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- EMDB-25044: Cryo-EM structure of the SHOC2:PP1C:MRAS complex -

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Entry
Database: EMDB / ID: EMD-25044
TitleCryo-EM structure of the SHOC2:PP1C:MRAS complex
Map dataFinal map
Sample
  • Complex: Ternary complex of SHOC2:PP1C:MRAS
    • Complex: SHOC2
      • Protein or peptide: Leucine-rich repeat protein SHOC-2
    • Complex: MRAS
      • Protein or peptide: Ras-related protein M-Ras
    • Complex: PP1C
      • Protein or peptide: Serine/threonine-protein phosphatase PP1-gamma catalytic subunit
  • Ligand: PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER
  • Ligand: MAGNESIUM ION
  • Ligand: MANGANESE (II) ION
KeywordsPhosphatase / leucine rich repeat / RAF / complex / SIGNALING PROTEIN
Function / homology
Function and homology information


cellular response to growth hormone stimulus / protein phosphatase type 1 complex / negative regulation of neural precursor cell proliferation / PTW/PP1 phosphatase complex / regulation of nucleocytoplasmic transport / GTP-dependent protein binding / nerve growth factor signaling pathway / protein phosphatase 1 binding / protein phosphatase regulator activity / lamin binding ...cellular response to growth hormone stimulus / protein phosphatase type 1 complex / negative regulation of neural precursor cell proliferation / PTW/PP1 phosphatase complex / regulation of nucleocytoplasmic transport / GTP-dependent protein binding / nerve growth factor signaling pathway / protein phosphatase 1 binding / protein phosphatase regulator activity / lamin binding / SHOC2 M1731 mutant abolishes MRAS complex function / Gain-of-function MRAS complexes activate RAF signaling / positive regulation of Ras protein signal transduction / microtubule organizing center / Maturation of hRSV A proteins / myosin phosphatase activity / protein serine/threonine phosphatase activity / glycogen metabolic process / protein-serine/threonine phosphatase / Triglyceride catabolism / entrainment of circadian clock by photoperiod / phosphatase activity / phosphoprotein phosphatase activity / cleavage furrow / blastocyst development / negative regulation of neuron differentiation / fibroblast growth factor receptor signaling pathway / Amplification of signal from unattached kinetochores via a MAD2 inhibitory signal / Mitotic Prometaphase / EML4 and NUDC in mitotic spindle formation / positive regulation of glial cell proliferation / Resolution of Sister Chromatid Cohesion / positive regulation of neuron differentiation / protein dephosphorylation / Downregulation of TGF-beta receptor signaling / small monomeric GTPase / G protein activity / cellular response to leukemia inhibitory factor / RHO GTPases Activate Formins / RAF activation / circadian regulation of gene expression / neuron differentiation / regulation of circadian rhythm / kinetochore / positive regulation of neuron projection development / Separation of Sister Chromatids / GDP binding / MAPK cascade / Circadian Clock / presynapse / midbody / actin cytoskeleton organization / spermatogenesis / protein phosphatase binding / mitochondrial outer membrane / Ras protein signal transduction / dendritic spine / nuclear speck / cell cycle / protein domain specific binding / cell division / focal adhesion / GTPase activity / glutamatergic synapse / protein-containing complex binding / nucleolus / GTP binding / protein kinase binding / signal transduction / protein-containing complex / mitochondrion / RNA binding / nucleoplasm / metal ion binding / nucleus / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Serine-threonine protein phosphatase, N-terminal / Serine-threonine protein phosphatase N-terminal domain / Serine/threonine specific protein phosphatases signature. / Protein phosphatase 2A homologues, catalytic domain. / Serine/threonine-specific protein phosphatase/bis(5-nucleosyl)-tetraphosphatase / Leucine-rich repeats, bacterial type / Leucine-rich repeat, SDS22-like subfamily / Calcineurin-like phosphoesterase domain, ApaH type / Calcineurin-like phosphoesterase / Metallo-dependent phosphatase-like ...Serine-threonine protein phosphatase, N-terminal / Serine-threonine protein phosphatase N-terminal domain / Serine/threonine specific protein phosphatases signature. / Protein phosphatase 2A homologues, catalytic domain. / Serine/threonine-specific protein phosphatase/bis(5-nucleosyl)-tetraphosphatase / Leucine-rich repeats, bacterial type / Leucine-rich repeat, SDS22-like subfamily / Calcineurin-like phosphoesterase domain, ApaH type / Calcineurin-like phosphoesterase / Metallo-dependent phosphatase-like / Small GTPase, Ras-type / small GTPase Ras family profile. / Ran (Ras-related nuclear proteins) /TC4 subfamily of small GTPases / Leucine rich repeat / Leucine-rich repeat, typical subtype / Leucine-rich repeats, typical (most populated) subfamily / Leucine-rich repeat profile. / Leucine-rich repeat / Rho (Ras homology) subfamily of Ras-like small GTPases / Ras subfamily of RAS small GTPases / Small GTPase / Ras family / Rab subfamily of small GTPases / Leucine-rich repeat domain superfamily / Small GTP-binding protein domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Ras-related protein M-Ras / Serine/threonine-protein phosphatase PP1-gamma catalytic subunit / Leucine-rich repeat protein SHOC-2
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.95 Å
AuthorsLiau NPD / Johnson MC
Funding support United States, 1 items
OrganizationGrant numberCountry
Other private United States
CitationJournal: Nature / Year: 2022
Title: Structural basis for SHOC2 modulation of RAS signalling.
Authors: Nicholas P D Liau / Matthew C Johnson / Saeed Izadi / Luca Gerosa / Michal Hammel / John M Bruning / Timothy J Wendorff / Wilson Phung / Sarah G Hymowitz / Jawahar Sudhamsu /
Abstract: The RAS-RAF pathway is one of the most commonly dysregulated in human cancers. Despite decades of study, understanding of the molecular mechanisms underlying dimerization and activation of the kinase ...The RAS-RAF pathway is one of the most commonly dysregulated in human cancers. Despite decades of study, understanding of the molecular mechanisms underlying dimerization and activation of the kinase RAF remains limited. Recent structures of inactive RAF monomer and active RAF dimer bound to 14-3-3 have revealed the mechanisms by which 14-3-3 stabilizes both RAF conformations via specific phosphoserine residues. Prior to RAF dimerization, the protein phosphatase 1 catalytic subunit (PP1C) must dephosphorylate the N-terminal phosphoserine (NTpS) of RAF to relieve inhibition by 14-3-3, although PP1C in isolation lacks intrinsic substrate selectivity. SHOC2 is as an essential scaffolding protein that engages both PP1C and RAS to dephosphorylate RAF NTpS, but the structure of SHOC2 and the architecture of the presumptive SHOC2-PP1C-RAS complex remain unknown. Here we present a cryo-electron microscopy structure of the SHOC2-PP1C-MRAS complex to an overall resolution of 3 Å, revealing a tripartite molecular architecture in which a crescent-shaped SHOC2 acts as a cradle and brings together PP1C and MRAS. Our work demonstrates the GTP dependence of multiple RAS isoforms for complex formation, delineates the RAS-isoform preference for complex assembly, and uncovers how the SHOC2 scaffold and RAS collectively drive specificity of PP1C for RAF NTpS. Our data indicate that disease-relevant mutations affect complex assembly, reveal the simultaneous requirement of two RAS molecules for RAF activation, and establish rational avenues for discovery of new classes of inhibitors to target this pathway.
History
DepositionSep 29, 2021-
Header (metadata) releaseApr 20, 2022-
Map releaseApr 20, 2022-
UpdateNov 29, 2023-
Current statusNov 29, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_25044.png

Downloads & links

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Map

FileDownload / File: emd_25044.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFinal map
Voxel sizeX=Y=Z: 0.838 Å
Density
Contour LevelBy AUTHOR: 0.05
Minimum - Maximum-0.24292484 - 0.516285
Average (Standard dev.)0.00024843993 (±0.013755233)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 214.528 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half map A

Fileemd_25044_half_map_1.map
AnnotationHalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B

Fileemd_25044_half_map_2.map
AnnotationHalf map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Ternary complex of SHOC2:PP1C:MRAS

EntireName: Ternary complex of SHOC2:PP1C:MRAS
Components
  • Complex: Ternary complex of SHOC2:PP1C:MRAS
    • Complex: SHOC2
      • Protein or peptide: Leucine-rich repeat protein SHOC-2
    • Complex: MRAS
      • Protein or peptide: Ras-related protein M-Ras
    • Complex: PP1C
      • Protein or peptide: Serine/threonine-protein phosphatase PP1-gamma catalytic subunit
  • Ligand: PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER
  • Ligand: MAGNESIUM ION
  • Ligand: MANGANESE (II) ION

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Supramolecule #1: Ternary complex of SHOC2:PP1C:MRAS

SupramoleculeName: Ternary complex of SHOC2:PP1C:MRAS / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 37 KDa

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Supramolecule #2: SHOC2

SupramoleculeName: SHOC2 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: MRAS

SupramoleculeName: MRAS / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #4: PP1C

SupramoleculeName: PP1C / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Leucine-rich repeat protein SHOC-2

MacromoleculeName: Leucine-rich repeat protein SHOC-2 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 65.156969 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: GSSSLGKEKD SKEKDPKVPS AKEREKEAKA SGGFGKESKE KEPKTKGKDA KDGKKDSSAA QPGVAFSVDN TIKRPNPAPG TRKKSSNAE VIKELNKCRE ENSMRLDLSK RSIHILPSSI KELTQLTELY LYSNKLQSLP AEVGCLVNLM TLALSENSLT S LPDSLDNL ...String:
GSSSLGKEKD SKEKDPKVPS AKEREKEAKA SGGFGKESKE KEPKTKGKDA KDGKKDSSAA QPGVAFSVDN TIKRPNPAPG TRKKSSNAE VIKELNKCRE ENSMRLDLSK RSIHILPSSI KELTQLTELY LYSNKLQSLP AEVGCLVNLM TLALSENSLT S LPDSLDNL KKLRMLDLRH NKLREIPSVV YRLDSLTTLY LRFNRITTVE KDIKNLSKLS MLSIRENKIK QLPAEIGELC NL ITLDVAH NQLEHLPKEI GNCTQITNLD LQHNELLDLP DTIGNLSSLS RLGLRYNRLS AIPRSLAKCS ALEELNLENN NIS TLPESL LSSLVKLNSL TLARNCFQLY PVGGPSQFST IYSLNMEHNR INKIPFGIFS RAKVLSKLNM KDNQLTSLPL DFGT WTSMV ELNLATNQLT KIPEDVSGLV SLEVLILSNN LLKKLPHGLG NLRKLRELDL EENKLESLPN EIAYLKDLQK LVLTN NQLT TLPRGIGHLT NLTHLGLGEN LLTHLPEEIG TLENLEELYL NDNPNLHSLP FELALCSKLS IMSIENCPLS HLPPQI VAG GPSFIIQFLK MQGPYRAMVG NS

UniProtKB: Leucine-rich repeat protein SHOC-2

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Macromolecule #2: Ras-related protein M-Ras

MacromoleculeName: Ras-related protein M-Ras / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: small monomeric GTPase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 24.028621 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: GSMATSAVPS DNLPTYKLVV VGDGGVGKSA LTIQFFQKIF VPDYDPTIED SYLKHTEIDN QWAILDVLDT AGQEEFSAMR EQYMRTGDG FLIVYSVTDK ASFEHVDRFH QLILRVKDRE SFPMILVANK VDLMHLRKIT REQGKEMATK HNIPYIETSA K DPPLNVDK ...String:
GSMATSAVPS DNLPTYKLVV VGDGGVGKSA LTIQFFQKIF VPDYDPTIED SYLKHTEIDN QWAILDVLDT AGQEEFSAMR EQYMRTGDG FLIVYSVTDK ASFEHVDRFH QLILRVKDRE SFPMILVANK VDLMHLRKIT REQGKEMATK HNIPYIETSA K DPPLNVDK AFHDLVRVIR QQIPEKSQKK KKKTKWRGDR ATGTHKLQCV IL

UniProtKB: Ras-related protein M-Ras

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Macromolecule #3: Serine/threonine-protein phosphatase PP1-gamma catalytic subunit

MacromoleculeName: Serine/threonine-protein phosphatase PP1-gamma catalytic subunit
type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO / EC number: protein-serine/threonine phosphatase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 37.174906 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: GSMADLDKLN IDSIIQRLLE VRGSKPGKNV QLQENEIRGL CLKSREIFLS QPILLELEAP LKICGDIHGQ YYDLLRLFEY GGFPPESNY LFLGDYVDRG KQSLETICLL LAYKIKYPEN FFLLRGNHEC ASINRIYGFY DECKRRYNIK LWKTFTDCFN C LPIAAIVD ...String:
GSMADLDKLN IDSIIQRLLE VRGSKPGKNV QLQENEIRGL CLKSREIFLS QPILLELEAP LKICGDIHGQ YYDLLRLFEY GGFPPESNY LFLGDYVDRG KQSLETICLL LAYKIKYPEN FFLLRGNHEC ASINRIYGFY DECKRRYNIK LWKTFTDCFN C LPIAAIVD EKIFCCHGGL SPDLQSMEQI RRIMRPTDVP DQGLLCDLLW SDPDKDVLGW GENDRGVSFT FGAEVVAKFL HK HDLDLIC RAHQVVEDGY EFFAKRQLVT LFSAPNYCGE FDNAGAMMSV DETLMCSFQI LKPAEKKKPN ATRPVTPPRG MIT KQAKK

UniProtKB: Serine/threonine-protein phosphatase PP1-gamma catalytic subunit

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Macromolecule #4: PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER

MacromoleculeName: PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER / type: ligand / ID: 4 / Number of copies: 1 / Formula: GCP
Molecular weightTheoretical: 521.208 Da
Chemical component information

ChemComp-G2P:
PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER / GMP-CPP, energy-carrying molecule analogue*YM

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #6: MANGANESE (II) ION

MacromoleculeName: MANGANESE (II) ION / type: ligand / ID: 6 / 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

Concentration0.19 mg/mL
BufferpH: 7.5
Component:
ConcentrationNameFormula
25.0 mMTris
100.0 mMNaClSodium chloride
1.0 mMTCEP
0.5 mMMgCl2
0.5 mMMnCl2
0.1 mMGMPPCP
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Support film - Material: GRAPHENE OXIDE / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 20 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: Used the "perpetually hydrated" method of applying graphene oxide. (Cheung et al., 2018).

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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: 1.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 105000
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Average electron dose: 64.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 3996056
Startup modelType of model: PDB ENTRY / Details: 1X1S (MRAS) 4MOV (PP1C) SHOC2 (this manuscript)
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.95 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 323910
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

1x1s

source_name: PDB, initial_model_type: experimental model

4mov

source_name: PDB, initial_model_type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-7sd0:
Cryo-EM structure of the SHOC2:PP1C:MRAS complex

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