / Gene: HSP90AB1, HSP90B, HSPC2, HSPCB / Production host: Saccharomyces cerevisiae (brewer's yeast) / Strain (production host): JEL1 / References: UniProt: P08238
#2: Protein Hsp90 co-chaperone Cdc37, N-terminally processed


Mass: 45352.223 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Cdc37 followed by HRV 3C cleavage site at C-terminal of construct.
Source: (gene. exp.) Homo sapiens (human) / Gene: CDC37, CDC37A / Production host: Saccharomyces cerevisiae (brewer's yeast) / Strain (production host): JEL1 / References: UniProt: Q16543
#3: Protein RAF proto-oncogene serine/threonine-protein kinase / Proto-oncogene c-RAF / cRaf / Raf-1


Mass: 34926.168 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: CRaf/Raf1 kinase domain followed by a LPESG linker, Strep Tag II sequence (WSHPQFEK) and a HRV 3C cleavage site (LEVLFQ).
Source: (gene. exp.) Homo sapiens (human) / Gene: RAF1, RAF / Production host: Saccharomyces cerevisiae (brewer's yeast) / Strain (production host): JEL1
References: UniProt: P04049, non-specific serine/threonine protein kinase
#4: Protein Serine/threonine-protein phosphatase 5 / PP5 / Protein phosphatase T / PP-T / PPT


Mass: 57184.730 Da / Num. of mol.: 1 / Mutation: H304A
Source method: isolated from a genetically manipulated source
Details: HRV 3C cleavage site followed by GS linker and PP5 sequence.
Source: (gene. exp.) Homo sapiens (human) / Gene: PPP5C, PPP5 / Plasmid: pQiq / Production host: Escherichia coli BL21 (bacteria)
References: UniProt: P53041, protein-serine/threonine phosphatase

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Non-polymers , 5 types, 8 molecules

#5: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
#6: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#7: Chemical ChemComp-K / POTASSIUM ION


Mass: 39.098 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: K
#8: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Comment: ATP, energy-carrying molecule*YM
#9: Chemical ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mn

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Details

Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeDetailsEntity IDParent-IDSource
1Hsp90:Cdc37:CRaf:PP5 complexCOMPLEXYeast purified Hsp90:Cdc37:CRaf complex incubated with e. Coli purified PP5 (mutant H304A). Sample then cross-linked with 0.05% glutaraldehyde for 15m at room temperature, and ran over S200 sizing column.#1-#40RECOMBINANT
2Protein Phosphatase 5 (H304A)COMPLEXE coli purified Protein Phosphatase 5, with inactivating H304A mutation.#31RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.304 MDaNO
210.057 MDaNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Homo sapiens (human)9606
32Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrainPlasmid
21Saccharomyces cerevisiae (brewer's yeast)4932JEL-183 nu yeast expression vector
32Escherichia coli (E. coli)562BL21pqiq vector
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESC8H18N2O4S1
250 mMKClKCl1
310 mMMagnesium ChlorideMgCl21
40.5 mMTCEPC9H15O6P1
51 mMEDTAC10H16N2O81
SpecimenConc.: 0.09 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 283.15 K
Details: 3uL OF SAMPLE, 10C, 100% HUMIDITY, 30S WAIT TIME, 3S BLOT TIME, -2 BLOT FORCE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 1800 nm / Nominal defocus min: 800 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 69 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of real images: 4160

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
EM software
IDNameVersionCategory
1cryoSPARC3.3.2particle selection
2SerialEM3.8-betaimage acquisition
4RELION3.1.3CTF correction
7ISOLDE1.0b3model fitting
8UCSF Chimera1.15model fitting
9UCSF ChimeraX1.2.5model fitting
10Rosetta3.11model fitting
11PHENIX1.20.1-4487model fitting
13PHENIX1.20.1-4487model refinement
14RELION3.1.3initial Euler assignment
15RELION3.1.3final Euler assignment
16RELION3.1.3classification
17RELION3.1.33D reconstruction
18UCSF ChimeraX1.2.53D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3730385 / Details: Gaussian blob particle picking in cryosparc.
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 545237 / Algorithm: FOURIER SPACE
Details: Three different maps were used for final composite map: Hsp90:Cdc37: 288k particles, 3.2A PP5 TPR: 215k particles, 3.3A PP5 catalytic domain: 43k particles, 3.8A Composite half maps were ...Details: Three different maps were used for final composite map: Hsp90:Cdc37: 288k particles, 3.2A PP5 TPR: 215k particles, 3.3A PP5 catalytic domain: 43k particles, 3.8A Composite half maps were used to get the final resolution in Relion PostProcessing. All original maps provided in the "Related entries" tab.
Num. of class averages: 3 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Details: This model was built using rigid body docking in Chimera and ChimeraX for all main chains, and RosettaCM to add remaining fragments not included in previous models. Refinement was done using ...Details: This model was built using rigid body docking in Chimera and ChimeraX for all main chains, and RosettaCM to add remaining fragments not included in previous models. Refinement was done using iterative Phenix and RosettaRelax, and finalized with ISOLDE.
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
15FWL

5fwl

15FWL1PDBexperimental model
21WAO

1wao

11WAO2PDBexperimental model
31S95

1s95

11S953PDBexperimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00618120
ELECTRON MICROSCOPYf_angle_d0.58124379
ELECTRON MICROSCOPYf_dihedral_angle_d12.9526970
ELECTRON MICROSCOPYf_chiral_restr0.052652
ELECTRON MICROSCOPYf_plane_restr0.0043135

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