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- EMDB-28987: Cryo-EM structure of p97:UBXD1 VIM-only state -

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Basic information

Entry
Database: EMDB / ID: EMD-28987
TitleCryo-EM structure of p97:UBXD1 VIM-only state
Map data
Sample
  • Complex: Complex of p97 and UBXD1
    • Protein or peptide: UBX domain-containing protein 6
    • Protein or peptide: Transitional endoplasmic reticulum ATPase
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
KeywordsAAA+ / chaperone / hydrolase
Function / homology
Function and homology information


positive regulation of Lys63-specific deubiquitinase activity / flavin adenine dinucleotide catabolic process / positive regulation of oxidative phosphorylation / VCP-NSFL1C complex / protein-DNA covalent cross-linking repair / endoplasmic reticulum stress-induced pre-emptive quality control / endosome to lysosome transport via multivesicular body sorting pathway / cellular response to arsenite ion / Derlin-1 retrotranslocation complex / BAT3 complex binding ...positive regulation of Lys63-specific deubiquitinase activity / flavin adenine dinucleotide catabolic process / positive regulation of oxidative phosphorylation / VCP-NSFL1C complex / protein-DNA covalent cross-linking repair / endoplasmic reticulum stress-induced pre-emptive quality control / endosome to lysosome transport via multivesicular body sorting pathway / cellular response to arsenite ion / Derlin-1 retrotranslocation complex / BAT3 complex binding / positive regulation of protein K63-linked deubiquitination / deubiquitinase activator activity / mitotic spindle disassembly / VCP-NPL4-UFD1 AAA ATPase complex / aggresome assembly / NADH metabolic process / regulation of protein localization to chromatin / vesicle-fusing ATPase / cellular response to misfolded protein / : / stress granule disassembly / K48-linked polyubiquitin modification-dependent protein binding / positive regulation of mitochondrial membrane potential / negative regulation of protein localization to chromatin / ERAD pathway / ubiquitin-modified protein reader activity / retrograde protein transport, ER to cytosol / regulation of aerobic respiration / ATPase complex / regulation of synapse organization / ubiquitin-specific protease binding / positive regulation of ATP biosynthetic process / ubiquitin-like protein ligase binding / autophagosome maturation / RHOH GTPase cycle / polyubiquitin modification-dependent protein binding / HSF1 activation / translesion synthesis / endoplasmic reticulum to Golgi vesicle-mediated transport / MHC class I protein binding / Protein methylation / interstrand cross-link repair / negative regulation of smoothened signaling pathway / : / Attachment and Entry / ATP metabolic process / endoplasmic reticulum unfolded protein response / lipid droplet / proteasome complex / viral genome replication / Josephin domain DUBs / N-glycan trimming in the ER and Calnexin/Calreticulin cycle / proteasomal protein catabolic process / ADP binding / Hh mutants are degraded by ERAD / positive regulation of protein-containing complex assembly / Defective CFTR causes cystic fibrosis / macroautophagy / Hedgehog ligand biogenesis / Translesion Synthesis by POLH / ABC-family proteins mediated transport / establishment of protein localization / autophagy / Aggrephagy / cytoplasmic stress granule / positive regulation of non-canonical NF-kappaB signal transduction / positive regulation of protein catabolic process / positive regulation of canonical Wnt signaling pathway / activation of cysteine-type endopeptidase activity involved in apoptotic process / azurophil granule lumen / KEAP1-NFE2L2 pathway / double-strand break repair / Ovarian tumor domain proteases / positive regulation of proteasomal ubiquitin-dependent protein catabolic process / E3 ubiquitin ligases ubiquitinate target proteins / site of double-strand break / cellular response to heat / late endosome membrane / Neddylation / ubiquitin-dependent protein catabolic process / early endosome membrane / proteasome-mediated ubiquitin-dependent protein catabolic process / protein phosphatase binding / secretory granule lumen / regulation of apoptotic process / ficolin-1-rich granule lumen / Attachment and Entry / protein ubiquitination / endosome / lysosomal membrane / protein domain specific binding / intracellular membrane-bounded organelle / DNA repair / centrosome / glutamatergic synapse / lipid binding / DNA damage response / ubiquitin protein ligase binding / Neutrophil degranulation / endoplasmic reticulum membrane
Similarity search - Function
UBXN6, PUB domain / Domain present in ubiquitin-regulatory proteins / PUB-like domain superfamily / PUB domain / PUB domain / UBX domain / UBX domain / UBX domain profile. / domain in protein kinases, N-glycanases and other nuclear proteins / AAA ATPase, CDC48 family ...UBXN6, PUB domain / Domain present in ubiquitin-regulatory proteins / PUB-like domain superfamily / PUB domain / PUB domain / UBX domain / UBX domain / UBX domain profile. / domain in protein kinases, N-glycanases and other nuclear proteins / 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 / Aspartate decarboxylase-like domain superfamily / 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-like domain superfamily / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Transitional endoplasmic reticulum ATPase / UBX domain-containing protein 6
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.95 Å
AuthorsBraxton JR / Tucker MR / Tse E / Southworth DR
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM138690 United States
CitationJournal: bioRxiv / Year: 2023
Title: The p97/VCP adapter UBXD1 drives AAA+ remodeling and ring opening through multi-domain tethered interactions.
Authors: Julian R Braxton / Chad R Altobelli / Maxwell R Tucker / Eric Tse / Aye C Thwin / Michelle R Arkin / Daniel R Southworth /
Abstract: p97/VCP is an essential cytosolic AAA+ ATPase hexamer that extracts and unfolds substrate polypeptides during protein homeostasis and degradation. Distinct sets of p97 adapters guide cellular ...p97/VCP is an essential cytosolic AAA+ ATPase hexamer that extracts and unfolds substrate polypeptides during protein homeostasis and degradation. Distinct sets of p97 adapters guide cellular functions but their roles in direct control of the hexamer are unclear. The UBXD1 adapter localizes with p97 in critical mitochondria and lysosome clearance pathways and contains multiple p97-interacting domains. We identify UBXD1 as a potent p97 ATPase inhibitor and report structures of intact p97:UBXD1 complexes that reveal extensive UBXD1 contacts across p97 and an asymmetric remodeling of the hexamer. Conserved VIM, UBX, and PUB domains tether adjacent protomers while a connecting strand forms an N-terminal domain lariat with a helix wedged at the interprotomer interface. An additional VIM-connecting helix binds along the second AAA+ domain. Together these contacts split the hexamer into a ring-open conformation. Structures, mutagenesis, and comparisons to other adapters further reveal how adapters containing conserved p97-remodeling motifs regulate p97 ATPase activity and structure.
History
DepositionDec 1, 2022-
Header (metadata) releaseJun 21, 2023-
Map releaseJun 21, 2023-
UpdateDec 20, 2023-
Current statusDec 20, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_28987.png

Downloads & links

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Map

FileDownload / File: emd_28987.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.834 Å
Density
Contour LevelBy AUTHOR: 0.05
Minimum - Maximum-0.37420237 - 0.99834615
Average (Standard dev.)-0.0017121261 (±0.036468294)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 333.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Sharpened map

Fileemd_28987_additional_1.map
AnnotationSharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_28987_half_map_1.map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Half map: #1

Fileemd_28987_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : Complex of p97 and UBXD1

EntireName: Complex of p97 and UBXD1
Components
  • Complex: Complex of p97 and UBXD1
    • Protein or peptide: UBX domain-containing protein 6
    • Protein or peptide: Transitional endoplasmic reticulum ATPase
  • Ligand: ADENOSINE-5'-DIPHOSPHATE

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Supramolecule #1: Complex of p97 and UBXD1

SupramoleculeName: Complex of p97 and UBXD1 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: UBX domain-containing protein 6

MacromoleculeName: UBX domain-containing protein 6 / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 49.823656 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MKKFFQEFKA DIKFKSAGPG QKLKESVGEK AHKEKPNQPA PRPPRQGPTN EAQMAAAAAL ARLEQKQSRA WGPTSQDTIR NQVRKELQA EATVSGSPEA PGTNVVSEPR EEGSAHLAVP GVYFTCPLTG ATLRKDQRDA CIKEAILLHF STDPVAASIM K IYTFNKDQ ...String:
MKKFFQEFKA DIKFKSAGPG QKLKESVGEK AHKEKPNQPA PRPPRQGPTN EAQMAAAAAL ARLEQKQSRA WGPTSQDTIR NQVRKELQA EATVSGSPEA PGTNVVSEPR EEGSAHLAVP GVYFTCPLTG ATLRKDQRDA CIKEAILLHF STDPVAASIM K IYTFNKDQ DRVKLGVDTI AKYLDNIHLH PEEEKYRKIK LQNKVFQERI NCLEGTHEFF EAIGFQKVLL PAQDQEDPEE FY VLSETTL AQPQSLERHK EQLLAAEPVR AKLDRQRRVF QPSPLASQFE LPGDFFNLTA EEIKREQRLR SEAVERLSVL RTK AMREKE EQRGLRKYNY TLLRVRLPDG CLLQGTFYAR ERLGAVYGFV REALQSDWLP FELLASGGQK LSEDENLALN ECGL VPSAL LTFSWDMAVL EDIKAAGAEP DSILKPELLS AIEKLL

UniProtKB: UBX domain-containing protein 6

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Macromolecule #2: Transitional endoplasmic reticulum ATPase

MacromoleculeName: Transitional endoplasmic reticulum ATPase / type: protein_or_peptide / ID: 2 / Number of copies: 6 / Enantiomer: LEVO / EC number: vesicle-fusing ATPase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 89.43682 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MASGADSKGD DLSTAILKQK NRPNRLIVDE AINEDNSVVS LSQPKMDELQ LFRGDTVLLK GKKRREAVCI VLSDDTCSDE KIRMNRVVR NNLRVRLGDV ISIQPCPDVK YGKRIHVLPI DDTVEGITGN LFEVYLKPYF LEAYRPIRKG DIFLVRGGMR A VEFKVVET ...String:
MASGADSKGD DLSTAILKQK NRPNRLIVDE AINEDNSVVS LSQPKMDELQ LFRGDTVLLK GKKRREAVCI VLSDDTCSDE KIRMNRVVR NNLRVRLGDV ISIQPCPDVK YGKRIHVLPI DDTVEGITGN LFEVYLKPYF LEAYRPIRKG DIFLVRGGMR A VEFKVVET DPSPYCIVAP DTVIHCEGEP IKREDEEESL NEVGYDDIGG CRKQLAQIKE MVELPLRHPA LFKAIGVKPP RG ILLYGPP GTGKTLIARA VANETGAFFF LINGPEIMSK LAGESESNLR KAFEEAEKNA PAIIFIDELD AIAPKREKTH GEV ERRIVS QLLTLMDGLK QRAHVIVMAA TNRPNSIDPA LRRFGRFDRE VDIGIPDATG RLEILQIHTK NMKLADDVDL EQVA NETHG HVGADLAALC SEAALQAIRK KMDLIDLEDE TIDAEVMNSL AVTMDDFRWA LSQSNPSALR ETVVEVPQVT WEDIG GLED VKRELQELVQ YPVEHPDKFL KFGMTPSKGV LFYGPPGCGK TLLAKAIANE CQANFISIKG PELLTMWFGE SEANVR EIF DKARQAAPCV LFFDELDSIA KARGGNIGDG GGAADRVINQ ILTEMDGMST KKNVFIIGAT NRPDIIDPAI LRPGRLD QL IYIPLPDEKS RVAILKANLR KSPVAKDVDL EFLAKMTNGF SGADLTEICQ RACKLAIRES IESEIRRERE RQTNPSAM E VEEDDPVPEI RRDHFEEAMR FARRSVSDND IRKYEMFAQT LQQSRGFGSF RFPSGNQGGA GPSQGSGGGT GGSVYTEDN DDDLYG

UniProtKB: Transitional endoplasmic reticulum ATPase

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Macromolecule #3: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 12 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM / Adenosine diphosphate

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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

Concentration1.89 mg/mL
BufferpH: 7.4
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 120 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.03 kPa / Details: Pelco easiGlow, 15 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: Wait time 10 sec, blot time 3 sec, blot force 0.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated magnification: 59952 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 105000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 22536 / Average exposure time: 2.024 sec. / Average electron dose: 43.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER / Details: Ab initio reconstruction from cryoSPARC
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final reconstructionApplied symmetry - Point group: C6 (6 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.95 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 100000

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Atomic model buiding 1

RefinementProtocol: FLEXIBLE FIT
Output model

PDB-8fcn:
Cryo-EM structure of p97:UBXD1 VIM-only state

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