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- EMDB-18506: Structure of the E2 Beryllium Fluoride Complex of the Autoinhibit... -

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Basic information

Entry
Database: EMDB / ID: EMD-18506
TitleStructure of the E2 Beryllium Fluoride Complex of the Autoinhibited Calcium ATPase ACA8
Map dataThe ACA8 E2P state map.
Sample
  • Complex: ACA8 beryllium fluoride complex
    • Protein or peptide: Calcium-transporting ATPase 8, plasma membrane-type
  • Ligand: MAGNESIUM ION
  • Ligand: BERYLLIUM TRIFLUORIDE ION
KeywordsHYDROLASE Calcium transporter P-type ATPase / HYDROLASE / TRANSPORT PROTEIN
Function / homology
Function and homology information


P-type Ca2+ transporter / P-type calcium transporter activity / plasmodesma / response to nematode / plastid / calmodulin binding / ATP hydrolysis activity / ATP binding / metal ion binding / plasma membrane
Similarity search - Function
Calcium-transporting P-type ATPase, N-terminal autoinhibitory domain / Ca2+-ATPase N terminal autoinhibitory domain / P-type ATPase, subfamily IIB / 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 / P-type ATPase, cytoplasmic domain N / P-type ATPase actuator domain ...Calcium-transporting P-type ATPase, N-terminal autoinhibitory domain / Ca2+-ATPase N terminal autoinhibitory domain / P-type ATPase, subfamily IIB / 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 / P-type ATPase, cytoplasmic domain N / 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
Calcium-transporting ATPase 8, plasma membrane-type
Similarity search - Component
Biological speciesArabidopsis thaliana (thale cress)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsThirup Larsen S / Karlsen Dannersoe J / Nissen P
Funding support Denmark, 1 items
OrganizationGrant numberCountry
Danish Council for Independent Research Denmark
CitationJournal: J Mol Biol / Year: 2024
Title: Conserved N-terminal Regulation of the ACA8 Calcium Pump with Two Calmodulin Binding Sites.
Authors: Sigrid Thirup Larsen / Josephine Karlsen Dannersø / Christine Juul Fælled Nielsen / Lisbeth Rosager Poulsen / Michael Palmgren / Poul Nissen /
Abstract: The autoinhibited plasma membrane calcium ATPase ACA8 from A. thaliana has an N-terminal autoinhibitory domain. Binding of calcium-loaded calmodulin at two sites located at residues 42-62 and 74-96 ...The autoinhibited plasma membrane calcium ATPase ACA8 from A. thaliana has an N-terminal autoinhibitory domain. Binding of calcium-loaded calmodulin at two sites located at residues 42-62 and 74-96 relieves autoinhibition of ACA8 activity. Through activity studies and a yeast complementation assay we investigated wild-type (WT) and N-terminally truncated ACA8 constructs (Δ20, Δ30, Δ35, Δ37, Δ40, Δ74 and Δ100) to explore the role of conserved motifs in the N-terminal segment preceding the calmodulin binding sites. Furthermore, we purified WT, Δ20- and Δ100-ACA8, tested activity in vitro and performed structural studies of purified Δ20-ACA8 stabilized in a lipid nanodisc to explore the mechanism of autoinhibition. We show that an N-terminal segment between residues 20 and 35 including conserved Phe32, upstream of the calmodulin binding sites, is important for autoinhibition and the activation by calmodulin. Cryo-EM structure determination at 3.3 Å resolution of a beryllium fluoride inhibited E2 form, and at low resolution for an E1 state combined with AlphaFold prediction provide a model for autoinhibition, consistent with the mutational studies.
History
DepositionSep 25, 2023-
Header (metadata) releaseOct 2, 2024-
Map releaseOct 2, 2024-
UpdateOct 23, 2024-
Current statusOct 23, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_18506.png

Downloads & links

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Map

FileDownload / File: emd_18506.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThe ACA8 E2P state map.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.05 Å/pix.
x 256 pix.
= 269.152 Å		1.05 Å/pix.
x 256 pix.
= 269.152 Å		1.05 Å/pix.
x 256 pix.
= 269.152 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.05138 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.236
Minimum - Maximum-1.025403 - 1.5201544
Average (Standard dev.)-0.0009660641 (±0.026002193)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 269.152 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_18506_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: The low resolution density of ACA8 in the...

Fileemd_18506_additional_1.map
AnnotationThe low resolution density of ACA8 in the E1 state, with density for the autoinhibitory domain
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_18506_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_18506_half_map_2.map
Projections & Slices
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Histogram

Histogram (log scale)

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Sample components

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Entire : ACA8 beryllium fluoride complex

EntireName: ACA8 beryllium fluoride complex
Components
  • Complex: ACA8 beryllium fluoride complex
    • Protein or peptide: Calcium-transporting ATPase 8, plasma membrane-type
  • Ligand: MAGNESIUM ION
  • Ligand: BERYLLIUM TRIFLUORIDE ION

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Supramolecule #1: ACA8 beryllium fluoride complex

SupramoleculeName: ACA8 beryllium fluoride complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Molecular weightTheoretical: 116 KDa

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Macromolecule #1: Calcium-transporting ATPase 8, plasma membrane-type

MacromoleculeName: Calcium-transporting ATPase 8, plasma membrane-type / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: P-type Ca2+ transporter
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Molecular weightTheoretical: 114.233523 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: KSEHADSDSD TFYIPSKNAS IERLQQWRKA ALVLNASRRF RYTLDLKKEQ ETREMRQKIR SHAHALLAAN RFMDMGRESG VEKTTGPAT PAGDFGITPE QLVIMSKDHN SGALEQYGGT QGLANLLKTN PEKGISGDDD DLLKRKTIYG SNTYPRKKGK G FLRFLWDA ...String:
KSEHADSDSD TFYIPSKNAS IERLQQWRKA ALVLNASRRF RYTLDLKKEQ ETREMRQKIR SHAHALLAAN RFMDMGRESG VEKTTGPAT PAGDFGITPE QLVIMSKDHN SGALEQYGGT QGLANLLKTN PEKGISGDDD DLLKRKTIYG SNTYPRKKGK G FLRFLWDA CHDLTLIILM VAAVASLALG IKTEGIKEGW YDGGSIAFAV ILVIVVTAVS DYKQSLQFQN LNDEKRNIHL EV LRGGRRV EISIYDIVVG DVIPLNIGNQ VPADGVLISG HSLALDESSM TGESKIVNKD ANKDPFLMSG CKVADGNGSM LVT GVGVNT EWGLLMASIS EDNGEETPLQ VRLNGVATFI GSIGLAVAAA VLVILLTRYF TGHTKDNNGG PQFVKGKTKV GHVI DDVVK VLTVAVTIVV VAVPEGLPLA VTLTLAYSMR KMMADKALVR RLSACETMGS ATTICSDKTG TLTLNQMTVV ESYAG GKKT DTEQLPATIT SLVVEGISQN TTGSIFVPEG GGDLEYSGSP TEKAILGWGV KLGMNFETAR SQSSILHAFP FNSEKK RGG VAVKTADGEV HVHWKGASEI VLASCRSYID EDGNVAPMTD DKASFFKNGI NDMAGRTLRC VALAFRTYEA EKVPTGE EL SKWVLPEDDL ILLAIVGIKD PCRPGVKDSV VLCQNAGVKV RMVTGDNVQT ARAIALECGI LSSDADLSEP TLIEGKSF R EMTDAERDKI SDKISVMGRS SPNDKLLLVQ SLRRQGHVVA VTGDGTNDAP ALHEADIGLA MGIAGTEVAK ESSDIIILD DNFASVVKVV RWGRSVYANI QKFIQFQLTV NVAALVINVV AAISSGDVPL TAVQLLWVNL IMDTLGALAL ATEPPTDHLM GRPPVGRKE PLITNIMWRN LLIQAIYQVS VLLTLNFRGI SILGLEHEVH EHATRVKNTI IFNAFVLCQA FNEFNARKPD E KNIFKGVI KNRLFMGIIV ITLVLQVIIV EFLGKFASTT KLNWKQWLIC VGIGVISWPL ALVGKFIPVP AAPISNKLKV LK FWGKKKN SSGEGSL

UniProtKB: Calcium-transporting ATPase 8, plasma membrane-type

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Macromolecule #2: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 2 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #3: BERYLLIUM TRIFLUORIDE ION

MacromoleculeName: BERYLLIUM TRIFLUORIDE ION / type: ligand / ID: 3 / Number of copies: 1 / Formula: BEF
Molecular weightTheoretical: 66.007 Da
Chemical component information

ChemComp-BEF:
BERYLLIUM TRIFLUORIDE ION

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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

Concentration0.67 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
200.0 mMKClpotassium chloride
50.0 mMTris-HCl
1.0 mMEGTA
1.0 mMBeFberyllium fluoride
3.0 mMMgCl2magnesium chloride
GridModel: C-flat-1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 283 K / Instrument: LEICA PLUNGER

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 130000
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 941952
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 193419
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC
FSC plot (resolution estimation)

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