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- EMDB-17044: Cryo-EM structure of P5A-ATPase CtSpf1 (E2.Pi state with endogeno... -

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Basic information

Entry
Database: EMDB / ID: EMD-17044
TitleCryo-EM structure of P5A-ATPase CtSpf1 (E2.Pi state with endogenous helix cargo bound)
Map data
Sample
  • Complex: CtSpf1 with AlF4 bound E2.Pi state
    • Complex: Unknown helix
      • Protein or peptide: Unknown helix
    • Complex: Cation-transporting ATPase-like protein
      • Protein or peptide: Cation-transporting ATPase-like protein
  • Ligand: TETRAFLUOROALUMINATE ION
  • Ligand: MAGNESIUM ION
  • Ligand: CALCIUM ION
Keywordstranslocase / MEMBRANE PROTEIN
Function / homology
Function and homology information


P-type ion transporter activity / ATPase-coupled monoatomic cation transmembrane transporter activity / intracellular calcium ion homeostasis / endoplasmic reticulum membrane / ATP hydrolysis activity / ATP binding
Similarity search - Function
: / P5A-ATPase, transmembrane helical hairpin / P-type ATPase, subfamily V / E1-E2 ATPase / P-type ATPase, haloacid dehalogenase domain / P-type ATPase, phosphorylation site / P-type ATPase, cytoplasmic domain N / E1-E2 ATPases phosphorylation site. / P-type ATPase, A domain superfamily / P-type ATPase ...: / P5A-ATPase, transmembrane helical hairpin / P-type ATPase, subfamily V / E1-E2 ATPase / P-type ATPase, haloacid dehalogenase domain / P-type ATPase, phosphorylation site / P-type ATPase, cytoplasmic domain N / E1-E2 ATPases phosphorylation site. / P-type ATPase, A domain superfamily / P-type ATPase / P-type ATPase, transmembrane domain superfamily / HAD superfamily / HAD-like superfamily
Similarity search - Domain/homology
Cation-transporting ATPase-like protein
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast) / Thermochaetoides thermophila (fungus)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.48 Å
AuthorsLi P / Gourdon P
Funding support Sweden, Denmark, 4 items
OrganizationGrant numberCountry
Knut and Alice Wallenberg Foundation2020.0194 Sweden
Swedish Research Council2022-01315 Sweden
The Crafoord Foundation20200739 Sweden
LundbeckfondenR346-2020-2019 Denmark
Citation
Journal: Nat Commun / Year: 2024
Title: The structure and function of P5A-ATPases.
Authors: Ping Li / Viktoria Bågenholm / Per Hägglund / Karin Lindkvist-Petersson / Kaituo Wang / Pontus Gourdon /
Abstract: Endoplasmic reticulum (ER) membrane resident P5A-ATPases broadly affect protein biogenesis and quality control, and yet their molecular function remains debated. Here, we report cryo-EM structures of ...Endoplasmic reticulum (ER) membrane resident P5A-ATPases broadly affect protein biogenesis and quality control, and yet their molecular function remains debated. Here, we report cryo-EM structures of a P5A-ATPase, CtSpf1, covering multiple transport intermediates of the E1 → E1-ATP → E1P-ADP → E1P → E2P → E2.P → E2 → E1 cycle. In the E2P and E2.P states a cleft spans the entire membrane, holding a polypeptide cargo molecule. The cargo includes an ER luminal extension, pinpointed as the C-terminus in the E2.P state, which reenters the membrane in E2P. The E1 structure harbors a cytosol-facing cavity that is blocked by an insertion we refer to as the Plug-domain. The Plug-domain is nestled to key ATPase features and is displaced in the E1P-ADP and E1P states. Collectively, our findings are compatible with a broad range of proteins as cargo, with the P5A-ATPases serving a role in membrane removal of helices, although insertion/secretion cannot be excluded, as well as with a mechanistic role of the Plug-domain.
#1: Journal: Acta Crystallogr., Sect. D: Biol. Crystallogr. / Year: 2018
Title: Real-space refinement in PHENIX for cryo-EM and crystallography
Authors: Li P / Gourdon P
History
DepositionApr 6, 2023-
Header (metadata) releaseOct 16, 2024-
Map releaseOct 16, 2024-
UpdateNov 20, 2024-
Current statusNov 20, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_17044.png

Downloads & links

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Map

FileDownload / File: emd_17044.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.85 Å/pix.
x 320 pix.
= 270.848 Å		0.85 Å/pix.
x 320 pix.
= 270.848 Å		0.85 Å/pix.
x 320 pix.
= 270.848 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.8464 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.13
Minimum - Maximum-0.7246241 - 1.1276898
Average (Standard dev.)0.0009527291 (±0.025518171)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 270.84802 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_17044_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_17044_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : CtSpf1 with AlF4 bound E2.Pi state

EntireName: CtSpf1 with AlF4 bound E2.Pi state
Components
  • Complex: CtSpf1 with AlF4 bound E2.Pi state
    • Complex: Unknown helix
      • Protein or peptide: Unknown helix
    • Complex: Cation-transporting ATPase-like protein
      • Protein or peptide: Cation-transporting ATPase-like protein
  • Ligand: TETRAFLUOROALUMINATE ION
  • Ligand: MAGNESIUM ION
  • Ligand: CALCIUM ION

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Supramolecule #1: CtSpf1 with AlF4 bound E2.Pi state

SupramoleculeName: CtSpf1 with AlF4 bound E2.Pi state / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2

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Supramolecule #2: Unknown helix

SupramoleculeName: Unknown helix / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)

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Supramolecule #3: Cation-transporting ATPase-like protein

SupramoleculeName: Cation-transporting ATPase-like protein / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Thermochaetoides thermophila (fungus)

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Macromolecule #1: Unknown helix

MacromoleculeName: Unknown helix / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 2.656265 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString:
(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)

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Macromolecule #2: Cation-transporting ATPase-like protein

MacromoleculeName: Cation-transporting ATPase-like protein / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Thermochaetoides thermophila (fungus)
Molecular weightTheoretical: 149.103156 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MAPLVDNPQI KSAELLRPLP LYQHAYVWPY VIVWPVFLRV YLTQELYDKY IGAQEWTFVW IISIVTFQTL TWLCTHWSVN LNALFTAKK ASSIEDAQLI KVIPVANAGA ADICKLVRDK VGDNKTNISF LFQKRRFLWY PERKAFSTLE FDIDAEPKPT L SKFQLSRG ...String:
MAPLVDNPQI KSAELLRPLP LYQHAYVWPY VIVWPVFLRV YLTQELYDKY IGAQEWTFVW IISIVTFQTL TWLCTHWSVN LNALFTAKK ASSIEDAQLI KVIPVANAGA ADICKLVRDK VGDNKTNISF LFQKRRFLWY PERKAFSTLE FDIDAEPKPT L SKFQLSRG IESEDELKRL EQHYGTNTFD IPVPTFTELF KEHAVAPFFV FQVFCVGLWL LDEYWYYSLF TLVMLVVFES TV VWQRQRT LTEFRSMSIK PYPIYVYRLG KWTEIQSDKL LPGDLVSVTR TKEDSGVACD MILVEGTAIV NEAMLSGEST PLL KDSIQL RPGDAVLEVD GLDKNSLLWG GTKVLQITHG TAEEERPKPA SGIPPPPDNG AMAVVTKTGF ETSQGSLVRT MIYS TERVS ANNTEALLFI LFLLVFALAA SWYVWDEGVR KDRKRSKLLL DCILIITSVV PPELPMELSL AVNTSLSALA KFAIF CTEP FRIPFAGRID VACFDKTGTL TGEDLVVEGI AGLGLGHSGT DTPKEADGAH TRMVSVHDAG METTLVLATA HALVKL DEG EIVGDPMEKA TLNALGWVLG KNDTLTSKPG NAASSGILGT VQIKRRFQFS SALKRQSSVA TITATEVKTG RKLRGSF VG VKGAPETIMK MLVTVPEHYE ETYKYFTRRG SRVLALAYKQ LTTEGELGAN KINDLKRESV EADLHFAGFL VLQCPLKE D AKQAVRMLNE SSHRVVMITG DNPLTAVHVA KEVEIVDRDV LILDAPEHSV YGEESLVWRS VDDKIRIDVD PTKPIDPEI LKTKDLCVTG YALNKFKGQV GWKSLLRYTW VYARVSPKQK EDILLGLKDM GYYTLMAGDG TNDVGALKQA HVGVALLNGT QEDLNRIAE HTRNQKMKEL YQKQVDLMAR WGQPPPPVPA MIAHLYPPGP SNPHYQKAME REAQKRGVTV EQLAKVNGTN V TSNPAGVQ QQSGQDAKKA KQVEAAKKAA NFADKLTSSL MEAEMDDEPP TLKLGDASVA APFTSKLRNV MAIPNILRQG RC TLVATIQ MYKILALNCL ISAYSLSVLY LEGIKFGDGQ ITISGMLMSV CFLSISRARS VEGLSKERPQ PNIFNFYIIG SIL GQFAVH VATLIYIAQL CDQIEPRTEV IDLEAEFKPS LLNSAVYLLQ LIQQISTFAV NYQGRPFRES LSENKGMFYG IVGV TAIAF ACSTEMLPEL NEAMKLVPFN ENFKTIMTTV MIIDFVACYV IEWVLKKLFS DLRARDIAER RPDQLERERV RKEKE AREK EEEEERKERE RIEAFERRLE EKRTRLVEAA AQREQQQQQW AQRR

UniProtKB: Cation-transporting ATPase-like protein

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Macromolecule #3: TETRAFLUOROALUMINATE ION

MacromoleculeName: TETRAFLUOROALUMINATE ION / type: ligand / ID: 3 / Number of copies: 1 / Formula: ALF
Molecular weightTheoretical: 102.975 Da
Chemical component information

ChemComp-ALF:
TETRAFLUOROALUMINATE ION

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #5: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 5 / Number of copies: 1 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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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

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.2 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.75 µm / Nominal defocus min: 0.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.48 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 185340
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: RANDOM ASSIGNMENT
FSC plot (resolution estimation)

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