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- EMDB-4875: Density map of GluA2cryst in the apo state -

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Basic information

Entry
Database: EMDB / ID: EMD-4875
TitleDensity map of GluA2cryst in the apo state
Map dataGluA2cryst in the apo form. Solubilized in DDM. Negative stain electron microscopy.
Sample
  • Complex: GluA2cryst in the apo state
Keywordsionotropic glutamate receptor / GluA2 / resting state / apo structure / MEMBRANE PROTEIN
Biological speciesRattus norvegicus (Norway rat)
Methodsingle particle reconstruction / negative staining / Resolution: 17.0 Å
AuthorsKrintel C / Dorosz J / Larsen AH / Thorsen TS / Venskutonyte R / Mirza O / Gajhede M / Boesen T / Kastrup JS
Funding support Denmark, 2 items
OrganizationGrant numberCountry
Danish Council for Independent Research Denmark
Lundbeckfonden Denmark
CitationJournal: FEBS J / Year: 2021
Title: Binding of a negative allosteric modulator and competitive antagonist can occur simultaneously at the ionotropic glutamate receptor GluA2.
Authors: Christian Krintel / Jerzy Dorosz / Andreas Haahr Larsen / Thor Seneca Thorsen / Raminta Venskutonytė / Osman Mirza / Michael Gajhede / Thomas Boesen / Jette Sandholm Kastrup /
Abstract: Ionotropic glutamate receptors are ligand-gated ion channels governing neurotransmission in the central nervous system. Three major types of antagonists are known for the AMPA-type receptor GluA2: ...Ionotropic glutamate receptors are ligand-gated ion channels governing neurotransmission in the central nervous system. Three major types of antagonists are known for the AMPA-type receptor GluA2: competitive, noncompetitive (i.e., negative allosteric modulators; NAMs) used for treatment of epilepsy, and uncompetitive antagonists. We here report a 4.65 Å resolution X-ray structure of GluA2, revealing that four molecules of the competitive antagonist ZK200775 and four molecules of the NAM GYKI53655 are capable of binding at the same time. Using negative stain electron microscopy, we show that GYKI53655 alone or ZK200775/GYKI53655 in combination predominantly results in compact receptor forms. The agonist AMPA provides a mixed population of compact and bulgy shapes of GluA2 not impacted by addition of GYKI53655. Taken together, this suggests that the two different mechanisms of antagonism that lead to channel closure are independent and that the distribution between bulgy and compact receptors primarily depends on the ligand bound in the glutamate binding site. DATABASE: The atomic coordinates and structure factors from the crystal structure determination have been deposited in the Protein Data Bank under accession code https://doi.org/10.2210/pdb6RUQ/pdb. The electron microscopy 3D reconstruction volumes have been deposited in EMDB (EMD-4875: Apo; EMD-4920: ZK200775/GYKI53655; EMD-4921: AMPA compact; EMD-4922: AMPA/GYKI53655 bulgy; EMD-4923: GYKI53655; EMD-4924: AMPA bulgy; EMD-4925: AMPA/GYKI53655 compact).
History
DepositionApr 16, 2019-
Header (metadata) releaseMay 20, 2020-
Map releaseMay 20, 2020-
UpdateJan 10, 2024-
Current statusJan 10, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0439
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.0439
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4875.png

Downloads & links

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Map

FileDownload / File: emd_4875.map.gz / Format: CCP4 / Size: 3.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationGluA2cryst in the apo form. Solubilized in DDM. Negative stain electron microscopy.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
3.14 Å/pix.
x 100 pix.
= 314. Å		3.14 Å/pix.
x 100 pix.
= 314. Å		3.14 Å/pix.
x 100 pix.
= 314. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 3.14 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0439 / Movie #1: 0.0439
Minimum - Maximum-0.048932955 - 0.11935481
Average (Standard dev.)0.0012561692 (±0.0109208245)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions100100100
Spacing100100100
CellA=B=C: 314.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.143.143.14
M x/y/z100100100
origin x/y/z0.0000.0000.000
length x/y/z314.000314.000314.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ320320320
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS100100100
D min/max/mean-0.0490.1190.001

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Supplemental data

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Sample components

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Entire : GluA2cryst in the apo state

EntireName: GluA2cryst in the apo state
Components
  • Complex: GluA2cryst in the apo state

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Supramolecule #1: GluA2cryst in the apo state

SupramoleculeName: GluA2cryst in the apo state / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: GluA2 solubilized in n-Dodecyl-beta-Maltoside Detergent (DDM) sequence: NSIQIGGLFPRGADQEYSAFRVGMVQFSTSEFRLTPHIDNLEVANSFAVTNAFCSQFSRGVYAIFGFYDKKSVNTITSFC ...Details: GluA2 solubilized in n-Dodecyl-beta-Maltoside Detergent (DDM) sequence: NSIQIGGLFPRGADQEYSAFRVGMVQFSTSEFRLTPHIDNLEVANSFAVTNAFCSQFSRGVYAIFGFYDKKSVNTITSFC GTLHVSFITPSFPTDGTHPFVIQMRPDLKGALLSLIEYYQWDKFAYLYDSDRGLSTLQAVLDSAAEKKWQVTAINVGNIN NDKKDETYRSLFQDLELKKERRVILDCERDKVNDIVDQVITIGKHVKGYHYIIANLGFTDGDLLKIQFGGAEVSGFQIVD YDDSLVSKFIERWSTLEEKEYPGAHTATIKYTSALTYDAVQVMTEAFRNLRKQRIEISRRGNAGDCLANPAVPWGQGVEI ERALKQVQVEGLSGNIKFDQNGKRINYTINIMELKTNGPRKIGYWSEVDKMVLTEDDTSGLEQKTVVVTTILESPYVMMK ANHAALAGNERYEGYCVDLAAEIAKHCGFKYKLTIVGDGKYGARDADTKIWNGMVGELVYGKADIAIAPLTITLVREEVI DFSKPFMSLGISIMIKKPQKSKPGVFSFLDPLAYEIWMCIVFAYIGVSVVLFLVSRFSPYEWHTEEFEDGRETQSSESTN EFGIFNSLWFSLGAFMQQGADISPRSLSGRIVGGVWWFFTLIIISSYTANLAAFLTVERMVSPIESAEDLSKQTEIAYGT LDSGSTKEFFRRSKIAVFDKMWTYMRSAEPSVFVRTTAEGVARVRKSKGKYAYLLESTMNEYIEQRKPCDTMKVGGNLDS KGYGIATPKGSSLGTPVNLAVLKLSEQGLLDKLKNKWWYDKGECGAKDSGSKEKTSALSLSNVAGVFYILVGGLGLAMLV ALIEFCYKSRAEAKRMKGLVPRG NSIQIGGLFPRGADQEYSAFRVGMVQFSTSEFRLTPHIDNLEVANSFAVTNAFCSQFSRGVYAIFGFYDKKSVNTITSFC GTLHVSFITPSFPTDGTHPFVIQMRPDLKGALLSLIEYYQWDKFAYLYDSDRGLSTLQAVLDSAAEKKWQVTAINVGNIN NDKKDETYRSLFQDLELKKERRVILDCERDKVNDIVDQVITIGKHVKGYHYIIANLGFTDGDLLKIQFGGAEVSGFQIVD YDDSLVSKFIERWSTLEEKEYPGAHTATIKYTSALTYDAVQVMTEAFRNLRKQRIEISRRGNAGDCLANPAVPWGQGVEI ERALKQVQVEGLSGNIKFDQNGKRINYTINIMELKTNGPRKIGYWSEVDKMVLTEDDTSGLEQKTVVVTTILESPYVMMK ANHAALAGNERYEGYCVDLAAEIAKHCGFKYKLTIVGDGKYGARDADTKIWNGMVGELVYGKADIAIAPLTITLVREEVI DFSKPFMSLGISIMIKKPQKSKPGVFSFLDPLAYEIWMCIVFAYIGVSVVLFLVSRFSPYEWHTEEFEDGRETQSSESTN EFGIFNSLWFSLGAFMQQGADISPRSLSGRIVGGVWWFFTLIIISSYTANLAAFLTVERMVSPIESAEDLSKQTEIAYGT LDSGSTKEFFRRSKIAVFDKMWTYMRSAEPSVFVRTTAEGVARVRKSKGKYAYLLESTMNEYIEQRKPCDTMKVGGNLDS KGYGIATPKGSSLGTPVNLAVLKLSEQGLLDKLKNKWWYDKGECGAKDSGSKEKTSALSLSNVAGVFYILVGGLGLAMLV ALIEFCYKSRAEAKRMKGLVPRG NSIQIGGLFPRGADQEYSAFRVGMVQFSTSEFRLTPHIDNLEVANSFAVTNAFCSQFSRGVYAIFGFYDKKSVNTITSFC GTLHVSFITPSFPTDGTHPFVIQMRPDLKGALLSLIEYYQWDKFAYLYDSDRGLSTLQAVLDSAAEKKWQVTAINVGNIN NDKKDETYRSLFQDLELKKERRVILDCERDKVNDIVDQVITIGKHVKGYHYIIANLGFTDGDLLKIQFGGAEVSGFQIVD YDDSLVSKFIERWSTLEEKEYPGAHTATIKYTSALTYDAVQVMTEAFRNLRKQRIEISRRGNAGDCLANPAVPWGQGVEI ERALKQVQVEGLSGNIKFDQNGKRINYTINIMELKTNGPRKIGYWSEVDKMVLTEDDTSGLEQKTVVVTTILESPYVMMK ANHAALAGNERYEGYCVDLAAEIAKHCGFKYKLTIVGDGKYGARDADTKIWNGMVGELVYGKADIAIAPLTITLVREEVI DFSKPFMSLGISIMIKKPQKSKPGVFSFLDPLAYEIWMCIVFAYIGVSVVLFLVSRFSPYEWHTEEFEDGRETQSSESTN EFGIFNSLWFSLGAFMQQGADISPRSLSGRIVGGVWWFFTLIIISSYTANLAAFLTVERMVSPIESAEDLSKQTEIAYGT LDSGSTKEFFRRSKIAVFDKMWTYMRSAEPSVFVRTTAEGVARVRKSKGKYAYLLESTMNEYIEQRKPCDTMKVGGNLDS KGYGIATPKGSSLGTPVNLAVLKLSEQGLLDKLKNKWWYDKGECGAKDSGSKEKTSALSLSNVAGVFYILVGGLGLAMLV ALIEFCYKSRAEAKRMKGLVPRG NSIQIGGLFPRGADQEYSAFRVGMVQFSTSEFRLTPHIDNLEVANSFAVTNAFCSQFSRGVYAIFGFYDKKSVNTITSFC GTLHVSFITPSFPTDGTHPFVIQMRPDLKGALLSLIEYYQWDKFAYLYDSDRGLSTLQAVLDSAAEKKWQVTAINVGNIN NDKKDETYRSLFQDLELKKERRVILDCERDKVNDIVDQVITIGKHVKGYHYIIANLGFTDGDLLKIQFGGAEVSGFQIVD YDDSLVSKFIERWSTLEEKEYPGAHTATIKYTSALTYDAVQVMTEAFRNLRKQRIEISRRGNAGDCLANPAVPWGQGVEI ERALKQVQVEGLSGNIKFDQNGKRINYTINIMELKTNGPRKIGYWSEVDKMVLTEDDTSGLEQKTVVVTTILESPYVMMK ANHAALAGNERYEGYCVDLAAEIAKHCGFKYKLTIVGDGKYGARDADTKIWNGMVGELVYGKADIAIAPLTITLVREEVI DFSKPFMSLGISIMIKKPQKSKPGVFSFLDPLAYEIWMCIVFAYIGVSVVLFLVSRFSPYEWHTEEFEDGRETQSSESTN EFGIFNSLWFSLGAFMQQGADISPRSLSGRIVGGVWWFFTLIIISSYTANLAAFLTVERMVSPIESAEDLSKQTEIAYGT LDSGSTKEFFRRSKIAVFDKMWTYMRSAEPSVFVRTTAEGVARVRKSKGKYAYLLESTMNEYIEQRKPCDTMKVGGNLDS KGYGIATPKGSSLGTPVNLAVLKLSEQGLLDKLKNKWWYDKGECGAKDSGSKEKTSALSLSNVAGVFYILVGGLGLAMLV ALIEFCYKSRAEAKRMKGLVPRG
Source (natural)Organism: Rattus norvegicus (Norway rat)
Molecular weightTheoretical: 370 KDa

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.1 mg/mL
BufferpH: 8
Component:
ConcentrationNameFormula
20.0 mMTris
150.0 mMNaCl
0.5 mg/mln-Dodecyl-beta-Maltoside (DDM)
StainingType: NEGATIVE / Material: Uranyl acetate / Details: sample stained with 2% uranyl acetate for 15 sec
GridMaterial: COPPER / Support film - Material: CARBON / Pretreatment - Type: GLOW DISCHARGE

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Electron microscopy

MicroscopeFEI TECNAI SPIRIT
Image recordingFilm or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Average electron dose: 10.0 e/Å2
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Tecnai Spirit / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 111710
Startup modelType of model: NONE
Final reconstructionNumber classes used: 5 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 17.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Details: RELION 3.0 was used for reconstruction. / Number images used: 40328
Initial angle assignmentType: OTHER / Software - Name: RELION (ver. 3.0)
Details: See: J. Struct Biol., 2012, 180, 519-530; doi: 10.1016/j.sjb.2012.09.006; RELION: Implementation of a Bayesian approach to cryo-EM structure determination; Sjors H.W.Scheres
Final angle assignmentType: OTHER / Software - Name: RELION (ver. 3.0)
Details: See: J. Struct Biol., 2012, 180, 519-530; doi: 10.1016/j.sjb.2012.09.006; RELION: Implementation of a Bayesian approach to cryo-EM structure determination; Sjors H.W.Scheres
Final 3D classificationNumber classes: 6 / Avg.num./class: 8066 / Software - Name: RELION (ver. 3.0)

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