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- EMDB-21986: Inward-facing Apo-open state of the glutamate transporter homolog... -

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Basic information

Entry
Database: EMDB / ID: EMD-21986
TitleInward-facing Apo-open state of the glutamate transporter homologue GltPh
Map data
SampleComplex of inward-facing Apo-open state of GltPh in MSP1E3 nanodisc:
Glutamate transporter homologue GltPh / ligand
Biological speciesPyrococcus horikoshii (archaea)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.52 Å
AuthorsWang X / Boudker O
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)R37NS085318 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)R01NS064357 United States
CitationJournal: Elife / Year: 2020
Title: Large domain movements through the lipid bilayer mediate substrate release and inhibition of glutamate transporters.
Authors: Xiaoyu Wang / Olga Boudker /
Abstract: Glutamate transporters are essential players in glutamatergic neurotransmission in the brain, where they maintain extracellular glutamate below cytotoxic levels and allow for rounds of transmission. ...Glutamate transporters are essential players in glutamatergic neurotransmission in the brain, where they maintain extracellular glutamate below cytotoxic levels and allow for rounds of transmission. The structural bases of their function are well established, particularly within a model archaeal homologue, sodium and aspartate symporter Glt. However, the mechanism of gating on the cytoplasmic side of the membrane remains ambiguous. We report Cryo-EM structures of Glt reconstituted into nanodiscs, including those structurally constrained in the cytoplasm-facing state and either apo, bound to sodium ions only, substrate, or blockers. The structures show that both substrate translocation and release involve movements of the bulky transport domain through the lipid bilayer. They further reveal a novel mode of inhibitor binding and show how solutes release is coupled to protein conformational changes. Finally, we describe how domain movements are associated with the displacement of bound lipids and significant membrane deformations, highlighting the potential regulatory role of the bilayer.
Validation ReportPDB-ID: 6x12

SummaryFull reportAbout validation report
History
DepositionMay 18, 2020-
Header (metadata) releaseNov 18, 2020-
Map releaseNov 18, 2020-
UpdateNov 18, 2020-
Current statusNov 18, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0388
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.0388
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6x12
  • Surface level: 0.0388
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_21986.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 256 pix.
= 274.752 Å
1.07 Å/pix.
x 256 pix.
= 274.752 Å
1.07 Å/pix.
x 256 pix.
= 274.752 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.07325 Å
Density
Contour LevelBy AUTHOR: 0.0388 / Movie #1: 0.0388
Minimum - Maximum-0.13179386 - 0.23327138
Average (Standard dev.)0.0000879221 (±0.0030091533)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 274.752 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.073251.073251.07325
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z274.752274.752274.752
α/β/γ90.00090.00090.000
start NX/NY/NZ714763
NX/NY/NZ169194164
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.1320.2330.000

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Supplemental data

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Sample components

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Entire Complex of inward-facing Apo-open state of GltPh in MSP1E3 nanodisc

EntireName: Complex of inward-facing Apo-open state of GltPh in MSP1E3 nanodisc
Number of components: 3

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Component #1: protein, Complex of inward-facing Apo-open state of GltPh in MSP1...

ProteinName: Complex of inward-facing Apo-open state of GltPh in MSP1E3 nanodisc
Recombinant expression: No
MassTheoretical: 134 kDa
SourceSpecies: Pyrococcus horikoshii (archaea)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #2: protein, Glutamate transporter homologue GltPh

ProteinName: Glutamate transporter homologue GltPh / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 44.643918 kDa
SourceSpecies: Pyrococcus horikoshii (archaea)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #3: ligand, [(2~{R})-1-[2-azanylethoxy(oxidanyl)phosphoryl]oxy-3-hexa...

LigandName: [(2~{R})-1-[2-azanylethoxy(oxidanyl)phosphoryl]oxy-3-hexadecanoyloxy-propan-2-yl] (~{Z})-octadec-9-enoate
Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 0.717996 kDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionpH: 7.4
Support filmunspecified
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 68.55 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: OTHER

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 148582
3D reconstructionResolution: 3.52 Å / Resolution method: FSC 0.143 CUT-OFF

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Atomic model buiding

Output model

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