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- PDB-8y1x: Cryo-EM structure of the aspartate:alanine antiporter AspT WT -

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Basic information

Entry
Database: PDB / ID: 8y1x
TitleCryo-EM structure of the aspartate:alanine antiporter AspT WT
ComponentsAspartate/alanine antiporter
KeywordsTRANSPORT PROTEIN / amino acid / antiporter / elevator mechanism
Function / homologyYidE/YbjL duplication / Aspartate-alanine antiporter / Predicted Permease Membrane Region / : / antiporter activity / identical protein binding / plasma membrane / ASPARTIC ACID / Aspartate/alanine antiporter
Function and homology information
Biological speciesTetragenococcus halophilus (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.68 Å
AuthorsNanatani, K. / Kanno, R. / Kawabata, T. / Watanabe, S. / Hidaka, M. / Yamanaka, T. / Toda, K. / Fujiki, T. / Kunii, K. / Miyamoto, A. ...Nanatani, K. / Kanno, R. / Kawabata, T. / Watanabe, S. / Hidaka, M. / Yamanaka, T. / Toda, K. / Fujiki, T. / Kunii, K. / Miyamoto, A. / Chiba, F. / Ogasawara, S. / Murata, T. / Humbel, B.M. / Inaba, K. / Mitsuoka, K. / Guan, L. / Abe, K. / Yamamoto, M. / Koshiba, S.
Funding support Japan, 5items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)16K07653 Japan
Japan Society for the Promotion of Science (JSPS)20K05779 Japan
Japan Society for the Promotion of Science (JSPS)23K05004 Japan
Japan Agency for Medical Research and Development (AMED)JP23ama121019 Japan
Japan Agency for Medical Research and Development (AMED)JP23ama121038 Japan
CitationJournal: To Be Published
Title: Cryo-EM structure and molecular mechanism of the aspartate:alanine antiporter AspT from Tetragenococcus halophilus
Authors: Nanatani, K. / Kanno, R. / Kawabata, T. / Watanabe, S. / Hidaka, M. / Yamanaka, T. / Toda, K. / Fujiki, T. / Kunii, K. / Miyamoto, A. / Chiba, F. / Ogasawara, S. / Murata, T. / Humbel, B.M. ...Authors: Nanatani, K. / Kanno, R. / Kawabata, T. / Watanabe, S. / Hidaka, M. / Yamanaka, T. / Toda, K. / Fujiki, T. / Kunii, K. / Miyamoto, A. / Chiba, F. / Ogasawara, S. / Murata, T. / Humbel, B.M. / Inaba, K. / Mitsuoka, K. / Guan, L. / Abe, K. / Yamamoto, M. / Koshiba, S.
History
DepositionJan 25, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jan 29, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Aspartate/alanine antiporter
B: Aspartate/alanine antiporter
hetero molecules


Theoretical massNumber of molelcules
Total (without water)114,7764
Polymers114,5102
Non-polymers2662
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Aspartate/alanine antiporter


Mass: 57255.129 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Tetragenococcus halophilus (bacteria) / Strain: D10 / Gene: aspT / Plasmid: pTrc99a / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): C43 / References: UniProt: Q8L3K8
#2: Chemical ChemComp-ASP / ASPARTIC ACID


Type: L-peptide linking / Mass: 133.103 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C4H7NO4 / Feature type: SUBJECT OF INVESTIGATION
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: aspartate:alanine antiporter AspT / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.058 MDa / Experimental value: NO
Source (natural)Organism: Tetragenococcus halophilus (bacteria) / Strain: D10
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: C43(DE3) / Plasmid: pTrc99a
Buffer solutionpH: 6
Buffer component
IDConc.NameFormulaBuffer-ID
120 mM2-(N-morpholino)ethanesulfonic acidMES1
2100 mMsodium chlorideNaCl1
30.003 %Lauryl maltose neopentyl glycolLMNG1
4100 mML-aspartic acidL-Asp1
SpecimenConc.: 2.83 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 80 % / Chamber temperature: 277 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: 96000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 30.6 sec. / Electron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 8 / Num. of real images: 12357

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Processing

EM software
IDNameVersionCategory
1crYOLOparticle selection
2EPUimage acquisition
4RELIONCTF correction
7Cootmodel fitting
9RELIONinitial Euler assignment
10cryoSPARCfinal Euler assignment
11cryoSPARCclassification
12cryoSPARC3D reconstruction
13PHENIX1.20.1_4487:model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1636712
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.68 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 125465 / Symmetry type: POINT
Atomic model buildingProtocol: BACKBONE TRACE
Atomic model buildingSource name: AlphaFold / Type: in silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0038218
ELECTRON MICROSCOPYf_angle_d0.6511162
ELECTRON MICROSCOPYf_dihedral_angle_d15.4392876
ELECTRON MICROSCOPYf_chiral_restr0.0461386
ELECTRON MICROSCOPYf_plane_restr0.0041382

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