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- EMDB-19473: p97 (VCP) double mutant - F266A F539A -

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Basic information

Entry
Database: EMDB / ID: EMD-19473
Titlep97 (VCP) double mutant - F266A F539A
Map dataPost-processed map
Sample
  • Complex: Tertiary complex of the hexameric p97 F266A F539A double mutant
    • Protein or peptide: Transitional endoplasmic reticulum ATPase
KeywordsHexameric complex / ATPase / Unfoldase / Protein Quality Control / Segregase / CHAPERONE
Function / homology
Function and homology information


cytoplasmic ubiquitin ligase complex / flavin adenine dinucleotide catabolic process / 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 / protein-DNA covalent cross-linking repair / Derlin-1 retrotranslocation complex / positive regulation of protein K63-linked deubiquitination ...cytoplasmic ubiquitin ligase complex / flavin adenine dinucleotide catabolic process / 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 / protein-DNA covalent cross-linking repair / Derlin-1 retrotranslocation complex / positive regulation of protein K63-linked deubiquitination / cytoplasm protein quality control / positive regulation of oxidative phosphorylation / aggresome assembly / deubiquitinase activator activity / ubiquitin-modified protein reader activity / regulation of protein localization to chromatin / mitotic spindle disassembly / cellular response to misfolded protein / VCP-NPL4-UFD1 AAA ATPase complex / ciliary transition zone / positive regulation of mitochondrial membrane potential / vesicle-fusing ATPase / K48-linked polyubiquitin modification-dependent protein binding / regulation of aerobic respiration / retrograde protein transport, ER to cytosol / stress granule disassembly / NAD+ metabolic process / ATPase complex / regulation of synapse organization / ubiquitin-specific protease binding / ciliary tip / positive regulation of ATP biosynthetic process / MHC class I protein binding / ubiquitin-like protein ligase binding / RHOH GTPase cycle / polyubiquitin modification-dependent protein binding / autophagosome maturation / endoplasmic reticulum to Golgi vesicle-mediated transport / negative regulation of hippo signaling / HSF1 activation / interstrand cross-link repair / ATP metabolic process / translesion synthesis / endoplasmic reticulum unfolded protein response / proteasomal protein catabolic process / negative regulation of protein localization to chromatin / Attachment and Entry / Protein methylation / ERAD pathway / lipid droplet / proteasome complex / viral genome replication / Josephin domain DUBs / N-glycan trimming in the ER and Calnexin/Calreticulin cycle / macroautophagy / negative regulation of smoothened signaling pathway / establishment of protein localization / Hh mutants are degraded by ERAD / positive regulation of protein-containing complex assembly / Hedgehog ligand biogenesis / Defective CFTR causes cystic fibrosis / positive regulation of non-canonical NF-kappaB signal transduction / Translesion Synthesis by POLH / ADP binding / autophagy / ABC-family proteins mediated transport / cytoplasmic stress granule / Aggrephagy / azurophil granule lumen / positive regulation of protein catabolic process / Ovarian tumor domain proteases / KEAP1-NFE2L2 pathway / positive regulation of canonical Wnt signaling pathway / double-strand break repair / positive regulation of proteasomal ubiquitin-dependent protein catabolic process / E3 ubiquitin ligases ubiquitinate target proteins / cellular response to heat / site of double-strand break / Neddylation / secretory granule lumen / regulation of apoptotic process / protein phosphatase binding / ubiquitin-dependent protein catabolic process / ficolin-1-rich granule lumen / proteasome-mediated ubiquitin-dependent protein catabolic process / Attachment and Entry / ciliary basal body / protein ubiquitination / intracellular membrane-bounded organelle / protein domain specific binding / DNA repair / DNA damage response / ubiquitin protein ligase binding / Neutrophil degranulation / lipid binding / endoplasmic reticulum membrane / perinuclear region of cytoplasm / glutamatergic synapse / endoplasmic reticulum / ATP hydrolysis activity
Similarity search - Function
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, 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 / 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
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsArie M / Matzov D / Karmona R / Szenkier N / Stanhill A / Navon A
Funding support Israel, 1 items
OrganizationGrant numberCountry
Israel Science Foundation2038/17 Israel
CitationJournal: iScience / Year: 2024
Title: A non-symmetrical p97 conformation initiates a multistep recruitment of Ufd1/Npl4.
Authors: Michal Arie / Donna Matzov / Rotem Karmona / Natalia Szenkier / Ariel Stanhill / Ami Navon /
Abstract: experiments and cryo-EM structures of p97 and its cofactor, Ufd1/Npl4 (UN), elucidated substrate processing. Yet, the structural transitions and the related ATPase cycle upon UN binding remain ... experiments and cryo-EM structures of p97 and its cofactor, Ufd1/Npl4 (UN), elucidated substrate processing. Yet, the structural transitions and the related ATPase cycle upon UN binding remain unresolved. We captured two discrete conformations: One in which D1 protomers are ATP bound, while the D2 subunits are in the ADP state, presumably required for substrate engagement with the D2 pore; and a heterologous nucleotide state within the D1 ring in which only two NTDs are in the "up" ATP state that favors UN binding. Further analysis suggests that initially, UN binds p97's non-symmetrical conformation, this association promotes a structural transition upon which five NTDs shift to an "up" state and are poised to bind ATP. The UBXL domain of Npl4 was captured bound to an NTD in the ADP state, demonstrating a conformation that may provide directionality to incoming substrate and introduce the flexibility needed for substrate processing.
History
DepositionJan 24, 2024-
Header (metadata) releaseMay 29, 2024-
Map releaseMay 29, 2024-
UpdateDec 25, 2024-
Current statusDec 25, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_19473.png

Downloads & links

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Map

FileDownload / File: emd_19473.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPost-processed map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.86 Å/pix.
x 280 pix.
= 240.8 Å		0.86 Å/pix.
x 280 pix.
= 240.8 Å		0.86 Å/pix.
x 280 pix.
= 240.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.86 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.244
Minimum - Maximum-0.5553403 - 0.9791154
Average (Standard dev.)0.0070939367 (±0.052801125)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 240.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Unsharpened map

Fileemd_19473_additional_1.map
AnnotationUnsharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1

Fileemd_19473_half_map_1.map
AnnotationHalf map 1
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2

Fileemd_19473_half_map_2.map
AnnotationHalf map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : Tertiary complex of the hexameric p97 F266A F539A double mutant

EntireName: Tertiary complex of the hexameric p97 F266A F539A double mutant
Components
  • Complex: Tertiary complex of the hexameric p97 F266A F539A double mutant
    • Protein or peptide: Transitional endoplasmic reticulum ATPase

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Supramolecule #1: Tertiary complex of the hexameric p97 F266A F539A double mutant

SupramoleculeName: Tertiary complex of the hexameric p97 F266A F539A double mutant
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: The mutant generated by the replacement of phenylalanine to alanine at positions 266 and 539.
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 530 KDa

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Macromolecule #1: Transitional endoplasmic reticulum ATPase

MacromoleculeName: Transitional endoplasmic reticulum ATPase / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO / EC number: vesicle-fusing ATPase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 89.284633 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 VANETGAFAF 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 CQANAISIKG 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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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration7 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
50.0 mMC8H18N2O4SHEPES
30.0 mMKClpotassium chloride
1.0 mMC4H10O2S2DTT
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 12
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV
Details: pre-blotting incubation time of 20 secounds and blot for 3.5 seconds with -1 blot force.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number real images: 11902 / Average electron dose: 38.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 3005242
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

5ftn

Final reconstructionApplied symmetry - Point group: C6 (6 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2) / Number images used: 79612
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1.2)
Final 3D classificationSoftware - Name: RELION (ver. 3.1.2)

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Atomic model buiding 1

Initial modelPDB ID:

5ftn


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementOverall B value: 111
Output model

PDB-8rs9:
p97 (VCP) double mutant - F266A F539A

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