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- PDB-8ztk: AtALMT9 with LMNG (cis2 class) -

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Basic information

Entry
Database: PDB / ID: 8ztk
TitleAtALMT9 with LMNG (cis2 class)
ComponentsAluminum-activated malate transporter 9
KeywordsMEMBRANE PROTEIN / Plant / Stomata / Vacuole / ALMT / Ion channel
Function / homologymalate transport / Aluminum-activated malate transporter / Aluminium activated malate transporter / plant-type vacuole membrane / monoatomic anion channel activity / Chem-LPP / Aluminum-activated malate transporter 9
Function and homology information
Biological speciesArabidopsis thaliana (thale cress)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.78 Å
AuthorsLee, Y. / Lee, S.
Funding support Korea, Republic Of, 3items
OrganizationGrant numberCountry
National Research Foundation (NRF, Korea)2022R1A2C100988211 Korea, Republic Of
National Research Foundation (NRF, Korea)RS-2023-00223552 Korea, Republic Of
National Research Foundation (NRF, Korea)2021M3A9I4022936 Korea, Republic Of
CitationJournal: Nat Commun / Year: 2025
Title: Structural basis for malate-driven, pore lipid-regulated activation of the Arabidopsis vacuolar anion channel ALMT9.
Authors: Yeongmok Lee / Elsa Demes-Causse / Jaemin Yoo / Seo Young Jang / Seoyeon Jung / Justyna Jaślan / Geum-Sook Hwang / Jejoong Yoo / Alexis De Angeli / Sangho Lee /
Abstract: In plant cells, ALMTs are key plasma and vacuolar membrane-localized anion channels regulating plant responses to the environment. Vacuolar ALMTs control anion accumulation in plant cells and, in ...In plant cells, ALMTs are key plasma and vacuolar membrane-localized anion channels regulating plant responses to the environment. Vacuolar ALMTs control anion accumulation in plant cells and, in guard cells, they regulate stomata aperture. The activation of vacuolar ALMTs depends on voltage and cytosolic malate, but the underlying molecular mechanisms remain elusive. Here we report the cryo-EM structures of ALMT9 from Arabidopsis thaliana (AtALMT9), a malate-activated vacuolar anion channel, in plugged and unplugged lipid-bound states. In all these states, membrane lipids interact with the ion conduction pathway of AtALMT9. We identify two unplugged states presenting two distinct pore width profiles. Combining structural and functional analysis we identified conserved residues involved in ion conduction and in the pore lipid interaction. Molecular dynamics simulations revealed a peculiar anion conduction mechanism in AtALMT9. We propose a voltage-dependent activation mechanism based on the competition between pore lipids and malate at the cytosolic entrance of the channel.
History
DepositionJun 7, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Mar 12, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Aluminum-activated malate transporter 9
B: Aluminum-activated malate transporter 9
hetero molecules


Theoretical massNumber of molelcules
Total (without water)135,5404
Polymers134,2422
Non-polymers1,2982
Water543
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Aluminum-activated malate transporter 9 / AtALMT9


Mass: 67121.008 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Arabidopsis thaliana (thale cress) / Gene: ALMT9, At3g18440, MYF24.16 / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q9LS46
#2: Chemical ChemComp-LPP / 2-(HEXADECANOYLOXY)-1-[(PHOSPHONOOXY)METHYL]ETHYL HEXADECANOATE / 1,2-DIPALMITOYL-SN-GLYCERO-3-PHOSPHATE / L-B,G-DIPALMITOYL-A-PHOSPHATIDIC ACID DISODIUM SALT / 3-SN-PHOSPHATIDIC ACID / 1,2-DIPALMITOYLDISODIUM SALT


Mass: 648.891 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C35H69O8P / Feature type: SUBJECT OF INVESTIGATION / Comment: phospholipid*YM
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Formula: H2O
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

ComponentName: AtALMT9 with LMNG / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.13 MDa / Experimental value: NO
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm) / Strain: Sf9
Buffer solutionpH: 7.6
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESC8H18N2O4S1
2150 mMSodium chlorideNaCl1
31 mMTCEPC9H15O6P1
40.005 % (w/v)LMNGC47H88O221
510 mMMalateC4H6O51
SpecimenConc.: 1.9 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Au-flat 1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 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: 105000 X / Nominal defocus max: 1900 nm / Nominal defocus min: 700 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 18 eV

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Processing

EM software
IDNameVersionCategory
1Topaz0.2.5aparticle selection
4cryoSPARC4.4CTF correction
7Coot0.9.8.1model fitting
9PHENIX1.20.1model refinement
10cryoSPARC4.4initial Euler assignment
11cryoSPARC4.4final Euler assignment
12cryoSPARC4.4classification
13cryoSPARC4.43D reconstruction
CTF correctionDetails: Patch CTF estimation / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2895997
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.78 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 76531 / Algorithm: FOURIER SPACE / Num. of class averages: 2 / Symmetry type: POINT
RefinementCross valid method: NONE

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