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- EMDB-29984: Hsp90 provides platform for CRaf dephosphorylation by PP5 -

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Basic information

Entry
Database: EMDB / ID: EMD-29984
TitleHsp90 provides platform for CRaf dephosphorylation by PP5
Map dataComposite map used for model fitting.
Sample
  • Complex: Hsp90:Cdc37:CRaf:PP5 complex
    • Complex: Protein Phosphatase 5 (H304A)
      • Protein or peptide: RAF proto-oncogene serine/threonine-protein kinase
    • Protein or peptide: Heat shock protein HSP 90-betaHeat shock response
    • Protein or peptide: Hsp90 co-chaperone Cdc37, N-terminally processed
    • Protein or peptide: Serine/threonine-protein phosphatase 5
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: POTASSIUM IONPotassium
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: MANGANESE (II) ION
Function / homology
Function and homology information


regulation of type II interferon-mediated signaling pathway / : / response to arachidonic acid / HSP90-CDC37 chaperone complex / positive regulation of cyclin-dependent protein kinase activity / death-inducing signaling complex assembly / positive regulation of mitophagy in response to mitochondrial depolarization / negative regulation of proteasomal protein catabolic process / Aryl hydrocarbon receptor signalling / peptidyl-serine dephosphorylation ...regulation of type II interferon-mediated signaling pathway / : / response to arachidonic acid / HSP90-CDC37 chaperone complex / positive regulation of cyclin-dependent protein kinase activity / death-inducing signaling complex assembly / positive regulation of mitophagy in response to mitochondrial depolarization / negative regulation of proteasomal protein catabolic process / Aryl hydrocarbon receptor signalling / peptidyl-serine dephosphorylation / peptidyl-threonine dephosphorylation / aryl hydrocarbon receptor complex / intermediate filament cytoskeleton organization / dynein axonemal particle / histone methyltransferase binding / type B pancreatic cell proliferation / protein kinase regulator activity / regulation of Rho protein signal transduction / positive regulation of protein localization to cell surface / SHOC2 M1731 mutant abolishes MRAS complex function / Gain-of-function MRAS complexes activate RAF signaling / Rap1 signalling / response to morphine / ATP-dependent protein binding / regulation of cell motility / protein folding chaperone complex / insulin secretion involved in cellular response to glucose stimulus / negative regulation of protein metabolic process / Negative feedback regulation of MAPK pathway / positive regulation of tau-protein kinase activity / post-transcriptional regulation of gene expression / myosin phosphatase activity / protein serine/threonine phosphatase activity / telomerase holoenzyme complex assembly / IFNG signaling activates MAPKs / GP1b-IX-V activation signalling / 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 / protein-serine/threonine phosphatase / ERBB2-ERBB3 signaling pathway / TPR domain binding / regulation of cell differentiation / face development / positive regulation of transforming growth factor beta receptor signaling pathway / pseudopodium / phosphatase activity / regulation of cyclin-dependent protein serine/threonine kinase activity / dendritic growth cone / somatic stem cell population maintenance / phosphoprotein phosphatase activity / neurotrophin TRK receptor signaling pathway / thyroid gland development / regulation of type I interferon-mediated signaling pathway / positive regulation of phosphoprotein phosphatase activity / Sema3A PAK dependent Axon repulsion / The NLRP3 inflammasome / regulation of protein ubiquitination / HSF1-dependent transactivation / telomere maintenance via telomerase / negative regulation of proteasomal ubiquitin-dependent protein catabolic process / response to unfolded protein / extrinsic apoptotic signaling pathway via death domain receptors / MAP kinase kinase kinase activity / protein targeting / chaperone-mediated protein complex assembly / HSF1 activation / Attenuation phase / cellular response to interleukin-4 / negative regulation of protein-containing complex assembly / RHOBTB2 GTPase cycle / Schwann cell development / Purinergic signaling in leishmaniasis infection / axonal growth cone / type II interferon-mediated signaling pathway / supramolecular fiber organization / DNA polymerase binding / negative regulation of extrinsic apoptotic signaling pathway via death domain receptors / 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 / activation of adenylate cyclase activity / response to muscle stretch / myelination / nitric-oxide synthase regulator activity / CD209 (DC-SIGN) signaling / Constitutive Signaling by Overexpressed ERBB2 / ESR-mediated signaling / protein dephosphorylation / 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 / Cdc37 Hsp90 binding 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. / Serine/threonine-specific protein phosphatase/bis(5-nucleosyl)-tetraphosphatase / Raf-like Ras-binding domain / Raf-like Ras-binding / Ras-binding domain (RBD) profile. / Raf-like Ras-binding domain / Diacylglycerol/phorbol-ester binding / Tetratricopeptide repeat 1 / Tetratricopeptide repeat / Phorbol esters/diacylglycerol binding domain (C1 domain) / Calcineurin-like phosphoesterase domain, ApaH type / Zinc finger phorbol-ester/DAG-type signature. / 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. / C1-like domain superfamily / HSP90, C-terminal domain / Heat shock protein Hsp90, N-terminal / 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 / 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
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsJaime-Garza M / Nowotny CA / Coutandin D / Wang F / Tabios M / Agard DA
Funding support United States, 4 items
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, 2023-
Header (metadata) releaseMay 3, 2023-
Map releaseMay 3, 2023-
UpdateMay 3, 2023-
Current statusMay 3, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29984.png

Downloads & links

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Map

FileDownload / File: emd_29984.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationComposite map used for model fitting.
Voxel sizeX=Y=Z: 0.835 Å
Density
Contour LevelBy AUTHOR: 0.00576
Minimum - Maximum-0.005436401 - 0.032438822
Average (Standard dev.)0.00025321546 (±0.0013087377)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-4-5-3
Dimensions320320320
Spacing320320320
CellA=B=C: 267.19998 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Postprocessed composite map which gave FSC curve file,...

Fileemd_29984_additional_1.map
AnnotationPostprocessed composite map which gave FSC curve file, map resolution, and was used for model fitting.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Composite half map.

Fileemd_29984_half_map_1.map
AnnotationComposite half map.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Composite half map.

Fileemd_29984_half_map_2.map
AnnotationComposite half map.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Hsp90:Cdc37:CRaf:PP5 complex

EntireName: Hsp90:Cdc37:CRaf:PP5 complex
Components
  • Complex: Hsp90:Cdc37:CRaf:PP5 complex
    • Complex: Protein Phosphatase 5 (H304A)
      • Protein or peptide: RAF proto-oncogene serine/threonine-protein kinase
    • Protein or peptide: Heat shock protein HSP 90-betaHeat shock response
    • Protein or peptide: Hsp90 co-chaperone Cdc37, N-terminally processed
    • Protein or peptide: Serine/threonine-protein phosphatase 5
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: POTASSIUM IONPotassium
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: MANGANESE (II) ION

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Supramolecule #1: Hsp90:Cdc37:CRaf:PP5 complex

SupramoleculeName: Hsp90:Cdc37:CRaf:PP5 complex / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1-#4
Details: Yeast 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.
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 57 KDa

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Supramolecule #2: Protein Phosphatase 5 (H304A)

SupramoleculeName: Protein Phosphatase 5 (H304A) / type: complex / ID: 2 / Chimera: Yes / Parent: 1 / Macromolecule list: #3
Details: E coli purified Protein Phosphatase 5, with inactivating H304A mutation.
Source (natural)Organism: Homo sapiens (human)

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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
Details: Hsp90 sequence with HRV 3C cleavage site and one residue glycine linker.
Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 83.595484 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: GPGMPEEVHH GEEEVETFAF QAEIAQLMSL IINTFYSNKE IFLRELISNA SDALDKIRYE SLTDPSKLDS GKELKIDIIP NPQERTLTL VDTGIGMTKA DLINNLGTIA KSGTKAFMEA LQAGADISMI GQFGVGFYSA YLVAEKVVVI TKHNDDEQYA W ESSAGGSF ...String:
GPGMPEEVHH GEEEVETFAF QAEIAQLMSL IINTFYSNKE IFLRELISNA SDALDKIRYE SLTDPSKLDS GKELKIDIIP NPQERTLTL VDTGIGMTKA DLINNLGTIA KSGTKAFMEA LQAGADISMI GQFGVGFYSA YLVAEKVVVI TKHNDDEQYA W ESSAGGSF TVRADHGEPI GRGTKVILHL KEDQTEYLEE RRVKEVVKKH SQFIGYPITL YLEKEREKEI SDDEAEEEKG EK EEEDKDD EEKPKIEDVG SDEEDDSGKD KKKKTKKIKE KYIDQEELNK TKPIWTRNPD DITQEEYGEF YKSLTNDWED HLA VKHFSV EGQLEFRALL FIPRRAPFDL FENKKKKNNI KLYVRRVFIM DSCDELIPEY LNFIRGVVDS EDLPLNISRE MLQQ SKILK VIRKNIVKKC LELFSELAED KENYKKFYEA FSKNLKLGIH EDSTNRRRLS ELLRYHTSQS GDEMTSLSEY VSRMK ETQK SIYYITGESK EQVANSAFVE RVRKRGFEVV YMTEPIDEYC VQQLKEFDGK SLVSVTKEGL ELPEDEEEKK KMEESK AKF ENLCKLMKEI LDKKVEKVTI SNRLVSSPCC IVTSTYGWTA NMERIMKAQA LRDNSTMGYM MAKKHLEINP DHPIVET LR QKAEADKNDK AVKDLVVLLF ETALLSSGFS LEDPQTHSNR IYRMIKLGLG IDEDEVAAEE PNAAVPDEIP PLEGDEDA S RMEEVD

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Macromolecule #2: Hsp90 co-chaperone Cdc37, N-terminally processed

MacromoleculeName: Hsp90 co-chaperone Cdc37, N-terminally processed / type: protein_or_peptide / ID: 2
Details: Cdc37 followed by HRV 3C cleavage site at C-terminal of construct.
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 45.352223 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MVDYSVWDHI EV(SEP)DDEDETH PNIDTASLFR WRHQARVERM EQFQKEKEEL DRGCRECKRK VAECQRKLKE LEVAEG GKA ELERLQAEAQ QLRKEERSWE QKLEEMRKKE KSMPWNVDTL SKDGFSKSMV NTKPEKTEED SEEVREQKHK TFVEKYE KQ IKHFGMLRRW ...String:
MVDYSVWDHI EV(SEP)DDEDETH PNIDTASLFR WRHQARVERM EQFQKEKEEL DRGCRECKRK VAECQRKLKE LEVAEG GKA ELERLQAEAQ QLRKEERSWE QKLEEMRKKE KSMPWNVDTL SKDGFSKSMV NTKPEKTEED SEEVREQKHK TFVEKYE KQ IKHFGMLRRW DDSQKYLSDN VHLVCEETAN YLVIWCIDLE VEEKCALMEQ VAHQTIVMQF ILELAKSLKV DPRACFRQ F FTKIKTADRQ YMEGFNDELE AFKERVRGRA KLRIEKAMKE YEEEERKKRL GPGGLDPVEV YESLPEELQK CFDVKDVQM LQDAISKMDP TDAKYHMQRC IDSGLWVPNS KASEAKEGEE AGPGDPLLEA VPKTGDEKDV SVLEVLFQ

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Macromolecule #3: RAF proto-oncogene serine/threonine-protein kinase

MacromoleculeName: RAF proto-oncogene serine/threonine-protein kinase / type: protein_or_peptide / ID: 3
Details: CRaf/Raf1 kinase domain followed by a LPESG linker, Strep Tag II sequence (WSHPQFEK) and a HRV 3C cleavage site (LEVLFQ).
Number of copies: 1 / Enantiomer: LEVO / EC number: non-specific serine/threonine protein kinase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 34.926168 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: GGRDSSYYWE IEASEVMLST RIGSGSFGTV YKGKWHGDVA VKILKVVDPT PEQFQAFRNE VAVLRKTRHV NILLFMGYMT KDNLAIVTQ WCEGSSLYKH LHVQETKFQM FQLIDIARQT AQGMDYLHAK NIIHRDMKSN NIFLHEGLTV KIGDFGLATV K SRWSGSQQ ...String:
GGRDSSYYWE IEASEVMLST RIGSGSFGTV YKGKWHGDVA VKILKVVDPT PEQFQAFRNE VAVLRKTRHV NILLFMGYMT KDNLAIVTQ WCEGSSLYKH LHVQETKFQM FQLIDIARQT AQGMDYLHAK NIIHRDMKSN NIFLHEGLTV KIGDFGLATV K SRWSGSQQ VEQPTGSVLW MAPEVIRMQD NNPFSFQSDV YSYGIVLYEL MTGELPYSHI NNRDQIIFMV GRGYASPDLS KL YKNCPKA MKRLVADCVK KVKEERPLFP QILSSIELLQ HSLPKINRLP ESGWSHPQFE KLEVLFQ

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Macromolecule #4: Serine/threonine-protein phosphatase 5

MacromoleculeName: Serine/threonine-protein phosphatase 5 / type: protein_or_peptide / ID: 4
Details: HRV 3C cleavage site followed by GS linker and PP5 sequence.
Number of copies: 1 / Enantiomer: LEVO / EC number: protein-serine/threonine phosphatase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 57.18473 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: GPGSMAMAEG ERTECAEPPR DEPPADGALK RAEELKTQAN DYFKAKDYEN AIKFYSQAIE LNPSNAIYYG NRSLAYLRTE CYGYALGDA TRAIELDKKY IKGYYRRAAS NMALGKFRAA LRDYETVVKV KPHDKDAKMK YQECNKIVKQ KAFERAIAGD E HKRSVVDS ...String:
GPGSMAMAEG ERTECAEPPR DEPPADGALK RAEELKTQAN DYFKAKDYEN AIKFYSQAIE LNPSNAIYYG NRSLAYLRTE CYGYALGDA TRAIELDKKY IKGYYRRAAS NMALGKFRAA LRDYETVVKV KPHDKDAKMK YQECNKIVKQ KAFERAIAGD E HKRSVVDS LDIESMTIED EYSGPKLEDG KVTISFMKEL MQWYKDQKKL HRKCAYQILV QVKEVLSKLS TLVETTLKET EK ITVCGDT HGQFYDLLNI FELNGLPSET NPYIFNGDFV DRGSFSVEVI LTLFGFKLLY PDHFHLLRGN AETDNMNQIY GFE GEVKAK YTAQMYELFS EVFEWLPLAQ CINGKVLIMH GGLFSEDGVT LDDIRKIERN RQPPDSGPMC DLLWSDPQPQ NGRS ISKRG VSCQFGPDVT KAFLEENNLD YIIRSHEVKA EGYEVAHGGR CVTVFSAPNY CDQMGNKASY IHLQGSDLRP QFHQF TAVP HPNVKPMAYA NTLLQLGMM

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Macromolecule #5: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 5 / Number of copies: 1 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM / Adenosine diphosphate

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Macromolecule #6: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 6 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #7: POTASSIUM ION

MacromoleculeName: POTASSIUM ION / type: ligand / ID: 7 / Number of copies: 2 / Formula: K
Molecular weightTheoretical: 39.098 Da

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Macromolecule #8: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 8 / Number of copies: 1 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM / Adenosine triphosphate

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Macromolecule #9: MANGANESE (II) ION

MacromoleculeName: MANGANESE (II) ION / type: ligand / ID: 9 / Number of copies: 2 / Formula: MN
Molecular weightTheoretical: 54.938 Da

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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

Concentration0.09 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHEPES
50.0 mMKClKCl
10.0 mMMgCl2Magnesium Chloride
0.5 mMC9H15O6PTCEP
1.0 mMC10H16N2O8EDTAEthylenediaminetetraacetic acid
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GRAPHENE OXIDE / Support film - topology: CONTINUOUS / Support film - Film thickness: 1.0 nm
VitrificationCryogen name: ETHANE / Chamber 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

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
SoftwareName: SerialEM (ver. 3.8-beta)
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number real images: 4160 / Average electron dose: 69.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 3730385 / Details: Gaussian blob particle picking in cryosparc.
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

5fwl


Details: 5FWL was used to fit the Hsp90 complex. 1WAO and 1S95 were used to fit the PP5 components.
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1.3)
Final 3D classificationSoftware - Name: RELION (ver. 3.1.3)
Details: Initial refinement on the Hsp90 component of the complex was followed up with focused classification with subtraction to obtain the separate maps used in this composite entry.
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1.3)
Final reconstructionNumber classes used: 3 / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF
Software: (Name: RELION (ver. 3.1.3), UCSF ChimeraX (ver. 1.2.5))
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.
Number images used: 545237
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model(Chain: PDB, experimental model, PDB, experimental model, PDB, experimental model)
SoftwareName: ISOLDE (ver. 1.0b3)
DetailsThis 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.
RefinementProtocol: RIGID BODY FIT
Output model

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

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