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- PDB-8qmp: Structure of the E2 Beryllium Fluoride Complex of the Autoinhibit... -

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Basic information

Entry
Database: PDB / ID: 8qmp
TitleStructure of the E2 Beryllium Fluoride Complex of the Autoinhibited Calcium ATPase ACA8
ComponentsCalcium-transporting ATPase 8, plasma membrane-type
KeywordsTRANSPORT PROTEIN / HYDROLASE Calcium transporter P-type ATPase / HYDROLASE
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
BERYLLIUM TRIFLUORIDE ION / Calcium-transporting ATPase 8, plasma membrane-type
Similarity search - Component
Biological speciesArabidopsis thaliana (thale cress)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsThirup Larsen, S. / Karlsen Dannersoe, J. / Nissen, P.
Funding support Denmark, 1items
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, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 2, 2024Provider: repository / Type: Initial release
Revision 1.1Oct 23, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Calcium-transporting ATPase 8, plasma membrane-type
hetero molecules


Theoretical massNumber of molelcules
Total (without water)114,3243
Polymers114,2341
Non-polymers902
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area280 Å2
ΔGint-7 kcal/mol
Surface area39340 Å2
MethodPISA

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Components

#1: Protein Calcium-transporting ATPase 8, plasma membrane-type / Ca(2+)-ATPase isoform 8


Mass: 114233.523 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Arabidopsis thaliana (thale cress) / Gene: ACA8, At5g57110, MUL3.5 / Production host: Saccharomyces cerevisiae (brewer's yeast) / Strain (production host): K616 / References: UniProt: Q9LF79, P-type Ca2+ transporter
#2: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#3: Chemical ChemComp-BEF / BERYLLIUM TRIFLUORIDE ION


Mass: 66.007 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: BeF3
Has ligand of interestN
Has protein modificationY

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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

ComponentName: ACA8 beryllium fluoride complex / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.116 MDa / Experimental value: NO
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Source (recombinant)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: K616 / Plasmid: pYES2
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
1200 mMpotassium chlorideKCl1
250 mMTris-HCl1
31 mMEGTA1
41 mMberyllium fluorideBeF1
53 mMmagnesium chlorideMgCl21
SpecimenConc.: 0.67 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: LEICA PLUNGER / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 283 K

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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 magnification: 130000 X / Nominal defocus max: 1800 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2PHENIX1.20.1_4487:model refinement
3EPUimage acquisition
5cryoSPARCCTF correction
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
12cryoSPARCclassification
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 941952
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 193419 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0037042
ELECTRON MICROSCOPYf_angle_d0.589557
ELECTRON MICROSCOPYf_dihedral_angle_d4.658964
ELECTRON MICROSCOPYf_chiral_restr0.0411154
ELECTRON MICROSCOPYf_plane_restr0.0061214

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