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

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Basic information

Entry
Database: PDB / ID: 11ve
TitleCryo-EM structure of substrate engaged p97-Ufd1-NPL4-Faf1 complex (State1)
Components
  • FAS-associated factor 1
  • Nuclear protein localization protein 4 homolog
  • Transitional endoplasmic reticulum ATPase
  • Ubiquitin recognition factor in ER-associated degradation protein 1
KeywordsHYDROLASE / ERAD / ubiquitin / AAA+ ATPase / unfoldase
Function / homology
Function and homology information


UFD1-NPL4 complex / Fas signaling pathway / negative regulation of RIG-I signaling pathway / CD95 death-inducing signaling complex / flavin adenine dinucleotide catabolic process / protein kinase regulator activity / VCP-NSFL1C complex / endoplasmic reticulum stress-induced pre-emptive quality control / endosome to lysosome transport via multivesicular body sorting pathway / BAT3 complex binding ...UFD1-NPL4 complex / Fas signaling pathway / negative regulation of RIG-I signaling pathway / CD95 death-inducing signaling complex / flavin adenine dinucleotide catabolic process / protein kinase regulator activity / VCP-NSFL1C complex / endoplasmic reticulum stress-induced pre-emptive quality control / endosome to lysosome transport via multivesicular body sorting pathway / BAT3 complex binding / cellular response to arsenite ion / cytoplasmic ubiquitin ligase complex / Derlin-1 retrotranslocation complex / positive regulation of protein K63-linked deubiquitination / protein-DNA covalent cross-linking repair / deubiquitinase activator activity / positive regulation of oxidative phosphorylation / cytoplasm protein quality control / regulation of protein localization to chromatin / ubiquitin-modified protein reader activity / cellular response to misfolded protein / mitotic spindle disassembly / VCP-NPL4-UFD1 AAA ATPase complex / positive regulation of mitochondrial membrane potential / regulation of protein catabolic process / vesicle-fusing ATPase / K48-linked polyubiquitin modification-dependent protein binding / nuclear outer membrane-endoplasmic reticulum membrane network / NAD+ metabolic process / regulation of aerobic respiration / retrograde protein transport, ER to cytosol / K63-linked polyubiquitin modification-dependent protein binding / stress granule disassembly / ATPase complex / negative regulation of type I interferon production / ubiquitin-specific protease binding / regulation of synapse organization / Golgi organization / polyubiquitin modification-dependent protein binding / positive regulation of ATP biosynthetic process / ubiquitin-like protein ligase binding / intracellular membrane-bounded organelle / RHOH GTPase cycle / skeletal system development / autophagosome maturation / MHC class I protein binding / HSF1 activation / endoplasmic reticulum to Golgi vesicle-mediated transport / negative regulation of hippo signaling / mitophagy / NF-kappaB binding / interstrand cross-link repair / ATP metabolic process / proteasome complex / protein unfolding / endoplasmic reticulum unfolded protein response / ribosome-associated ubiquitin-dependent protein catabolic process / Attachment and Entry / ERAD pathway / heat shock protein binding / Protein methylation / translesion synthesis / negative regulation of smoothened signaling pathway / positive regulation of DNA replication / viral genome replication / negative regulation of protein localization to chromatin / macroautophagy / negative regulation of canonical NF-kappaB signal transduction / rescue of stalled cytosolic ribosome / lipid droplet / Josephin domain DUBs / autophagy / ubiquitin binding / establishment of protein localization / proteasomal protein catabolic process / N-glycan trimming in the ER and Calnexin/Calreticulin cycle / positive regulation of protein-containing complex assembly / positive regulation of non-canonical NF-kappaB signal transduction / ADP binding / Hh mutants are degraded by ERAD / Dengue Virus Genome Translation and Replication / Translesion Synthesis by POLH / Hedgehog ligand biogenesis / Defective CFTR causes cystic fibrosis / AMPK-induced ERAD and lysosome mediated degradation of PD-L1(CD274) / ABC-family protein mediated transport / cytoplasmic stress granule / Ribosome Quality Control (RQC) complex extracts and degrades nascent peptide / double-strand break repair / Aggrephagy / positive regulation of canonical Wnt signaling pathway / positive regulation of protein catabolic process / azurophil granule lumen / Ovarian tumor domain proteases / positive regulation of proteasomal ubiquitin-dependent protein catabolic process / nuclear envelope / KEAP1-NFE2L2 pathway / site of double-strand break / cellular response to heat / E3 ubiquitin ligases ubiquitinate target proteins
Similarity search - Function
Nuclear pore localisation protein Npl4, ubiquitin-like domain / Nuclear pore localisation protein NPL4 / FAS-associated factor 1-like, UBX domain / FAF1, UBA-like domain / : / Fas-associated factor 1 / UAS / : / UAS / Ubiquitin fusion degradation protein Ufd1-like ...Nuclear pore localisation protein Npl4, ubiquitin-like domain / Nuclear pore localisation protein NPL4 / FAS-associated factor 1-like, UBX domain / FAF1, UBA-like domain / : / Fas-associated factor 1 / UAS / : / UAS / 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 / Domain present in ubiquitin-regulatory proteins / UBX domain / UBX domain / UBX domain profile. / UBA-like domain / Zinc finger domain / AAA ATPase, CDC48 family / CDC48, N-terminal subdomain / Cell division protein 48 (CDC48), N-terminal domain / Cell division protein 48 (CDC48) N-terminal domain / CDC48, domain 2 / Cell division protein 48 (CDC48) domain 2 / Cell division protein 48 (CDC48), domain 2 / : / CDC48 domain 2-like superfamily / Zinc finger RanBP2 type profile. / Zinc finger, RanBP2-type superfamily / Zinc finger RanBP2-type signature. / Zinc finger, RanBP2-type / Aspartate decarboxylase-like domain superfamily / AAA ATPase, AAA+ lid domain / AAA+ lid domain / MPN domain / MPN domain profile. / ATPase, AAA-type, conserved site / AAA-protein family signature. / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / Thioredoxin-like superfamily / Ubiquitin-like domain superfamily / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / ADENOSINE-5'-TRIPHOSPHATE / Transitional endoplasmic reticulum ATPase / Nuclear protein localization protein 4 homolog / Ubiquitin recognition factor in ER-associated degradation protein 1 / FAS-associated factor 1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.85 Å
AuthorsLiao, Z. / Arkinson, C. / Andreas, M.
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 13, 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
M: FAS-associated factor 1
O: FAS-associated factor 1
P: Ubiquitin recognition factor in ER-associated degradation protein 1
A: Transitional endoplasmic reticulum ATPase
B: Transitional endoplasmic reticulum ATPase
C: Transitional endoplasmic reticulum ATPase
D: Transitional endoplasmic reticulum ATPase
E: Transitional endoplasmic reticulum ATPase
F: Transitional endoplasmic reticulum ATPase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)697,12924
Polymers691,63210
Non-polymers5,49714
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

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Protein , 4 types, 10 molecules GMOPABCDEF

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


Mass: 68202.016 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 (E. coli) / References: UniProt: Q8TAT6
#2: Protein FAS-associated factor 1 / hFAF1 / UBX domain-containing protein 12 / UBX domain-containing protein 3A


Mass: 20495.000 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: FAF1, UBXD12, UBXN3A, CGI-03 / Production host: Escherichia coli (E. coli) / References: UniProt: Q9UNN5
#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 (E. coli) / References: UniProt: Q92890
#4: Protein
Transitional endoplasmic reticulum ATPase / TER ATPase / 15S Mg(2+)-ATPase p97 subunit / Valosin-containing protein / VCP


Mass: 91177.781 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: VCP, HEL-220, HEL-S-70 / Production host: Escherichia coli (E. coli) / References: UniProt: P55072, vesicle-fusing ATPase

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Non-polymers , 3 types, 14 molecules

#5: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
#7: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 9 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM

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Details

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: substrate engaged p97-Ufd1-NPL4-Faf1 complex / Type: COMPLEX / Entity ID: #2-#4 / Source: RECOMBINANT
Molecular weightValue: 734 kDa/nm / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.6
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
VitrificationCryogen name: ETHANE

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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 defocus max: 1600 nm / Nominal defocus min: 800 nm
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)

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2PHENIX2.0_5936model refinement
3SerialEMimage acquisition
5cryoSPARCCTF correction
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
12cryoSPARCclassification
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.85 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 99404 / Symmetry type: POINT
RefinementHighest resolution: 3.85 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00336019
ELECTRON MICROSCOPYf_angle_d0.66748866
ELECTRON MICROSCOPYf_dihedral_angle_d9.465292
ELECTRON MICROSCOPYf_chiral_restr0.0455541
ELECTRON MICROSCOPYf_plane_restr0.0056474

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