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- PDB-8op8: Cryo-EM structure of P5A-ATPase CtSpf1 (E2.Pi state with endogeno... -

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Basic information

Entry
Database: PDB / ID: 8op8
TitleCryo-EM structure of P5A-ATPase CtSpf1 (E2.Pi state with endogenous helix cargo bound)
Components
  • Cation-transporting ATPase-like protein
  • Unknown helix
KeywordsMEMBRANE PROTEIN / translocase
Function / homology
Function and homology information


membrane protein dislocase activity / P-type ion transporter activity / ATPase-coupled monoatomic cation transmembrane transporter activity / intracellular calcium ion homeostasis / endoplasmic reticulum membrane / ATP hydrolysis activity / ATP binding / metal ion binding
Similarity search - Function
: / : / P5A-ATPase, transmembrane helical hairpin / P-type ATPase, subfamily V / P-type ATPase actuator domain / 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 ...: / : / P5A-ATPase, transmembrane helical hairpin / P-type ATPase, subfamily V / P-type ATPase actuator domain / 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
TETRAFLUOROALUMINATE ION / Cation-transporting ATPase-like protein
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Thermochaetoides thermophila (fungus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.48 Å
AuthorsLi, P. / Gourdon, P.
Funding support Sweden, Denmark, 4items
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, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 16, 2024Provider: repository / Type: Initial release
Revision 1.1Nov 20, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
B: Unknown helix
A: Cation-transporting ATPase-like protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)151,9275
Polymers151,7592
Non-polymers1673
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein/peptide Unknown helix


Mass: 2656.265 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Production host: Saccharomyces cerevisiae (brewer's yeast)
#2: Protein Cation-transporting ATPase-like protein


Mass: 149103.156 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermochaetoides thermophila (fungus) / Gene: CTHT_0032200 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: G0S4Z4
#3: Chemical ChemComp-ALF / TETRAFLUOROALUMINATE ION


Mass: 102.975 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: AlF4 / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION
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

Component
IDNameTypeEntity IDParent-IDSource
1CtSpf1 with AlF4 bound E2.Pi stateCOMPLEX#1-#20RECOMBINANT
2Unknown helixCOMPLEX#11RECOMBINANT
3Cation-transporting ATPase-like proteinCOMPLEX#21RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Saccharomyces cerevisiae (brewer's yeast)4932
33Thermochaetoides thermophila (fungus)209285
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Saccharomyces cerevisiae (brewer's yeast)4932
33Saccharomyces cerevisiae (brewer's yeast)4932
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2750 nm / Nominal defocus min: 500 nm
Image recordingElectron dose: 50.2 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.20.1_4487refinement
PHENIX1.20.1_4487refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.48 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 185340 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 131.04 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00259452
ELECTRON MICROSCOPYf_angle_d0.514512828
ELECTRON MICROSCOPYf_chiral_restr0.0421500
ELECTRON MICROSCOPYf_plane_restr0.00441615
ELECTRON MICROSCOPYf_dihedral_angle_d3.03441273

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