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- EMDB-38390: Cryo-EM structure of a frog VMAT2 in an apo conformation -

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Basic information

Entry
Database: EMDB / ID: EMD-38390
TitleCryo-EM structure of a frog VMAT2 in an apo conformation
Map dataXlVMAT2 WT.
Sample
  • Complex: XlVMAT2 WT
    • Protein or peptide: XlVMAT2
KeywordsVesicular monoamine transporter / SLC18A2 / MFS / fold / TRANSPORT PROTEIN
Biological speciesXenopus laevis (African clawed frog)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.0 Å
AuthorsLyu Y / Fu C / Ma H / Sun Z / Su Z / Zhou X
Funding support China, 1 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)31770783 China
CitationJournal: Nat Commun / Year: 2024
Title: Engineering of a mammalian VMAT2 for cryo-EM analysis results in non-canonical protein folding.
Authors: Ying Lyu / Chunting Fu / Haiyun Ma / Zhaoming Su / Ziyi Sun / Xiaoming Zhou /
Abstract: Vesicular monoamine transporter 2 (VMAT2) belongs to the major facilitator superfamily (MFS), and mediates cytoplasmic monoamine packaging into presynaptic vesicles. Here, we present two cryo-EM ...Vesicular monoamine transporter 2 (VMAT2) belongs to the major facilitator superfamily (MFS), and mediates cytoplasmic monoamine packaging into presynaptic vesicles. Here, we present two cryo-EM structures of VMAT2, with a frog VMAT2 adopting a canonical MFS fold and an engineered sheep VMAT2 adopting a non-canonical fold. Both VMAT2 proteins mediate uptake of a selective fluorescent VMAT2 substrate into cells. Molecular docking, substrate binding and transport analysis reveal potential substrate binding mechanism in VMAT2. Meanwhile, caution is advised when interpreting engineered membrane protein structures.
History
DepositionDec 20, 2023-
Header (metadata) releaseAug 14, 2024-
Map releaseAug 14, 2024-
UpdateAug 14, 2024-
Current statusAug 14, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_38390.png

Downloads & links

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Map

FileDownload / File: emd_38390.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationXlVMAT2 WT.
Voxel sizeX=Y=Z: 0.85 Å
Density
Contour LevelBy AUTHOR: 0.1
Minimum - Maximum-0.4508062 - 0.65106213
Average (Standard dev.)0.00073189446 (±0.014960106)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 217.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half map B.

Fileemd_38390_half_map_1.map
AnnotationHalf map B.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A.

Fileemd_38390_half_map_2.map
AnnotationHalf map A.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : XlVMAT2 WT

EntireName: XlVMAT2 WT
Components
  • Complex: XlVMAT2 WT
    • Protein or peptide: XlVMAT2

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Supramolecule #1: XlVMAT2 WT

SupramoleculeName: XlVMAT2 WT / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Xenopus laevis (African clawed frog)

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Macromolecule #1: XlVMAT2

MacromoleculeName: XlVMAT2 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Xenopus laevis (African clawed frog)
Molecular weightTheoretical: 55.624984 KDa
Recombinant expressionOrganism: Komagataella pastoris (fungus)
SequenceString: MSMTEFTLLR WLREGRQSRK LILLIVFIAL LLDNMLLTVV VPIIPSYLYS MTHESNNTHD GSIPPAAPSG FHSIFSYYDN TTVITTNFT SSDQLQQSAL TPTTSPSLAT PPASADCPKA DSSLLNENVK VGLLFASKAT VQLLTNPFIG PMTNRIGYQI P MFAGFCIM ...String:
MSMTEFTLLR WLREGRQSRK LILLIVFIAL LLDNMLLTVV VPIIPSYLYS MTHESNNTHD GSIPPAAPSG FHSIFSYYDN TTVITTNFT SSDQLQQSAL TPTTSPSLAT PPASADCPKA DSSLLNENVK VGLLFASKAT VQLLTNPFIG PMTNRIGYQI P MFAGFCIM FVSTIMFAFS GTYTLLFIAR SLQGIGSSCS SVAGMGMLAS VYTDDEERGN AMGVALGGLA MGVLVGPPFG SI LYEFVGK TAPFIVLAVL VLFDGALQLF VLQPSRVQPE SQTGTPLLTL IRDPYILIAA GSICFANMAI AMLEPALPIW MME TMCSRK WQLGVAFLPA SISYLLGTNI FGPLAHKMGR WLCAFIGMIM VGISIICVPF ARNIYGLIAP NFGVGFAIGM VDSS MMPIM GYLVDLRHVS VYGSVYAIAD VAFCMGFAFG PSAGGAIAKS IGFPWLMTII GVVDILFAPL CLFLRSPPAR EEKMA ILMD HKCPVKTKMY TQNSGQPYYT GEEEESESDE

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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

Concentration8.0 mg/mL
BufferpH: 6 / Details: 150 mm NaCl, 20 mM MES-Na pH 6.0, 0.4 mM DDM
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: blot for 3 s before plunging.
DetailsThis sample was monodisperse.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.6 µm / Nominal defocus min: 0.9 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER / Details: AlphaFold-predicted XlVMAT2 model
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.3) / Number images used: 107961
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4.0)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 4.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
Output model

PDB-8xiu:
Cryo-EM structure of a frog VMAT2 in an apo conformation

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