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- EMDB-33713: CryoEM structure of SPCA1a in E1-Ca-AMPPCP state subclass 3 -

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Basic information

Entry
Database: EMDB / ID: EMD-33713
TitleCryoEM structure of SPCA1a in E1-Ca-AMPPCP state subclass 3
Map dataprimary map for SPCA1a in E1-Ca-AMPPCP state, subclass3
Sample
  • Complex: A complex of SPCA1a and Megabody in E1-Ca-AMPPCP state, subclass3
    • Complex: SPCA1a
      • Protein or peptide: Calcium-transporting ATPase type 2C member 1
    • Complex: Megabody
      • Protein or peptide: Nanobody head piece of megabody
  • Ligand: PHOSPHOMETHYLPHOSPHONIC ACID ADENYLATE ESTER
  • Ligand: CALCIUM IONCalcium
Function / homology
Function and homology information


Golgi calcium ion homeostasis / Golgi calcium ion transport / P-type manganese transporter activity / trans-Golgi network membrane organization / manganese ion transport / intracellular manganese ion homeostasis / P-type Ca2+ transporter / P-type calcium transporter activity / calcium-dependent cell-cell adhesion via plasma membrane cell adhesion molecules / positive regulation of Golgi to plasma membrane protein transport ...Golgi calcium ion homeostasis / Golgi calcium ion transport / P-type manganese transporter activity / trans-Golgi network membrane organization / manganese ion transport / intracellular manganese ion homeostasis / P-type Ca2+ transporter / P-type calcium transporter activity / calcium-dependent cell-cell adhesion via plasma membrane cell adhesion molecules / positive regulation of Golgi to plasma membrane protein transport / cis-Golgi network membrane / Golgi cisterna membrane / Ion transport by P-type ATPases / epidermis development / calcium ion transmembrane transport / trans-Golgi network / intracellular calcium ion homeostasis / calcium ion transport / manganese ion binding / actin cytoskeleton organization / positive regulation of canonical NF-kappaB signal transduction / Golgi membrane / calcium ion binding / Golgi apparatus / endoplasmic reticulum / ATP hydrolysis activity / ATP binding / membrane / metal ion binding / plasma membrane
Similarity search - Function
P-type ATPase, subfamily IIA, PMR1-type / Cation-transporting P-type ATPase, C-terminal / Cation transporting ATPase, C-terminus / Cation transporter/ATPase, N-terminus / Cation-transporting P-type ATPase, N-terminal / Cation transporter/ATPase, N-terminus / Cation transport ATPase (P-type) / E1-E2 ATPase / P-type ATPase, haloacid dehalogenase domain / P-type ATPase, phosphorylation site ...P-type ATPase, subfamily IIA, PMR1-type / Cation-transporting P-type ATPase, C-terminal / Cation transporting ATPase, C-terminus / Cation transporter/ATPase, N-terminus / Cation-transporting P-type ATPase, N-terminal / Cation transporter/ATPase, N-terminus / Cation transport ATPase (P-type) / 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
Calcium-transporting ATPase type 2C member 1
Similarity search - Component
Biological speciesHomo sapiens (human) / Vicugna pacos (alpaca)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.14 Å
AuthorsChen Z / Watanabe S / Inaba K
Funding support Japan, 2 items
OrganizationGrant numberCountry
Japan Agency for Medical Research and Development (AMED)21gm1410006h0001, JP19am0101115 Japan
Japan Society for the Promotion of Science (JSPS)18H03978, 21H04758, 21H05247, 21K15036 Japan
CitationJournal: Sci Adv / Year: 2023
Title: Cryo-EM structures of human SPCA1a reveal the mechanism of Ca/Mn transport into the Golgi apparatus.
Authors: Zhenghao Chen / Satoshi Watanabe / Hironori Hashida / Michio Inoue / Yasukazu Daigaku / Masahide Kikkawa / Kenji Inaba /
Abstract: Secretory pathway Ca/Mn ATPase 1 (SPCA1) actively transports cytosolic Ca and Mn into the Golgi lumen, playing a crucial role in cellular calcium and manganese homeostasis. Detrimental mutations of ...Secretory pathway Ca/Mn ATPase 1 (SPCA1) actively transports cytosolic Ca and Mn into the Golgi lumen, playing a crucial role in cellular calcium and manganese homeostasis. Detrimental mutations of the gene encoding SPCA1 cause Hailey-Hailey disease. Here, using nanobody/megabody technologies, we determined cryo-electron microscopy structures of human SPCA1a in the ATP and Ca/Mn-bound (E1-ATP) state and the metal-free phosphorylated (E2P) state at 3.1- to 3.3-Å resolutions. The structures revealed that Ca and Mn share the same metal ion-binding pocket with similar but notably different coordination geometries in the transmembrane domain, corresponding to the second Ca-binding site in sarco/endoplasmic reticulum Ca-ATPase (SERCA). In the E1-ATP to E2P transition, SPCA1a undergoes similar domain rearrangements to those of SERCA. Meanwhile, SPCA1a shows larger conformational and positional flexibility of the second and sixth transmembrane helices, possibly explaining its wider metal ion specificity. These structural findings illuminate the unique mechanisms of SPCA1a-mediated Ca/Mn transport.
History
DepositionJun 28, 2022-
Header (metadata) releaseMar 22, 2023-
Map releaseMar 22, 2023-
UpdateMar 22, 2023-
Current statusMar 22, 2023Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_33713.png

Downloads & links

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Map

FileDownload / File: emd_33713.map.gz / Format: CCP4 / Size: 34.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationprimary map for SPCA1a in E1-Ca-AMPPCP state, subclass3
Voxel sizeX=Y=Z: 0.985 Å
Density
Contour LevelBy AUTHOR: 0.3188
Minimum - Maximum-1.6275156 - 2.4282079
Average (Standard dev.)0.004968397 (±0.04942527)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions208208208
Spacing208208208
CellA=B=C: 204.88 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_33713_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_33713_half_map_1.map
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: #1

Fileemd_33713_half_map_2.map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Sample components

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Entire : A complex of SPCA1a and Megabody in E1-Ca-AMPPCP state, subclass3

EntireName: A complex of SPCA1a and Megabody in E1-Ca-AMPPCP state, subclass3
Components
  • Complex: A complex of SPCA1a and Megabody in E1-Ca-AMPPCP state, subclass3
    • Complex: SPCA1a
      • Protein or peptide: Calcium-transporting ATPase type 2C member 1
    • Complex: Megabody
      • Protein or peptide: Nanobody head piece of megabody
  • Ligand: PHOSPHOMETHYLPHOSPHONIC ACID ADENYLATE ESTER
  • Ligand: CALCIUM IONCalcium

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Supramolecule #1: A complex of SPCA1a and Megabody in E1-Ca-AMPPCP state, subclass3

SupramoleculeName: A complex of SPCA1a and Megabody in E1-Ca-AMPPCP state, subclass3
type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1-#2

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Supramolecule #2: SPCA1a

SupramoleculeName: SPCA1a / type: complex / ID: 2 / Chimera: Yes / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: Megabody

SupramoleculeName: Megabody / type: complex / ID: 3 / Chimera: Yes / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Vicugna pacos (alpaca)

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Macromolecule #1: Calcium-transporting ATPase type 2C member 1

MacromoleculeName: Calcium-transporting ATPase type 2C member 1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: P-type Ca2+ transporter
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 103.574203 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MGGVAMPGAE DDVVRENLYF QGKDGLAAMK VARFQKIPNG ENETMIPVLT SKKASELPVS EVASILQADL QNGLNKCEVS HRRAFHGWN EFDISEDEPL WKKYISQFKN PLIMLLLASA VISVLMHQFD DAVSITVAIL IVVTVAFVQE YRSEKSLEEL S KLVPPECH ...String:
MGGVAMPGAE DDVVRENLYF QGKDGLAAMK VARFQKIPNG ENETMIPVLT SKKASELPVS EVASILQADL QNGLNKCEVS HRRAFHGWN EFDISEDEPL WKKYISQFKN PLIMLLLASA VISVLMHQFD DAVSITVAIL IVVTVAFVQE YRSEKSLEEL S KLVPPECH CVREGKLEHT LARDLVPGDT VCLSVGDRVP ADLRLFEAVD LSIDESSLTG ETTPCSKVTA PQPAATNGDL AS RSNIAFM GTLVRCGKAK GVVIGTGENS EFGEVFKMMQ AEEAPKTPLQ KSMDLLGKQL SFYSFGIIGI IMLVGWLLGK DIL EMFTIS VSLAVAAIPE GLPIVVTVTL ALGVMRMVKK RAIVKKLPIV ETLGCCNVIC SDKTGTLTKN EMTVTHIFTS DGLH AEVTG VGYNQFGEVI VDGDVVHGFY NPAVSRIVEA GCVCNDAVIR NNTLMGKPTE GALIALAMKM GLDGLQQDYI RKAEY PFSS EQKWMAVKCV HRTQQDRPEI CFMKGAYEQV IKYCTTYQSK GQTLTLTQQQ RDVYQQEKAR MGSAGLRVLA LASGPE LGQ LTFLGLVGII DPPRTGVKEA VTTLIASGVS IKMITGDSQE TAVAIASRLG LYSKTSQSVS GEEIDAMDVQ QLSQIVP KV AVFYRASPRH KMKIIKSLQK NGSVVAMTGD GVNDAVALKA ADIGVAMGQT GTDVCKEAAD MILVDDDFQT IMSAIEEG K GIYNNIKNFV RFQLSTSIAA LTLISLATLM NFPNPLNAMQ ILWINIIMDG PPAQSLGVEP VDKDVIRKPP RNWKDSILT KNLILKILVS SIIIVCGTLF VFWRELRDNV ITPRDTTMTF TCFVFFDMFN ALSSRSQTKS VFEIGLCSNR MFCYAVLGSI MGQLLVIYF PPLQKVFQTE SLSILDLLFL LGLTSSVCIV AEIIKKVERS REKIQKHVSS TSSSFLEV

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Macromolecule #2: Nanobody head piece of megabody

MacromoleculeName: Nanobody head piece of megabody / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Vicugna pacos (alpaca)
Molecular weightTheoretical: 13.900263 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
QVQLQESGGG LVQAGGSLRL SCAASGSIFG ADWMGWYRQA PGKEREFVAG IGHGASTYYA DSVKGRFTIS RDNAKNTVYL QMNSLKPED TAVYYCAVQY TQGWSGQYRS YDSLLYWGQG TQVTVSSGS

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Macromolecule #3: PHOSPHOMETHYLPHOSPHONIC ACID ADENYLATE ESTER

MacromoleculeName: PHOSPHOMETHYLPHOSPHONIC ACID ADENYLATE ESTER / type: ligand / ID: 3 / Number of copies: 1 / Formula: ACP
Molecular weightTheoretical: 505.208 Da
Chemical component information

ChemComp-ACP:
PHOSPHOMETHYLPHOSPHONIC ACID ADENYLATE ESTER / AMP-PCP, energy-carrying molecule analogue*YM

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Macromolecule #4: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 4 / 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: 6
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeJEOL CRYO ARM 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1.6 µm / Nominal defocus min: 0.8 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.14 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 102741
FSC plot (resolution estimation)

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