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- EMDB-76026: Cryo-EM structure of substrate engaged p97-Ufd1-NPL4-Faf1 complex... -

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Entry
Database: EMDB / ID: EMD-76026
TitleCryo-EM structure of substrate engaged p97-Ufd1-NPL4-Faf1 complex (NPL4 focused)
Map data
Sample
  • Complex: Cryo-EM structure of substrate engaged p97-Ufd1-NPL4-Faf1 complex (NPL4 focused)
    • Protein or peptide: Nuclear protein localization protein 4 homolog
    • Protein or peptide: Ubiquitin
    • Protein or peptide: Ubiquitin recognition factor in ER-associated degradation protein 1
  • Ligand: ZINC ION
KeywordsERAD / ubiquitin / AAA+ ATPase / unfoldase / HYDROLASE
Function / homology
Function and homology information


UFD1-NPL4 complex / negative regulation of RIG-I signaling pathway / VCP-NPL4-UFD1 AAA ATPase complex / cellular response to misfolded protein / K48-linked polyubiquitin modification-dependent protein binding / nuclear outer membrane-endoplasmic reticulum membrane network / K63-linked polyubiquitin modification-dependent protein binding / retrograde protein transport, ER to cytosol / negative regulation of type I interferon production / Golgi organization ...UFD1-NPL4 complex / negative regulation of RIG-I signaling pathway / VCP-NPL4-UFD1 AAA ATPase complex / cellular response to misfolded protein / K48-linked polyubiquitin modification-dependent protein binding / nuclear outer membrane-endoplasmic reticulum membrane network / K63-linked polyubiquitin modification-dependent protein binding / retrograde protein transport, ER to cytosol / negative regulation of type I interferon production / Golgi organization / polyubiquitin modification-dependent protein binding / ERAD pathway / Maturation of protein E / Maturation of protein E / ER Quality Control Compartment (ERQC) / Myoclonic epilepsy of Lafora / FLT3 signaling by CBL mutants / IRAK2 mediated activation of TAK1 complex / Alpha-protein kinase 1 signaling pathway / Glycogen synthesis / IRAK1 recruits IKK complex / IRAK1 recruits IKK complex upon TLR7/8 or 9 stimulation / Prevention of phagosomal-lysosomal fusion / Endosomal Sorting Complex Required For Transport (ESCRT) / Membrane binding and targetting of GAG proteins / Negative regulation of FLT3 / Regulation of TBK1, IKKε (IKBKE)-mediated activation of IRF3, IRF7 / PTK6 Regulates RTKs and Their Effectors AKT1 and DOK1 / Regulation of TBK1, IKKε-mediated activation of IRF3, IRF7 upon TLR3 ligation / IRAK2 mediated activation of TAK1 complex upon TLR7/8 or 9 stimulation / Constitutive Signaling by NOTCH1 HD Domain Mutants / NOTCH2 Activation and Transmission of Signal to the Nucleus / TICAM1,TRAF6-dependent induction of TAK1 complex / TICAM1-dependent activation of IRF3/IRF7 / APC/C:Cdc20 mediated degradation of Cyclin B / Downregulation of ERBB4 signaling / Regulation of FZD by ubiquitination / APC-Cdc20 mediated degradation of Nek2A / p75NTR recruits signalling complexes / InlA-mediated entry of Listeria monocytogenes into host cells / TRAF6 mediated IRF7 activation in TLR7/8 or 9 signaling / NF-kB is activated and signals survival / TRAF6-mediated induction of TAK1 complex within TLR4 complex / Regulation of pyruvate metabolism / Pexophagy / Downregulation of ERBB2:ERBB3 signaling / Regulation of innate immune responses to cytosolic DNA / NRIF signals cell death from the nucleus / Regulation of PTEN localization / VLDLR internalisation and degradation / Activated NOTCH1 Transmits Signal to the Nucleus / Synthesis of active ubiquitin: roles of E1 and E2 enzymes / Translesion synthesis by REV1 / TICAM1, RIP1-mediated IKK complex recruitment / Regulation of BACH1 activity / Translesion synthesis by POLK / InlB-mediated entry of Listeria monocytogenes into host cell / JNK (c-Jun kinases) phosphorylation and activation mediated by activated human TAK1 / MAP3K8 (TPL2)-dependent MAPK1/3 activation / Activation of IRF3, IRF7 mediated by TBK1, IKKε (IKBKE) / Downregulation of TGF-beta receptor signaling / Translesion synthesis by POLI / Josephin domain DUBs / Gap-filling DNA repair synthesis and ligation in GG-NER / IKK complex recruitment mediated by RIP1 / PINK1-PRKN Mediated Mitophagy / TGF-beta receptor signaling in EMT (epithelial to mesenchymal transition) / Regulation of activated PAK-2p34 by proteasome mediated degradation / TNFR1-induced NF-kappa-B signaling pathway / TCF dependent signaling in response to WNT / skeletal system development / Regulation of NF-kappa B signaling / activated TAK1 mediates p38 MAPK activation / Autodegradation of Cdh1 by Cdh1:APC/C / APC/C:Cdc20 mediated degradation of Securin / ubiquitin binding / Asymmetric localization of PCP proteins / N-glycan trimming in the ER and Calnexin/Calreticulin cycle / Negative regulators of DDX58/IFIH1 signaling / Ubiquitin-dependent degradation of Cyclin D / NOTCH3 Activation and Transmission of Signal to the Nucleus / Regulation of signaling by CBL / Fanconi Anemia Pathway / Negative regulation of FGFR3 signaling / Peroxisomal protein import / SCF-beta-TrCP mediated degradation of Emi1 / NIK-->noncanonical NF-kB signaling / AUF1 (hnRNP D0) binds and destabilizes mRNA / TNFR2 non-canonical NF-kB pathway / Deactivation of the beta-catenin transactivating complex / Stabilization of p53 / Negative regulation of FGFR2 signaling / Assembly of the pre-replicative complex / Negative regulation of FGFR4 signaling / Vpu mediated degradation of CD4 / Downregulation of SMAD2/3:SMAD4 transcriptional activity / Negative regulation of FGFR1 signaling / Cdc20:Phospho-APC/C mediated degradation of Cyclin A / Termination of translesion DNA synthesis / EGFR downregulation
Similarity search - Function
Nuclear pore localisation protein Npl4, ubiquitin-like domain / Nuclear pore localisation protein NPL4 / Ubiquitin fusion degradation protein Ufd1-like / Ufd1-like, Nn domain / : / : / Ubiquitin fusion degradation protein UFD1, N-terminal subdomain 1 / Ubiquitin fusion degradation protein UFD1, N-terminal subdomain 2 / NPL4, zinc-binding putative / NPL4 family, putative zinc binding region ...Nuclear pore localisation protein Npl4, ubiquitin-like domain / Nuclear pore localisation protein NPL4 / Ubiquitin fusion degradation protein Ufd1-like / Ufd1-like, Nn domain / : / : / Ubiquitin fusion degradation protein UFD1, N-terminal subdomain 1 / Ubiquitin fusion degradation protein UFD1, N-terminal subdomain 2 / NPL4, zinc-binding putative / NPL4 family, putative zinc binding region / Nuclear pore localisation protein NPL4, C-terminal / Nuclear protein localization protein 4 / NPL4 family / Zinc finger domain / Zinc finger RanBP2 type profile. / Zinc finger, RanBP2-type superfamily / Zinc finger RanBP2-type signature. / Zinc finger, RanBP2-type / MPN domain / MPN domain profile. / : / Ubiquitin domain signature. / Ubiquitin conserved site / Ubiquitin domain / Ubiquitin family / Ubiquitin homologues / Ubiquitin domain profile. / Ubiquitin-like domain / Ubiquitin-like domain superfamily
Similarity search - Domain/homology
Polyubiquitin-C / Nuclear protein localization protein 4 homolog / Ubiquitin recognition factor in ER-associated degradation protein 1
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.28 Å
AuthorsLiao Z / Martin A
Funding support United States, 1 items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: bioRxiv / Year: 2025
Title: Faf1 accelerates p97-mediated protein unfolding by promoting ubiquitin engagement.
Authors: Zengwei Liao / Connor Arkinson / Andreas Martin /
Abstract: P97/VCP is a protein unfoldase of the AAA+ ATPase family that plays essential roles in numerous cellular processes, including ER-associated degradation and DNA replication. P97 utilizes various ...P97/VCP is a protein unfoldase of the AAA+ ATPase family that plays essential roles in numerous cellular processes, including ER-associated degradation and DNA replication. P97 utilizes various cofactors to process different substrates. For unfolding of proteins that are modified with K48-linked ubiquitin chains, p97 works with the heterodimeric cofactor Ufd1-Npl4, and the cofactor Faf1 was shown to enhance this activity in the context of replisome disassembly, yet the underlying mechanisms remain unknown. Here, we employ an reconstituted system with human components for biochemical experiments, mutational studies, FRET-based assays, and cryo-EM structure determination to reveal that Faf1 plays a generic role in accelerating ubiquitin-dependent substrate processing by promoting the unfolding of an initiator ubiquitin and its engagement by the ATPase motor. Faf1 thereby uses its p97-bound C-terminal UBX domain to anchor a long helix that braces the UT3 domain of Ufd1 and apparently stabilizes the Ufd1-Npl4 cofactor for ubiquitin unfolding. Our findings demonstrate how p97 works simultaneously with several cofactors to facilitate the unfolding of ubiquitinated proteins, indicating more complex regulatory mechanisms for substrate selection than for the simpler Cdc48 ortholog in yeast.
History
DepositionMar 11, 2026-
Header (metadata) releaseApr 22, 2026-
Map releaseApr 22, 2026-
UpdateApr 29, 2026-
Current statusApr 29, 2026Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_76026.png

Downloads & links

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Map

FileDownload / File: emd_76026.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.48 Å/pix.
x 256 pix.
= 378.24 Å		1.48 Å/pix.
x 256 pix.
= 378.24 Å		1.48 Å/pix.
x 256 pix.
= 378.24 Å

Surface

Projections

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.4775 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.115
Minimum - Maximum-0.45561382 - 0.9694738
Average (Standard dev.)0.00088520563 (±0.025022699)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 378.24 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_76026_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_76026_half_map_2.map
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : Cryo-EM structure of substrate engaged p97-Ufd1-NPL4-Faf1 complex...

EntireName: Cryo-EM structure of substrate engaged p97-Ufd1-NPL4-Faf1 complex (NPL4 focused)
Components
  • Complex: Cryo-EM structure of substrate engaged p97-Ufd1-NPL4-Faf1 complex (NPL4 focused)
    • Protein or peptide: Nuclear protein localization protein 4 homolog
    • Protein or peptide: Ubiquitin
    • Protein or peptide: Ubiquitin recognition factor in ER-associated degradation protein 1
  • Ligand: ZINC ION

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Supramolecule #1: Cryo-EM structure of substrate engaged p97-Ufd1-NPL4-Faf1 complex...

SupramoleculeName: Cryo-EM structure of substrate engaged p97-Ufd1-NPL4-Faf1 complex (NPL4 focused)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 700 kDa/nm

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Macromolecule #1: Nuclear protein localization protein 4 homolog

MacromoleculeName: Nuclear protein localization protein 4 homolog / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 68.373172 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: MGGGAESIII RVQSPDGVKR ITATKRETAA TFLKKVAKEF GFQNNGFSVY INRNKTGEIT ASSNKSLNLL KIKHGDLLFL FPSSLAGPS SEMETSVPPG FKVFGAPNVV EDEIDQYLSK QDGKIYRSRD PQLCRHGPLG KCVHCVPLEP FDEDYLNHLE P PVKHMSFH ...String:
MGGGAESIII RVQSPDGVKR ITATKRETAA TFLKKVAKEF GFQNNGFSVY INRNKTGEIT ASSNKSLNLL KIKHGDLLFL FPSSLAGPS SEMETSVPPG FKVFGAPNVV EDEIDQYLSK QDGKIYRSRD PQLCRHGPLG KCVHCVPLEP FDEDYLNHLE P PVKHMSFH AYIRKLTGGA DKGKFVALEN ISCKIKSGCE GHLPWPNGIC TKCQPSAITL NRQKYRHVDN IMFENHTVAD RF LDFWRKT GNQHFGYLYG RYTEHKDIPL GIRAEVAAIY EPPQIGTQNS LELLEDPKAE VVDEIAAKLG LRKVGWIFTD LVS EDTRKG TVRYSRNKDT YFLSSEECIT AGDFQNKHPN MCRLSPDGHF GSKFVTAVAT GGPDNQVHFE GYQVSNQCMA LVRD ECLLP CKDAPELGYA KESSSEQYVP DVFYKDVDKF GNEITQLARP LPVEYLIIDI TTTFPKDPVY TFSISQNPFP IENRD VLGE TQDFHSLATY LSQNTSSVFL DTISDFHLLL FLVTNEVMPL QDSISLLLEA VRTRNEELAQ TWKRSEQWAT IEQLCS TVG GQLPGLHEYG AVGGSTHTAT AAMWACQHCT FMNQPGTGHC EMCSLPRT

UniProtKB: Nuclear protein localization protein 4 homolog

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Macromolecule #2: Ubiquitin

MacromoleculeName: Ubiquitin / type: protein_or_peptide / ID: 2 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 8.576831 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MQIFVKTLTG KTITLEVEPS DTIENVKAKI QDKEGIPPDQ QRLIFAGKQL EDGRTLSDYN IQKESTLHLV LRLRGG

UniProtKB: Polyubiquitin-C

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Macromolecule #3: Ubiquitin recognition factor in ER-associated degradation protein 1

MacromoleculeName: Ubiquitin recognition factor in ER-associated degradation protein 1
type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 35.373188 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: MFSFNMFDHP IPRVFQNRFS TQYRCFSVSM LAGPNDRSDV EKGGKIIMPP SALDQLSRLN ITYPMLFKLT NKNSDRMTHC GVLEFVADE GICYLPHWMM QNLLLEEGGL VQVESVNLQV ATYSKFQPQS PDFLDITNPK AVLENALRNF ACLTTGDVIA I NYNEKIYE ...String:
MFSFNMFDHP IPRVFQNRFS TQYRCFSVSM LAGPNDRSDV EKGGKIIMPP SALDQLSRLN ITYPMLFKLT NKNSDRMTHC GVLEFVADE GICYLPHWMM QNLLLEEGGL VQVESVNLQV ATYSKFQPQS PDFLDITNPK AVLENALRNF ACLTTGDVIA I NYNEKIYE LRVMETKPDK AVSIIECDMN VDFDAPLGYK EPERQVQHEE STEGEADHSG YAGELGFRAF SGSGNRLDGK KK GVEPSPS PIKPGDIKRG IPNYEFKLGK ITFIRNSRPL VKKVEEDEAG GRFVAFSGEG QSLRKKGRKP HHHHHH

UniProtKB: Ubiquitin recognition factor in ER-associated degradation protein 1

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Macromolecule #4: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 4 / Number of copies: 2 / Formula: ZN
Molecular weightTheoretical: 65.409 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

BufferpH: 7.6
Details: 50 mM HEPES, pH 7.6, 150 mM KCl, 5 mM MgCl2, 2 mM ATP
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 2
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Number grids imaged: 2 / Number real images: 17244 / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.6 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

CTF correctionSoftware - Name: cryoSPARC / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.28 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 20565
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationSoftware - Name: cryoSPARC

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