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- EMDB-15846: p97-SPI substrate complex on subunit E -

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Basic information

Entry
Database: EMDB / ID: EMD-15846
Titlep97-SPI substrate complex on subunit E
Map datamap of p97 with SPI substrate on subunit E
Sample
  • Complex: p97 SPI substrate complex on subunit E
    • Protein or peptide: p97
    • Protein or peptide: SDS22
    • Protein or peptide: PP1 gamma
    • Protein or peptide: I3
    • Protein or peptide: p37 adaptor
KeywordsAAA+ ATPase / p97 / VCP / Cdc48 / unfoldase / protein phosphatase-1 / protein maturation / CHAPERONE
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.7 Å
AuthorsSaibil H / van den Boom J / Marini G / Meyer H
Funding support United Kingdom, 3 items
OrganizationGrant numberCountry
Wellcome Trust106249/Z/14/Z United Kingdom
Wellcome Trust202679/Z/16/Z United Kingdom
Wellcome Trust206166/Z/17/Z United Kingdom
CitationJournal: EMBO J / Year: 2023
Title: Structural basis of ubiquitin-independent PP1 complex disassembly by p97.
Authors: Johannes van den Boom / Guendalina Marini / Hemmo Meyer / Helen R Saibil /
Abstract: The AAA+-ATPase p97 (also called VCP or Cdc48) unfolds proteins and disassembles protein complexes in numerous cellular processes, but how substrate complexes are loaded onto p97 and disassembled is ...The AAA+-ATPase p97 (also called VCP or Cdc48) unfolds proteins and disassembles protein complexes in numerous cellular processes, but how substrate complexes are loaded onto p97 and disassembled is unclear. Here, we present cryo-EM structures of p97 in the process of disassembling a protein phosphatase-1 (PP1) complex by extracting an inhibitory subunit from PP1. We show that PP1 and its partners SDS22 and inhibitor-3 (I3) are loaded tightly onto p97, surprisingly via a direct contact of SDS22 with the p97 N-domain. Loading is assisted by the p37 adapter that bridges two adjacent p97 N-domains underneath the substrate complex. A stretch of I3 is threaded into the central channel of the spiral-shaped p97 hexamer, while other elements of I3 are still attached to PP1. Thus, our data show how p97 arranges a protein complex between the p97 N-domain and central channel, suggesting a hold-and-extract mechanism for p97-mediated disassembly.
History
DepositionSep 19, 2022-
Header (metadata) releaseJun 7, 2023-
Map releaseJun 7, 2023-
UpdateJul 26, 2023-
Current statusJul 26, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15846.png

Downloads & links

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Map

FileDownload / File: emd_15846.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationmap of p97 with SPI substrate on subunit E
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.1 Å/pix.
x 320 pix.
= 352. Å		1.1 Å/pix.
x 320 pix.
= 352. Å		1.1 Å/pix.
x 320 pix.
= 352. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.1 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0026
Minimum - Maximum-0.011687032 - 0.02235697
Average (Standard dev.)0.000013260365 (±0.0010098238)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 352.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half map 2

Fileemd_15846_half_map_1.map
AnnotationHalf map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1

Fileemd_15846_half_map_2.map
AnnotationHalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : p97 SPI substrate complex on subunit E

EntireName: p97 SPI substrate complex on subunit E
Components
  • Complex: p97 SPI substrate complex on subunit E
    • Protein or peptide: p97
    • Protein or peptide: SDS22
    • Protein or peptide: PP1 gamma
    • Protein or peptide: I3
    • Protein or peptide: p37 adaptor

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Supramolecule #1: p97 SPI substrate complex on subunit E

SupramoleculeName: p97 SPI substrate complex on subunit E / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: p97

MacromoleculeName: p97 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO / EC number: vesicle-fusing ATPase
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: MHHHHHHASG ADSKGDDLST AILKQKNRPN RLIVDEAINE DNSVVSLSQP KMDELQLFRG DTVLLKGKKR REAVCIVLSD DTCSDEKIRM NRVVRNNLRV RLGDVISIQP CPDVKYGKRI HVLPIDDTVE GITGNLFEVY LKPYFLEAYR PIRKGDIFLV RGGMRAVEFK ...String:
MHHHHHHASG ADSKGDDLST AILKQKNRPN RLIVDEAINE DNSVVSLSQP KMDELQLFRG DTVLLKGKKR REAVCIVLSD DTCSDEKIRM NRVVRNNLRV RLGDVISIQP CPDVKYGKRI HVLPIDDTVE GITGNLFEVY LKPYFLEAYR PIRKGDIFLV RGGMRAVEFK VVETDPSPYC IVAPDTVIHC EGEPIKREDE EESLNEVGYD DIGGCRKQLA QIKEMVELPL RHPALFKAIG VKPPRGILLY GPPGTGKTLI ARAVANETGA FFFLINGPEI MSKLAGESES NLRKAFEEAE KNAPAIIFID ELDAIAPKRE KTHGEVERRI VSQLLTLMDG LKQRAHVIVM AATNRPNSID PALRRFGRFD REVDIGIPDA TGRLEILQIH TKNMKLADDV DLEQVANETH GHVGADLAAL CSEAALQAIR KKMDLIDLED ETIDAEVMNS LAVTMDDFRW ALSQSNPSAL RETVVEVPQV TWEDIGGLED VKRELQELVQ YPVEHPDKFL KFGMTPSKGV LFYGPPGCGK TLLAKAIANE CQANFISIKG PELLTMWFGE SEANVREIFD KARQAAPCVL FFDELDSIAK ARGGNIGDGG GAADRVINQI LTEMDGMSTK KNVFIIGATN RPDIIDPAIL RPGRLDQLIY IPLPDEKSRV AILKANLRKS PVAKDVDLEF LAKMTNGFSG ADLTEICQRA CKLAIRESIE SEIRRERERQ TNPSAMEVEE DDPVPEIRRD HFEEAMRFAR RSVSDNDIRK YEMFAQTLQQ SRGFGSFRFP SGNQGGAGPS QGSGGGTGGS VYTEDNDDDL YG

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Macromolecule #2: SDS22

MacromoleculeName: SDS22 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: MAAERGAGQQ QSQEMMEVDR RVESEESGDE EGKKHSSGIV ADLSEQSLKD GEERGEEDPE EEHELPVDME TINLDRDAED VDLNHYRIGK IEGFEVLKKV KTLCLRQNLI KCIENLEELQ SLRELDLYDN QIKKIENLEA LTELEILDIS FNLLRNIEGV DKLTRLKKLF ...String:
MAAERGAGQQ QSQEMMEVDR RVESEESGDE EGKKHSSGIV ADLSEQSLKD GEERGEEDPE EEHELPVDME TINLDRDAED VDLNHYRIGK IEGFEVLKKV KTLCLRQNLI KCIENLEELQ SLRELDLYDN QIKKIENLEA LTELEILDIS FNLLRNIEGV DKLTRLKKLF LVNNKISKIE NLSNLHQLQM LELGSNRIRA IENIDTLTNL ESLFLGKNKI TKLQNLDALT NLTVLSMQSN RLTKIEGLQN LVNLRELYLS HNGIEVIEGL ENNNKLTMLD IASNRIKKIE NISHLTELQE FWMNDNLLES WSDLDELKGA RSLETVYLER NPLQKDPQYR RKVMLALPSV RQIDATFVRF

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Macromolecule #3: PP1 gamma

MacromoleculeName: PP1 gamma / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO / EC number: protein-serine/threonine phosphatase
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: MADLDKLNID SIIQRLLEVR GSKPGKNVQL QENEIRGLCL KSREIFLSQP ILLELEAPLK ICGDIHGQYY DLLRLFEYGG FPPESNYLFL GDYVDRGKQS LETICLLLAY KIKYPENFFL LRGNHECASI NRIYGFYDEC KRRYNIKLWK TFTDCFNCLP IAAIVDEKIF ...String:
MADLDKLNID SIIQRLLEVR GSKPGKNVQL QENEIRGLCL KSREIFLSQP ILLELEAPLK ICGDIHGQYY DLLRLFEYGG FPPESNYLFL GDYVDRGKQS LETICLLLAY KIKYPENFFL LRGNHECASI NRIYGFYDEC KRRYNIKLWK TFTDCFNCLP IAAIVDEKIF CCHGGLSPDL QSMEQIRRIM RPTDVPDQGL LCDLLWSDPD KDVLGWGEND RGVSFTFGAE VVAKFLHKHD LDLICRAHQV VEDGYEFFAK RQLVTLFSAP NYCGEFDNAG AMMSVDETLM CSFQILKPAE KKKPNATRPV TPPRGMITKQ AKK

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Macromolecule #4: I3

MacromoleculeName: I3 / type: protein_or_peptide / ID: 4 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' ni (bacteria)
SequenceString:
MSYYHHHHHH DYDIPTTENL YFQGAMGSMA EAGAGLSETV TETTVTVTTE PENRSLTIKL RKRKPEKKVE WTSDTVDNEH MGRRSSKCCC IYEKPRAFGE SSTESDEEEE EGCGHTHCVR GHRKGRRRAT LGPTPTTPPQ PPDPSQPPPG PMQH

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Macromolecule #5: p37 adaptor

MacromoleculeName: p37 adaptor / type: protein_or_peptide / ID: 5 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: GPLGSMAEGG RAEPEEQERG SSRPRPPSAR DLQLALAELY EDEMKCKSSK PDRSTPATCR SPRTPPHRLY SGDHKYDGLH IVQPPTGKIV NELFKEAREH GAVPLNEATR SSREDKTKSF TGGGYRLGNS FYKRSEYIYG ENQLQDVQVL LKLWRNGFSL DDGELRPYSD ...String:
GPLGSMAEGG RAEPEEQERG SSRPRPPSAR DLQLALAELY EDEMKCKSSK PDRSTPATCR SPRTPPHRLY SGDHKYDGLH IVQPPTGKIV NELFKEAREH GAVPLNEATR SSREDKTKSF TGGGYRLGNS FYKRSEYIYG ENQLQDVQVL LKLWRNGFSL DDGELRPYSD PTNAQFLESV KRGETPLELQ RLVHGAQVNL DMEDHQDQEY IKPRLRFKAF SGEGQKLGSL TPEIVSTPSS PEEEDKSILN AAVLIDDSMP TTKIQIRLAD GSRLVQRFNS THRILDVRDF IVRSRPEFAT TDFILVTSFP SKELTDETVT LQEADILNTV ILQQLK

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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
GridModel: C-flat-1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 85 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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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: 3.3000000000000003 µm / Nominal defocus min: 1.8 µm / Nominal magnification: 81000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 30 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average exposure time: 3.0 sec. / Average electron dose: 48.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationSoftware - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final reconstructionResolution.type: BY AUTHOR / Resolution: 6.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 82968
FSC plot (resolution estimation)

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