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- EMDB-18517: p97 (VCP) mutant - F539A with its adaptor -

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Basic information

Entry
Database: EMDB / ID: EMD-18517
Titlep97 (VCP) mutant - F539A with its adaptor
Map dataPost-processed map
Sample
  • Complex: Tertiary complex of the hexameric p97 F539A mutant with its adaptor
    • Protein or peptide: Transitional endoplasmic reticulum ATPase
    • Protein or peptide: Nuclear protein localization protein 4 homolog
KeywordsHexameric complex / ATPase / Unfoldase / Protein Quality Control / Segregase / CHAPERONE
Biological speciesHomo sapiens (human) / Rattus (rat)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 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
DepositionSep 27, 2023-
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_18517.png

Downloads & links

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Map

FileDownload / File: emd_18517.map.gz / Format: CCP4 / Size: 178 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.)
0.79 Å/pix.
x 360 pix.
= 285.48 Å		0.79 Å/pix.
x 360 pix.
= 285.48 Å		0.79 Å/pix.
x 360 pix.
= 285.48 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.793 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.00797
Minimum - Maximum-0.058161628 - 0.09663817
Average (Standard dev.)0.00021431719 (±0.0026036876)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 285.47998 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Unsharpened map

Fileemd_18517_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 B

Fileemd_18517_half_map_1.map
AnnotationHalf-map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map A

Fileemd_18517_half_map_2.map
AnnotationHalf-map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Tertiary complex of the hexameric p97 F539A mutant with its adaptor

EntireName: Tertiary complex of the hexameric p97 F539A mutant with its adaptor
Components
  • Complex: Tertiary complex of the hexameric p97 F539A mutant with its adaptor
    • Protein or peptide: Transitional endoplasmic reticulum ATPase
    • Protein or peptide: Nuclear protein localization protein 4 homolog

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Supramolecule #1: Tertiary complex of the hexameric p97 F539A mutant with its adaptor

SupramoleculeName: Tertiary complex of the hexameric p97 F539A mutant with its adaptor
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: The mutant generated by the replacement of phenylalanine to alanine at position 539
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Transitional endoplasmic reticulum ATPase

MacromoleculeName: Transitional endoplasmic reticulum ATPase / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
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 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

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Macromolecule #2: Nuclear protein localization protein 4 homolog

MacromoleculeName: Nuclear protein localization protein 4 homolog / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Rattus (rat)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MAEGTIIRVQ SPDGVKRITA TKRETAATFL KKVAKEFGFQ NNGFSVYINR NKTGEITASS SKSLHLLKI KHGDLLFLFP SSLAGPSSEM ETSTSVGLKA FGAPHVVEDE IDQYLSKQDG K IYRSRDPQ LCRHGPLGKC VHCVPLEPFD EDYLNHLEPP VKHMSFHAYI ...String:
MAEGTIIRVQ SPDGVKRITA TKRETAATFL KKVAKEFGFQ NNGFSVYINR NKTGEITASS SKSLHLLKI KHGDLLFLFP SSLAGPSSEM ETSTSVGLKA FGAPHVVEDE IDQYLSKQDG K IYRSRDPQ LCRHGPLGKC VHCVPLEPFD EDYLNHLEPP VKHMSFHAYI RKLTGGADKG KF VALENIS CKIKSGCEGH LPWPNGICTK CQPSAITLNR QKYRHVDNIM FENHTVADRF LDF WRKTGN QHFGYLYGRY TEHKDIPLGI RAEVAAIYEP PQIGTQNSLE LLEDPKAEVV DEIA SKLGL RKVGWIFTDL VSEDTRKGTV RYSRNKDTYF LSSEECITAG DFQNKHPNIC RLSPD GHFG SKFVTAVATG GPDNQVHFEG YQVSNQCMAL VRDECLLPCK DAPELGYAKE SSSEQY VPD VFYKDIDKFG NEITQLARPL PVEYLIIDIT TTFPKDPVYT FSISQNPFPI ENRDVLG ET QDFHSLATYL SQNTSSVFLD TISDFHLLLF LVTNEVMPLQ DSISLLLEAV RTRNEELA Q TWKKSEQWAT IEQLCSTVGV QLPGLHEFGA VGGSARAATS AMWACQHCTF MNQPGTGHC EMCSLPRT

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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

Concentration3.5 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
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV
Details: pre-blotting incubation time of 20 seconds 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: 11807 / Average electron dose: 43.0 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: 1.8 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: 2448068
Startup modelType of model: EMDB MAP
EMDB ID:

20730

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1.3) / Number images used: 399769
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1.3)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1.3)
Final 3D classificationSoftware - Name: RELION (ver. 3.1.3)

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Atomic model buiding 1

Initial modelPDB ID:

0730


Chain - Source name: Other / Chain - Initial model type: experimental model / Details: FROM EMDB
RefinementOverall B value: 104

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