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- PDB-31op: CryoEM structure of substrate engaged p97 in complex with Ufd1/Np... -

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Basic information

Entry
Database: PDB / ID: 31op
TitleCryoEM structure of substrate engaged p97 in complex with Ufd1/Npl4 and C-terminal FAF1- State II
Components
  • FAS-associated factor 1
  • Nuclear protein localization protein 4 homolog
  • Polyubiquitin-B
  • Transitional endoplasmic reticulum ATPase
  • Ubiquitin recognition factor in ER-associated degradation protein 1
KeywordsTRANSLOCASE / Complex / ATPase / Protein unfolding / Cell cycle
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 / VCP-NSFL1C complex / protein kinase regulator activity / intracellular protein-containing complex / endoplasmic reticulum stress-induced pre-emptive quality control / endosome to lysosome transport via multivesicular body sorting pathway ...UFD1-NPL4 complex / Fas signaling pathway / negative regulation of RIG-I signaling pathway / CD95 death-inducing signaling complex / flavin adenine dinucleotide catabolic process / VCP-NSFL1C complex / protein kinase regulator activity / intracellular protein-containing 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 / peptidyl-lysine trimethylation / cytoplasm protein quality control / regulation of protein localization to chromatin / ubiquitin-modified protein reader activity / RQC complex / cellular response to misfolded protein / mitotic spindle disassembly / VCP-NPL4-UFD1 AAA ATPase complex / mitochondrion transport along microtubule / positive regulation of mitochondrial membrane potential / positive regulation of protein monoubiquitination / 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 / K63-linked polyubiquitin modification-dependent protein binding / retrograde protein transport, ER to cytosol / stress granule disassembly / negative regulation of type I interferon production / ubiquitin-specific protease binding / polyubiquitin modification-dependent protein binding / ATPase complex / regulation of synapse organization / Golgi organization / positive regulation of ATP biosynthetic process / ubiquitin-like protein ligase binding / RHOH GTPase cycle / intracellular membrane-bounded organelle / skeletal system development / autophagosome maturation / MHC class I protein binding / HSF1 activation / positive regulation of intrinsic apoptotic signaling pathway by p53 class mediator / negative regulation of hippo signaling / endoplasmic reticulum to Golgi vesicle-mediated transport / NF-kappaB binding / mitophagy / interstrand cross-link repair / ATP metabolic process / protein unfolding / endoplasmic reticulum unfolded protein response / heat shock protein binding / Attachment and Entry / ribosome-associated ubiquitin-dependent protein catabolic process / ERAD pathway / Protein methylation / neuron projection morphogenesis / translesion synthesis / proteasome complex / Maturation of protein E / Maturation of protein E / ER Quality Control Compartment (ERQC) / Myoclonic epilepsy of Lafora / regulation of mitochondrial membrane potential / FLT3 signaling by CBL mutants / regulation of neuron apoptotic process / 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 / positive regulation of DNA replication / Regulation of TBK1, IKKε (IKBKE)-mediated activation of IRF3, IRF7 / Negative regulation of FLT3 / 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 / viral genome replication / NOTCH2 Activation and Transmission of Signal to the Nucleus / TICAM1,TRAF6-dependent induction of TAK1 complex / PTK6 Regulates RTKs and Their Effectors AKT1 and DOK1 / TICAM1-dependent activation of IRF3/IRF7 / APC/C:Cdc20 mediated degradation of Cyclin B / positive regulation of protein ubiquitination / Downregulation of ERBB4 signaling / APC-Cdc20 mediated degradation of Nek2A / regulation of proteasomal protein catabolic process
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 profile. / UBX domain / 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 / Zinc finger RanBP2 type profile. / Zinc finger, RanBP2-type superfamily / Zinc finger RanBP2-type signature. / CDC48 domain 2-like superfamily / Zinc finger, RanBP2-type / Aspartate decarboxylase-like domain 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 / BERYLLIUM TRIFLUORIDE ION / Polyubiquitin-B / 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: 3.5 Å
AuthorsDrakonaki, A. / van den Boom, J. / Kroening, A. / Meyer, H. / Gatsogiannis, C.
Funding support Germany, 5items
OrganizationGrant numberCountry
German Research Foundation (DFG)424228829 Germany
German Research Foundation (DFG)509479817 Germany
German Research Foundation (DFG)496113311 Germany
German Research Foundation (DFG)DFG INST 211/667-1 Germany
German Research Foundation (DFG)INST-208767513-1_FUGG Germany
CitationJournal: Nat Commun / Year: 2026
Title: Structural dynamics of substrate processing by the p97-Ufd1-Npl4-FAF1 machinery.
Authors: Athina Drakonaki / Johannes van den Boom / Alexander Kröning / Hemmo Meyer / Christos Gatsogiannis /
Abstract: The AAA-ATPase VCP/p97 unfolds ubiquitylated proteins in key signaling and cellular homeostasis pathways, yet the mechanisms by which Ufd1-Npl4 and FAF1 adapters handle ubiquitin chains to coordinate ...The AAA-ATPase VCP/p97 unfolds ubiquitylated proteins in key signaling and cellular homeostasis pathways, yet the mechanisms by which Ufd1-Npl4 and FAF1 adapters handle ubiquitin chains to coordinate substrate loading and processing remain unclear. Our cryo-EM study of p97-Ufd1-Npl4 and FAF1 engaging a substrate reveal interactions that drive substrate processing. While Npl4 is capped by distal ubiquitins, and the initiator ubiquitin can be traced from the Npl4 groove to the ATPase channel at near-atomic resolution, we find that the Npl4 tower adaptively responds to the configurational changes in the ATPase motor's spiral staircase. A helix in FAF1 together with the Ufd1-UT3 domain engage proximal ubiquitins while providing the necessary structural plasticity to accommodate this motion and deliver ubiquitins to Npl4's groove. Our study reveals the intricate, dynamic interplay between p97, Ufd1-Npl4, and FAF1 in efficient processing of ubiquitylated substrates.
History
DepositionJun 16, 2026Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 4, 2026Provider: repository / Type: Initial release
Revision 1.0Nov 4, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
J: Polyubiquitin-B
K: Polyubiquitin-B
G: Nuclear protein localization protein 4 homolog
I: Polyubiquitin-B
F: Transitional endoplasmic reticulum ATPase
E: Transitional endoplasmic reticulum ATPase
D: Transitional endoplasmic reticulum ATPase
C: Transitional endoplasmic reticulum ATPase
B: Transitional endoplasmic reticulum ATPase
A: Transitional endoplasmic reticulum ATPase
N: Polyubiquitin-B
H: Ubiquitin recognition factor in ER-associated degradation protein 1
L: Polyubiquitin-B
M: Polyubiquitin-B
T: FAS-associated factor 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)730,45647
Polymers724,42615
Non-polymers6,03032
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, 15 molecules JKINLMGFEDCBAHT

#1: Protein
Polyubiquitin-B


Mass: 8576.831 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: UBB / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: P0CG47
#2: 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 BL21 (bacteria) / References: UniProt: Q8TAT6
#3: Protein
Transitional endoplasmic reticulum ATPase / TER ATPase / 15S Mg(2+)-ATPase p97 subunit / Valosin-containing protein / VCP


Mass: 91665.227 Da / Num. of mol.: 6 / Mutation: A232E, E578Q
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 BL21 (bacteria) / References: UniProt: P55072, vesicle-fusing ATPase
#4: Protein Ubiquitin recognition factor in ER-associated degradation protein 1 / Ubiquitin fusion degradation protein 1 / UB fusion protein 1


Mass: 34544.316 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 (bacteria) / References: UniProt: Q92890
#5: Protein FAS-associated factor 1 / hFAF1 / UBX domain-containing protein 12 / UBX domain-containing protein 3A


Mass: 20227.676 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: FAF1, UBXD12, UBXN3A, CGI-03 / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: Q9UNN5

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Non-polymers , 3 types, 32 molecules

#6: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
#7: Chemical
ChemComp-BEF / BERYLLIUM TRIFLUORIDE ION


Mass: 66.007 Da / Num. of mol.: 10 / Source method: obtained synthetically / Formula: BeF3 / Feature type: SUBJECT OF INVESTIGATION
#8: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 10 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION

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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 in complex with Ufd1/Npl4 and only-C-term FAF1
Type: CELL / Entity ID: #1-#5 / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli BL21 (bacteria)
Buffer solutionpH: 7.5 / Details: pH 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPES1
2100 mMPotassium chlorideKCl1
35 mMMagnesium chlorideMgCl21
40.5 mMTCEP1
51 mMADP1
61 mMBeryllium sulfateBeSO41
74 mMSodium fluorideNaF1
SpecimenConc.: 12 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK II / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 286.15 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: 270000 X / Nominal defocus max: 2600 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Film or detector model: TFS FALCON 4i (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 279701
EM imaging opticsEnergyfilter name: TFS Selectris X / Energyfilter slit width: 10 eV
Image scansWidth: 4096 / Height: 4096

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Processing

EM software
IDNameVersionCategoryDetails (eV)
1cryoSPARCv3.3-v4.6.2particle selection
2EPU3.2.0.4776image acquisitionEPU was used to automatically collect EER movies
4cryoSPARCv3.3-v4.6.2CTF correction
10cryoSPARCv3.3-v4.6.2initial Euler assignment
11cryoSPARCv3.3-v4.6.2final Euler assignment
12cryoSPARCv3.3-v4.6.2classification
13cryoSPARCv3.3-v4.6.23D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
Particle selectionNum. of particles selected: 10568986
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 64905 / Symmetry type: POINT

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