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- EMDB-38389: Cryo-EM structure of sheep VMAT2 dimer in an atypical fold -

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Basic information

Entry
Database: EMDB / ID: EMD-38389
TitleCryo-EM structure of sheep VMAT2 dimer in an atypical fold
Map dataOaVMAT2 TM8/9 replaced by BRIL.
Sample
  • Complex: OaVMAT2 TM8/9 loop replaced by BRIL
    • Protein or peptide: OaVMAT2-BRIL
KeywordsVesicular monoamine transporter / SLC18A2 / MFS / Apo / Conformation / TRANSPORT PROTEIN
Biological speciesOvis aries (sheep)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 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_38389.png

Downloads & links

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Map

FileDownload / File: emd_38389.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationOaVMAT2 TM8/9 replaced by BRIL.
Voxel sizeX=Y=Z: 0.85 Å
Density
Contour LevelBy AUTHOR: 0.15
Minimum - Maximum-0.53700006 - 0.9610242
Average (Standard dev.)0.0017556006 (±0.024670253)
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_38389_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_38389_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 : OaVMAT2 TM8/9 loop replaced by BRIL

EntireName: OaVMAT2 TM8/9 loop replaced by BRIL
Components
  • Complex: OaVMAT2 TM8/9 loop replaced by BRIL
    • Protein or peptide: OaVMAT2-BRIL

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Supramolecule #1: OaVMAT2 TM8/9 loop replaced by BRIL

SupramoleculeName: OaVMAT2 TM8/9 loop replaced by BRIL / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Ovis aries (sheep)

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Macromolecule #1: OaVMAT2-BRIL

MacromoleculeName: OaVMAT2-BRIL / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Ovis aries (sheep)
Molecular weightTheoretical: 68.592688 KDa
Recombinant expressionOrganism: Komagataella pastoris (fungus)
SequenceString: MALSELALLR RLQESRHSRK LVLFIVFLAL LLDNMLLTVV VPIIPSYLYS IEHEKDALEI QTAKPGLTAS APGSFQNIFS YYDNSTMVT GNNTDHLQGP LVHEATTQRM VTNSSSAPSD CPSEDKDLLN ENVQVGLLFA SKATVQLLTN PFIGLLTNRI G YPIPMFTG ...String:
MALSELALLR RLQESRHSRK LVLFIVFLAL LLDNMLLTVV VPIIPSYLYS IEHEKDALEI QTAKPGLTAS APGSFQNIFS YYDNSTMVT GNNTDHLQGP LVHEATTQRM VTNSSSAPSD CPSEDKDLLN ENVQVGLLFA SKATVQLLTN PFIGLLTNRI G YPIPMFTG FCIMFISTVM FAFSRSYAFL LFARSLQGIG SSCSSVAGMG MLASVYTDDE ERGNAMGIAL GGLAMGVLVG PP FGSVLYE FVGKTAPFLV LAALVLLDGA IQLFVLQPSR VQPESQKGTP LTTLLRDPYI LIAAGSICFA NMGIAMLEPA LPI WMMETM CSHKWQLGVA FLPASISYLI GTNVFGILAR RQLADLEDNW ETLNDNLKVI EKADNAAQVK DALTKMRAAA LDAQ KATPP KLEDKSPDSP EMKDFRHGFD ILVGQIDDAL KLANEGKVKE AQAAAEQLKT TRNAYIQKYL ERARSTLRWL CALLG MIIV GMSILCIPLA KNIYGLIAPN FGVGFAIGMV DSSMMPIMGY LVDLRHVSVY GSVYAIADVA FCMGYAIGPS AGGAIA KAI GFPWLMTIIG IIDILFAPLC FFLRSPPAKE EKMAILMDHN CPIKTKMYTQ NSSQSHPIGE DEDSESD

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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: 7.4 / Details: 150 mm NaCl, 20 mM HEPES-Na pH 7.4, 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: 62.64 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: 1.0 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

Particle selectionNumber selected: 1064611
Startup modelType of model: OTHER / Details: AlphaFold-predicted OaVMAT2 model
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.3) / Number images used: 304899
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-8xit:
Cryo-EM structure of sheep VMAT2 dimer in an atypical fold

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