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Yorodumi- PDB-8qmp: Structure of the E2 Beryllium Fluoride Complex of the Autoinhibit... -
+Open data
-Basic information
Entry | Database: PDB / ID: 8qmp | ||||||
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Title | Structure of the E2 Beryllium Fluoride Complex of the Autoinhibited Calcium ATPase ACA8 | ||||||
Components | Calcium-transporting ATPase 8, plasma membrane-type | ||||||
Keywords | TRANSPORT PROTEIN / HYDROLASE Calcium transporter P-type ATPase / HYDROLASE | ||||||
Function / homology | Function and homology information response to nematode / P-type Ca2+ transporter / P-type calcium transporter activity / plasmodesma / plastid / calmodulin binding / ATP hydrolysis activity / ATP binding / metal ion binding / plasma membrane Similarity search - Function | ||||||
Biological species | Arabidopsis thaliana (thale cress) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å | ||||||
Authors | Thirup Larsen, S. / Karlsen Dannersoe, J. / Nissen, P. | ||||||
Funding support | Denmark, 1items
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Citation | Journal: 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 |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8qmp.cif.gz | 342.3 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8qmp.ent.gz | 277.2 KB | Display | PDB format |
PDBx/mmJSON format | 8qmp.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 8qmp_validation.pdf.gz | 1.3 MB | Display | wwPDB validaton report |
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Full document | 8qmp_full_validation.pdf.gz | 1.3 MB | Display | |
Data in XML | 8qmp_validation.xml.gz | 39.9 KB | Display | |
Data in CIF | 8qmp_validation.cif.gz | 58.6 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/qm/8qmp ftp://data.pdbj.org/pub/pdb/validation_reports/qm/8qmp | HTTPS FTP |
-Related structure data
Related structure data | 18506MC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | 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 |
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#2: Chemical | ChemComp-MG / |
#3: Chemical | ChemComp-BEF / |
Has ligand of interest | N |
Has protein modification | Y |
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: ACA8 beryllium fluoride complex / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT | ||||||||||||||||||||||||||||||
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Molecular weight | Value: 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 solution | pH: 7.5 | ||||||||||||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 0.67 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | ||||||||||||||||||||||||||||||
Specimen support | Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: C-flat-1.2/1.3 | ||||||||||||||||||||||||||||||
Vitrification | Instrument: LEICA PLUNGER / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 283 K |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 1800 nm / Nominal defocus min: 800 nm |
Image recording | Electron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) |
-Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 941952 | ||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 193419 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||
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