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- PDB-8xit: Cryo-EM structure of sheep VMAT2 dimer in an atypical fold -

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Basic information

Entry
Database: PDB / ID: 8xit
TitleCryo-EM structure of sheep VMAT2 dimer in an atypical fold
ComponentsOaVMAT2-BRIL
KeywordsTRANSPORT PROTEIN / Vesicular monoamine transporter / SLC18A2 / MFS / Apo / Conformation
Biological speciesOvis aries (sheep)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsLyu, Y. / Fu, C. / Ma, H. / Sun, Z. / Su, Z. / Zhou, X.
Funding support China, 1items
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, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Aug 14, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: OaVMAT2-BRIL
B: OaVMAT2-BRIL


Theoretical massNumber of molelcules
Total (without water)137,1852
Polymers137,1852
Non-polymers00
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 OaVMAT2-BRIL


Mass: 68592.688 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Ovis aries (sheep) / Production host: Komagataella pastoris (fungus)

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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: OaVMAT2 TM8/9 loop replaced by BRIL / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Ovis aries (sheep)
Source (recombinant)Organism: Komagataella pastoris (fungus)
Buffer solutionpH: 7.4 / Details: 150 mm NaCl, 20 mM HEPES-Na pH 7.4, 0.4 mM DDM
SpecimenConc.: 8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse.
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: blot for 3 s before plunging

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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 defocus max: 1600 nm / Nominal defocus min: 1000 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 62.64 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameVersionCategory
7Coot0.9.3model fitting
9RELION4initial Euler assignment
10RELION4final Euler assignment
12cryoSPARC4.33D reconstruction
13PHENIX1.19.1model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1064611
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 304899 / Symmetry type: POINT
Atomic model buildingSource name: AlphaFold / Type: in silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0036124
ELECTRON MICROSCOPYf_angle_d0.6228296
ELECTRON MICROSCOPYf_dihedral_angle_d3.846826
ELECTRON MICROSCOPYf_chiral_restr0.04990
ELECTRON MICROSCOPYf_plane_restr0.0051012

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