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- EMDB-29973: PP5 bound to Hsp90:Cdc37:CRaf complex -

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Basic information

Entry
Database: EMDB / ID: EMD-29973
TitlePP5 bound to Hsp90:Cdc37:CRaf complex
Map dataPP5 bound to Hsp90:Cdc37:CRaf complex.
Sample
  • Complex: Hsp90:Cdc37:CRaf complex bound to PP5.
    • Complex: Protein Phosphatase 5 (H304A)
      • Protein or peptide: Serine/threonine-protein phosphatase 5
    • Protein or peptide: Heat shock protein HSP 90-beta
    • Protein or peptide: Hsp90 co-chaperone Cdc37
    • Protein or peptide: RAF proto-oncogene serine/threonine-protein kinase
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.0 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 6, 2023-
Header (metadata) releaseApr 26, 2023-
Map releaseApr 26, 2023-
UpdateMay 3, 2023-
Current statusMay 3, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29973.png

Downloads & links

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Map

FileDownload / File: emd_29973.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPP5 bound to Hsp90:Cdc37:CRaf complex.
Voxel sizeX=Y=Z: 0.835 Å
Density
Contour LevelBy AUTHOR: 0.0072
Minimum - Maximum-0.0062973644 - 0.026073098
Average (Standard dev.)0.000103575505 (±0.0013736554)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
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, sharpened map.

Fileemd_29973_additional_1.map
AnnotationPostProcessed, sharpened map.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map before postprocessing.

Fileemd_29973_half_map_1.map
AnnotationHalf map before postprocessing.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map before postprocessing.

Fileemd_29973_half_map_2.map
AnnotationHalf map before postprocessing.
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 complex bound to PP5.

EntireName: Hsp90:Cdc37:CRaf complex bound to PP5.
Components
  • Complex: Hsp90:Cdc37:CRaf complex bound to PP5.
    • Complex: Protein Phosphatase 5 (H304A)
      • Protein or peptide: Serine/threonine-protein phosphatase 5
    • Protein or peptide: Heat shock protein HSP 90-beta
    • Protein or peptide: Hsp90 co-chaperone Cdc37
    • Protein or peptide: RAF proto-oncogene serine/threonine-protein kinase

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Supramolecule #1: Hsp90:Cdc37:CRaf complex bound to PP5.

SupramoleculeName: Hsp90:Cdc37:CRaf complex bound to PP5. / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: all
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 ...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. Particles were then classified to yield a Hsp90:Cdc37:CRaf:PP5 complex.
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: #4
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: HRV 3C cleavage site, followed by a glycine linker and the Hsp90B sequence.
Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: GPGMPEEVHH GEEEVETFAF QAEIAQLMSL IINTFYSNKE IFLRELISNA SDALDKIRYE SLTDPSKLDS GKELKIDIIP NPQERTLTLV DTGIGMTKAD LINNLGTIAK SGTKAFMEAL QAGADISMIG QFGVGFYSAY LVAEKVVVIT KHNDDEQYAW ESSAGGSFTV ...String:
GPGMPEEVHH GEEEVETFAF QAEIAQLMSL IINTFYSNKE IFLRELISNA SDALDKIRYE SLTDPSKLDS GKELKIDIIP NPQERTLTLV DTGIGMTKAD LINNLGTIAK SGTKAFMEAL QAGADISMIG QFGVGFYSAY LVAEKVVVIT KHNDDEQYAW ESSAGGSFTV RADHGEPIGR GTKVILHLKE DQTEYLEERR VKEVVKKHSQ FIGYPITLYL EKEREKEISD DEAEEEKGEK EEEDKDDEEK PKIEDVGSDE EDDSGKDKKK KTKKIKEKYI DQEELNKTKP IWTRNPDDIT QEEYGEFYKS LTNDWEDHLA VKHFSVEGQL EFRALLFIPR RAPFDLFENK KKKNNIKLYV RRVFIMDSCD ELIPEYLNFI RGVVDSEDLP LNISREMLQQ SKILKVIRKN IVKKCLELFS ELAEDKENYK KFYEAFSKNL KLGIHEDSTN RRRLSELLRY HTSQSGDEMT SLSEYVSRMK ETQKSIYYIT GESKEQVANS AFVERVRKRG FEVVYMTEPI DEYCVQQLKE FDGKSLVSVT KEGLELPEDE EEKKKMEESK AKFENLCKLM KEILDKKVEK VTISNRLVSS PCCIVTSTYG WTANMERIMK AQALRDNSTM GYMMAKKHLE INPDHPIVET LRQKAEADKN DKAVKDLVVL LFETALLSSG FSLEDPQTHS NRIYRMIKLG LGIDEDEVAA EEPNAAVPDE IPPLEGDEDA SRMEEVD

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Macromolecule #2: Hsp90 co-chaperone Cdc37

MacromoleculeName: Hsp90 co-chaperone Cdc37 / type: protein_or_peptide / ID: 2
Details: Cdc37 sequence followed by HRV 3C cleavage site (LEVLFQ)
Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MVDYSVWDHI EVSDDEDETH PNIDTASLFR WRHQARVERM EQFQKEKEEL DRGCRECKRK VAECQRKLKE LEVAEGGKAE LERLQAEAQQ LRKEERSWEQ KLEEMRKKEK SMPWNVDTLS KDGFSKSMVN TKPEKTEEDS EEVREQKHKT FVEKYEKQIK HFGMLRRWDD ...String:
MVDYSVWDHI EVSDDEDETH PNIDTASLFR WRHQARVERM EQFQKEKEEL DRGCRECKRK VAECQRKLKE LEVAEGGKAE LERLQAEAQQ LRKEERSWEQ KLEEMRKKEK SMPWNVDTLS KDGFSKSMVN TKPEKTEEDS EEVREQKHKT FVEKYEKQIK HFGMLRRWDD SQKYLSDNVH LVCEETANYL VIWCIDLEVE EKCALMEQVA HQTIVMQFIL ELAKSLKVDP RACFRQFFTK IKTADRQYME GFNDELEAFK ERVRGRAKLR IEKAMKEYEE EERKKRLGPG GLDPVEVYES LPEELQKCFD VKDVQMLQDA ISKMDPTDAK YHMQRCIDSG LWVPNSKASE AKEGEEAGPG DPLLEAVPKT GDEKDVSVLE VLFQ

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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: Truncated CRaf (RAF-1) kinase domain followed by Strep Tag II sequence (WSHPQFEK) and HRV 3C cleavage site (LEVLFQ)
Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: GGRDSSYYWE IEASEVMLST RIGSGSFGTV YKGKWHGDVA VKILKVVDPT PEQFQAFRNE VAVLRKTRHV NILLFMGYMT KDNLAIVTQW CEGSSLYKHL HVQETKFQMF QLIDIARQTA QGMDYLHAKN IIHRDMKSNN IFLHEGLTVK IGDFGLATVK SRWSGSQQVE ...String:
GGRDSSYYWE IEASEVMLST RIGSGSFGTV YKGKWHGDVA VKILKVVDPT PEQFQAFRNE VAVLRKTRHV NILLFMGYMT KDNLAIVTQW CEGSSLYKHL HVQETKFQMF QLIDIARQTA QGMDYLHAKN IIHRDMKSNN IFLHEGLTVK IGDFGLATVK SRWSGSQQVE QPTGSVLWMA PEVIRMQDNN PFSFQSDVYS YGIVLYELMT GELPYSHINN RDQIIFMVGR GYASPDLSKL YKNCPKAMKR LVADCVKKVK EERPLFPQIL SSIELLQHSL PKINRLPESG WSHPQFEKLE VLFQ

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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.
Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: GPGSMAMAEG ERTECAEPPR DEPPADGALK RAEELKTQAN DYFKAKDYEN AIKFYSQAIE LNPSNAIYYG NRSLAYLRTE CYGYALGDAT RAIELDKKYI KGYYRRAASN MALGKFRAAL RDYETVVKVK PHDKDAKMKY QECNKIVKQK AFERAIAGDE HKRSVVDSLD ...String:
GPGSMAMAEG ERTECAEPPR DEPPADGALK RAEELKTQAN DYFKAKDYEN AIKFYSQAIE LNPSNAIYYG NRSLAYLRTE CYGYALGDAT RAIELDKKYI KGYYRRAASN MALGKFRAAL RDYETVVKVK PHDKDAKMKY QECNKIVKQK AFERAIAGDE HKRSVVDSLD IESMTIEDEY SGPKLEDGKV TISFMKELMQ WYKDQKKLHR KCAYQILVQV KEVLSKLSTL VETTLKETEK ITVCGDTHGQ FYDLLNIFEL NGLPSETNPY IFNGDFVDRG SFSVEVILTL FGFKLLYPDH FHLLRGNAET DNMNQIYGFE GEVKAKYTAQ MYELFSEVFE WLPLAQCING KVLIMHGGLF SEDGVTLDDI RKIERNRQPP DSGPMCDLLW SDPQPQNGRS ISKRGVSCQF GPDVTKAFLE ENNLDYIIRS HEVKAEGYEV AHGGRCVTVF SAPNYCDQMG NKASYIHLQG SDLRPQFHQF TAVPHPNVKP MAYANTLLQL GMM

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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
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHEPES
50.0 mMKClKCl
10.0 mMMgCl2Magnesium Chloride
0.5 mMC9H15O6PTCEP
1.0 mMC10H16N2O8EDTA
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 / Instrument: FEI VITROBOT MARK IV
Details: Sample Vol: 3UL Temp: 10C Humidity: 100% WAIT TIME: 30S BLOT TIME: 3S BLOT FORCE: -2.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 4160 / Average electron dose: 69.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / 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
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.
Final reconstructionNumber classes used: 1 / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 4.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1.3) / Number images used: 21829
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1.3)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1.3)
Final 3D classificationSoftware - Name: RELION (ver. 3.1.3)
FSC plot (resolution estimation)

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