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- EMDB-51912: Bacterial LAT transporter BASC in complex with L-Ala and NB53 -

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Basic information

Entry
Database: EMDB / ID: EMD-51912
TitleBacterial LAT transporter BASC in complex with L-Ala and NB53
Map data
Sample
  • Complex: BASC-NB53 complex
    • Protein or peptide: Putative amino acid/polyamine transport protein
    • Protein or peptide: Nanobody NB53
  • Ligand: ALANINE
KeywordsTransporter / nanobody / aminoacid transporter / MEMBRANE PROTEIN
Function / homology: / L-amino acid transmembrane transporter activity / Amino acid/polyamine transporter I / Amino acid permease / membrane / Putative amino acid/polyamine transport protein
Function and homology information
Biological speciesCarnobacterium sp. (bacteria) / Carnobacterium sp. AT7 (bacteria) / Lama glama (llama)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsFort J / Palacin M
Funding support Spain, 2 items
OrganizationGrant numberCountry
Ministerio de Ciencia e Innovacion (MCIN)PID2021- 122802OB-I00 Spain
Generalitat de Catalunya2021 SGR 01281 Spain
CitationJournal: PNAS Nexus / Year: 2025
Title: The conserved lysine residue in transmembrane helix 5 is pivotal for the cytoplasmic gating of the L-amino acid transporters.
Authors: Joana Fort / Adrià Nicolàs-Aragó / Luca Maggi / Maria Martinez-Molledo / Despoina Kapiki / Paula González-Novoa / Patricia Gómez-Gejo / Niels Zijlstra / Susanna Bodoy / Els Pardon / Jan ...Authors: Joana Fort / Adrià Nicolàs-Aragó / Luca Maggi / Maria Martinez-Molledo / Despoina Kapiki / Paula González-Novoa / Patricia Gómez-Gejo / Niels Zijlstra / Susanna Bodoy / Els Pardon / Jan Steyaert / Oscar Llorca / Modesto Orozco / Thorben Cordes / Manuel Palacín /
Abstract: L-Amino acid transporters (LATs) play a key role in a wide range of physiological processes. Defects in LATs can lead to neurological disorders and aminoacidurias, while the overexpression of these ...L-Amino acid transporters (LATs) play a key role in a wide range of physiological processes. Defects in LATs can lead to neurological disorders and aminoacidurias, while the overexpression of these transporters is related to cancer. BasC is a bacterial LAT transporter with an APC fold. In this study, to monitor the cytoplasmic motion of BasC, we developed a single-molecule Förster resonance energy transfer assay that can characterize the conformational states of the intracellular gate in solution at room temperature. Based on combined biochemical and biophysical data and molecular dynamics simulations, we propose a model in which the conserved lysine residue in TM5 supports TM1a to explore both open and closed states within the cytoplasmic gate under apo conditions. This equilibrium can be altered by substrates, mutation of conserved lysine 154 in TM5, or a transport-blocking nanobody interacting with TM1a. Overall, these findings provide insights into the transport mechanism of BasC and highlight the significance of the lysine residue in TM5 in the cytoplasmic gating of LATs.
History
DepositionOct 26, 2024-
Header (metadata) releaseJan 1, 2025-
Map releaseJan 1, 2025-
UpdateFeb 19, 2025-
Current statusFeb 19, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_51912.png

Downloads & links

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Map

FileDownload / File: emd_51912.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.51 Å/pix.
x 512 pix.
= 261.12 Å		0.51 Å/pix.
x 512 pix.
= 261.12 Å		0.51 Å/pix.
x 512 pix.
= 261.12 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.51 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.017
Minimum - Maximum-0.046697933 - 0.10788762
Average (Standard dev.)0.000039861967 (±0.0018824129)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 261.12 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_51912_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_51912_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 : BASC-NB53 complex

EntireName: BASC-NB53 complex
Components
  • Complex: BASC-NB53 complex
    • Protein or peptide: Putative amino acid/polyamine transport protein
    • Protein or peptide: Nanobody NB53
  • Ligand: ALANINE

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Supramolecule #1: BASC-NB53 complex

SupramoleculeName: BASC-NB53 complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Carnobacterium sp. (bacteria)
Molecular weightTheoretical: 45 kDa/nm

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Macromolecule #1: Putative amino acid/polyamine transport protein

MacromoleculeName: Putative amino acid/polyamine transport protein / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Carnobacterium sp. AT7 (bacteria)
Molecular weightTheoretical: 46.696609 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MKEVSGITAL TVVVGTVIGA GIFFKPTAVY GAAGAPGLGL LAWFVAGIIT IAGGLTVAEI GTIYPQTGGM MIYLEKVYGR WLGFLVGWA QMVIYYPANI AALAIIFATQ FVNLFALSDS TIVPTAILTS IFLMGVNFLG TKYSGWIQTL ATILKLIPLV V IIVAGLLY ...String:
MKEVSGITAL TVVVGTVIGA GIFFKPTAVY GAAGAPGLGL LAWFVAGIIT IAGGLTVAEI GTIYPQTGGM MIYLEKVYGR WLGFLVGWA QMVIYYPANI AALAIIFATQ FVNLFALSDS TIVPTAILTS IFLMGVNFLG TKYSGWIQTL ATILKLIPLV V IIVAGLLY PGGGVIRLVP FSVETHPVLT SFGSALIATL FAYDGWINVG TLAGEMKNPG KMLPKVIIGG LSIVMAVYLL TN IAYLFVL DSSQLAGTDT PAALVASHLF EGIGSKLVTI GILISVFGGI NGYIISGLRV PYALATQKML PFSDWFARIN PKT NLPING GLVMLGIAIV MILTGQFNQL TDLIVFVIWF FITLTFIAVI ILRKTQPDIE RPYRVPFYPV IPLIAIIGGL YIIF NTLIV QPKNAFIGIL LTLIGIPIYF YCKKKYGSPE

UniProtKB: Putative amino acid/polyamine transport protein

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Macromolecule #2: Nanobody NB53

MacromoleculeName: Nanobody NB53 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Lama glama (llama)
Molecular weightTheoretical: 14.257789 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MAQVQLVESG GGLVQAGGSL RLSCAASGIA FSRMSMAWYR QDPGKQRALV ARITNDGSTY YDDSVKGRFT ISRDNAKNTV HLQMNSLKP EDTAVYYCNA QLVAWSENYW GQGTQVTVSS HHHHHHEPEA

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Macromolecule #3: ALANINE

MacromoleculeName: ALANINE / type: ligand / ID: 3 / Number of copies: 1 / Formula: ALA
Molecular weightTheoretical: 89.093 Da
Chemical component information

ChemComp-ALA:
ALANINE

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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

Concentration2.9 mg/mL
BufferpH: 7.4
GridModel: C-flat-1.2/1.3 / Material: GRAPHENE OXIDE / Mesh: 300
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 38963 / Average exposure time: 0.022 sec. / Average electron dose: 1.04 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.4 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Details32312 images selected after Ctfs and Motion correct.
Particle selectionNumber selected: 2000000
Details: particle picked using CS template picker with low resolution volume abtained previously
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6f2w

Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v4.4.1) / Software - details: NU refinement / Number images used: 324910
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cryoSPARC (ver. v4.4.1) / Software - details: Ab-initio
Final angle assignmentType: PROJECTION MATCHING / Software - Name: cryoSPARC (ver. v4.4.1) / Software - details: NU-refinement
Final 3D classificationNumber classes: 4 / Avg.num./class: 320000 / Software - Name: cryoSPARC (ver. v4.4.1) / Software - details: 3D classification

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