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- PDB-11sy: Cryo-EM structure of substrate engaged p97-Ufd1-NPL4-Faf1 complex... -

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Basic information

Entry
Database: PDB / ID: 11sy
TitleCryo-EM structure of substrate engaged p97-Ufd1-NPL4-Faf1 complex (NPL4 focused)
Components
  • Nuclear protein localization protein 4 homolog
  • Ubiquitin
  • Ubiquitin recognition factor in ER-associated degradation protein 1
KeywordsHYDROLASE / ERAD / ubiquitin / AAA+ ATPase / unfoldase
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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.28 Å
AuthorsLiao, Z. / Martin, A.
Funding support United States, 1items
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, 2026Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 22, 2026Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
G: Nuclear protein localization protein 4 homolog
H: Ubiquitin
I: Ubiquitin
J: Ubiquitin
K: Ubiquitin
P: Ubiquitin recognition factor in ER-associated degradation protein 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)138,1858
Polymers138,0546
Non-polymers1312
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Nuclear protein localization protein 4 homolog / Protein NPL4


Mass: 68373.172 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: NPLOC4, KIAA1499, NPL4
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: Q8TAT6
#2: Protein
Ubiquitin


Mass: 8576.831 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: UBC / Production host: Escherichia coli (E. coli) / References: UniProt: P0CG48
#3: Protein Ubiquitin recognition factor in ER-associated degradation protein 1 / Ubiquitin fusion degradation protein 1 / UB fusion protein 1


Mass: 35373.188 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: UFD1, UFD1L
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: Q92890
#4: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Cryo-EM structure of substrate engaged p97-Ufd1-NPL4-Faf1 complex (NPL4 focused)
Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightValue: 700 kDa/nm / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.6
Details: 50 mM HEPES, pH 7.6, 150 mM KCl, 5 mM MgCl2, 2 mM ATP
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 1600 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 17244

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2PHENIX2.0_5936model refinement
5cryoSPARCCTF correction
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
12cryoSPARCclassification
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4.28 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 20565 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 206.82 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00236141
ELECTRON MICROSCOPYf_angle_d0.57598298
ELECTRON MICROSCOPYf_chiral_restr0.0432930
ELECTRON MICROSCOPYf_plane_restr0.00431080
ELECTRON MICROSCOPYf_dihedral_angle_d4.388808

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