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

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Basic information

Entry
Database: PDB / ID: 11ta
TitleCryo-EM structure of substrate engaged p97-Ufd1-NPL4-Faf1 complex (motor focused)
Components
  • FAS-associated factor 1
  • Nuclear protein localization protein 4 homolog
  • Transitional endoplasmic reticulum ATPase
  • 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 / 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 / protein-DNA covalent cross-linking repair / Derlin-1 retrotranslocation complex / positive regulation of protein K63-linked deubiquitination / ATPase complex / deubiquitinase activator activity / response to insecticide / positive regulation of oxidative phosphorylation / cytoplasm protein quality control / aggresome assembly / ubiquitin-modified protein reader activity / regulation of protein localization to chromatin / 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 / K63-linked polyubiquitin modification-dependent protein binding / ciliary transition zone / Enterobacterial factors antagonize host defense / PD-L1(CD274) glycosylation and translocation to plasma membrane / regulation of aerobic respiration / NAD+ metabolic process / retrograde protein transport, ER to cytosol / ribosome-associated ubiquitin-dependent protein catabolic process / stress granule disassembly / negative regulation of type I interferon production / ubiquitin-specific protease binding / Golgi organization / regulation of synapse organization / negative regulation of myoblast fusion / PELO:HBS1L and ABCE1 dissociate a ribosome on a non-stop mRNA / Dengue Virus Genome Translation and Replication / positive regulation of ATP biosynthetic process / Maturation of DENV proteins / ZNF598 and the Ribosome-associated Quality Trigger (RQT) complex dissociate a ribosome stalled on a no-go mRNA / intracellular membrane-bounded organelle / ubiquitin-like protein ligase binding / RHOH GTPase cycle / MHC class I protein binding / autophagosome maturation / Peptide chain elongation / Selenocysteine synthesis / Formation of a pool of free 40S subunits / negative regulation of hippo signaling / Eukaryotic Translation Termination / endoplasmic reticulum to Golgi vesicle-mediated transport / HSF1 activation / SRP-dependent cotranslational protein targeting to membrane / Response of EIF2AK4 (GCN2) to amino acid deficiency / AMPK-induced ERAD and lysosome mediated degradation of PD-L1(CD274) / polyubiquitin modification-dependent protein binding / Viral mRNA Translation / NF-kappaB binding / Nonsense Mediated Decay (NMD) independent of the Exon Junction Complex (EJC) / interstrand cross-link repair / GTP hydrolysis and joining of the 60S ribosomal subunit / ATP metabolic process / L13a-mediated translational silencing of Ceruloplasmin expression / Major pathway of rRNA processing in the nucleolus and cytosol / GSK3B-mediated proteasomal degradation of PD-L1(CD274) / SPOP-mediated proteasomal degradation of PD-L1(CD274) / Nonsense Mediated Decay (NMD) enhanced by the Exon Junction Complex (EJC) / Attachment and Entry / skeletal system development / endoplasmic reticulum unfolded protein response / Protein methylation / ERAD pathway / heat shock protein binding / ciliary tip / translesion synthesis / Ribosome Quality Control (RQC) complex extracts and degrades nascent peptide / mRNA Polyadenylation / negative regulation of protein localization to chromatin / rescue of stalled cytosolic ribosome / Maturation of protein E / Maturation of protein E / lipid droplet / 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
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 / Cell division protein 48 (CDC48), N-terminal domain / : / CDC48, N-terminal subdomain / 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 / Ribosomal L40e family / Ribosomal_L40e / Ribosomal protein L40e / Ribosomal protein L40e superfamily / MPN domain / MPN domain profile. / AAA ATPase, AAA+ lid domain / AAA+ lid domain / ATPase, AAA-type, conserved site / AAA-protein family signature. / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / : / Ubiquitin domain signature. / Ubiquitin conserved site / Ubiquitin domain / Ubiquitin family / Ubiquitin homologues / Ubiquitin domain profile. / Ubiquitin-like domain / 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 / Ubiquitin-ribosomal protein eL40 fusion protein / 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: 3.58 Å
AuthorsLiao, Z. / Arkinson, C. / 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
I: Ubiquitin
K: Ubiquitin
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)714,45426
Polymers708,95712
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 , 5 types, 12 molecules GIKMOPABCDEF

#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.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: UBA52, UBCEP2
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: P62987
#3: 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
Details: FH-UBX fragment / Source: (gene. exp.) Homo sapiens (human) / Gene: FAF1, UBXD12, UBXN3A, CGI-03
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: Q9UNN5
#4: 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
#5: 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) / Strain (production host): rosetta 2 de3 plyss / References: UniProt: P55072, vesicle-fusing ATPase

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Non-polymers , 3 types, 14 molecules

#6: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#7: 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
#8: 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: Cryo-EM structure of substrate engaged p97-Ufd1-NPL4-Faf1 complex (motor focused)
Type: COMPLEX / Entity ID: #1-#5 / 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: 3.58 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 102875 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 101.19 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.005638577
ELECTRON MICROSCOPYf_angle_d0.711152177
ELECTRON MICROSCOPYf_chiral_restr0.04595855
ELECTRON MICROSCOPYf_plane_restr0.00486851
ELECTRON MICROSCOPYf_dihedral_angle_d9.83175513

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