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- EMDB-29741: Native GABA-A receptor from the mouse brain, ortho-alpha1-alpha3-... -

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Basic information

Entry
Database: EMDB / ID: EMD-29741
TitleNative GABA-A receptor from the mouse brain, ortho-alpha1-alpha3-beta2-gamma2 subtype, in complex with GABA and allopregnanolone
Map data
Sample
  • Complex: Native GABAA Receptor isolated from mouse brain containing one alpha1 subunit, in complex with 8E3-Fab, GABA, and allopregnanolone
    • Protein or peptide: Gamma-aminobutyric acid receptor subunit alpha-3
    • Protein or peptide: Gamma-aminobutyric acid receptor subunit beta-2
    • Protein or peptide: Gamma-aminobutyric acid receptor subunit alpha-1
    • Protein or peptide: Gamma-aminobutyric acid receptor subunit gamma-2
    • Protein or peptide: Heavy Chain of 8E3 Fab
    • Protein or peptide: Light Chain of 8E3 Fab
  • Ligand: [(2R)-2-octanoyloxy-3-[oxidanyl-[(1R,2R,3S,4R,5R,6S)-2,3,6-tris(oxidanyl)-4,5-diphosphonooxy-cyclohexyl]oxy-phosphoryl]oxy-propyl] octanoate
  • Ligand: allopregnanolone
  • Ligand: GAMMA-AMINO-BUTANOIC ACID
Keywordsligand-gated ion channel / cys-loop receptor / neurotransmitter receptor / MEMBRANE PROTEIN
Function / homology
Function and homology information


GABA receptor activation / diazepam binding / inhibitory synapse / organic cyclic compound binding / GABA receptor complex / inhibitory extracellular ligand-gated monoatomic ion channel activity / benzodiazepine receptor activity / cellular response to histamine / inner ear receptor cell development / GABA-gated chloride ion channel activity ...GABA receptor activation / diazepam binding / inhibitory synapse / organic cyclic compound binding / GABA receptor complex / inhibitory extracellular ligand-gated monoatomic ion channel activity / benzodiazepine receptor activity / cellular response to histamine / inner ear receptor cell development / GABA-gated chloride ion channel activity / GABA-A receptor activity / GABA-A receptor complex / inhibitory synapse assembly / innervation / synaptic transmission, GABAergic / postsynaptic specialization membrane / gamma-aminobutyric acid signaling pathway / neurotransmitter receptor activity / adult behavior / cochlea development / chloride channel activity / chloride channel complex / GABA-ergic synapse / transmembrane transporter complex / neuron development / regulation of postsynaptic membrane potential / presynaptic active zone membrane / chloride transmembrane transport / dendrite membrane / ligand-gated monoatomic ion channel activity involved in regulation of presynaptic membrane potential / post-embryonic development / transmitter-gated monoatomic ion channel activity involved in regulation of postsynaptic membrane potential / cytoplasmic vesicle membrane / cytoplasmic vesicle / postsynapse / chemical synaptic transmission / neuron projection / axon / glutamatergic synapse / synapse / plasma membrane
Similarity search - Function
Gamma-aminobutyric-acid A receptor, alpha 3 subunit / Gamma-aminobutyric-acid A receptor, gamma 2 subunit / Gamma-aminobutyric acid receptor subunit gamma-1/4 / : / Gamma-aminobutyric-acid A receptor, alpha 1 subunit / Gamma-aminobutyric-acid A receptor, beta subunit / : / Gamma-aminobutyric-acid A receptor, alpha subunit / Gamma-aminobutyric acid A receptor/Glycine receptor alpha / Neurotransmitter-gated ion-channel, conserved site ...Gamma-aminobutyric-acid A receptor, alpha 3 subunit / Gamma-aminobutyric-acid A receptor, gamma 2 subunit / Gamma-aminobutyric acid receptor subunit gamma-1/4 / : / Gamma-aminobutyric-acid A receptor, alpha 1 subunit / Gamma-aminobutyric-acid A receptor, beta subunit / : / Gamma-aminobutyric-acid A receptor, alpha subunit / Gamma-aminobutyric acid A receptor/Glycine receptor alpha / Neurotransmitter-gated ion-channel, conserved site / Neurotransmitter-gated ion-channels signature. / Neurotransmitter-gated ion-channel transmembrane domain / Neurotransmitter-gated ion-channel transmembrane region / Neurotransmitter-gated ion-channel transmembrane domain superfamily / Neuronal acetylcholine receptor / Neurotransmitter-gated ion-channel / Neurotransmitter-gated ion-channel ligand-binding domain / Neurotransmitter-gated ion-channel ligand-binding domain superfamily / Neurotransmitter-gated ion-channel ligand binding domain
Similarity search - Domain/homology
Gamma-aminobutyric acid receptor subunit gamma-2 / Gamma-aminobutyric acid receptor subunit alpha-3 / Gamma-aminobutyric acid receptor subunit alpha-1 / Gamma-aminobutyric acid receptor subunit beta-2
Similarity search - Component
Biological speciesMus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.64 Å
AuthorsSun C / Gouaux E
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)5R01GM100400-08 United States
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Acta Crystallogr D Struct Biol / Year: 2018
Title: Real-space refinement in PHENIX for cryo-EM and crystallography.
Authors: Pavel V Afonine / Billy K Poon / Randy J Read / Oleg V Sobolev / Thomas C Terwilliger / Alexandre Urzhumtsev / Paul D Adams /
Abstract: This article describes the implementation of real-space refinement in the phenix.real_space_refine program from the PHENIX suite. The use of a simplified refinement target function enables very fast ...This article describes the implementation of real-space refinement in the phenix.real_space_refine program from the PHENIX suite. The use of a simplified refinement target function enables very fast calculation, which in turn makes it possible to identify optimal data-restraint weights as part of routine refinements with little runtime cost. Refinement of atomic models against low-resolution data benefits from the inclusion of as much additional information as is available. In addition to standard restraints on covalent geometry, phenix.real_space_refine makes use of extra information such as secondary-structure and rotamer-specific restraints, as well as restraints or constraints on internal molecular symmetry. The re-refinement of 385 cryo-EM-derived models available in the Protein Data Bank at resolutions of 6 Å or better shows significant improvement of the models and of the fit of these models to the target maps.
History
DepositionFeb 13, 2023-
Header (metadata) releaseSep 20, 2023-
Map releaseSep 20, 2023-
UpdateNov 15, 2023-
Current statusNov 15, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29741.png

Downloads & links

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Map

FileDownload / File: emd_29741.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.831 Å
Density
Contour LevelBy AUTHOR: 0.09
Minimum - Maximum-0.30326787 - 0.7304868
Average (Standard dev.)0.00039912152 (±0.018936358)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 299.16 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_29741_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_29741_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_29741_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Native GABAA Receptor isolated from mouse brain containing one al...

EntireName: Native GABAA Receptor isolated from mouse brain containing one alpha1 subunit, in complex with 8E3-Fab, GABA, and allopregnanolone
Components
  • Complex: Native GABAA Receptor isolated from mouse brain containing one alpha1 subunit, in complex with 8E3-Fab, GABA, and allopregnanolone
    • Protein or peptide: Gamma-aminobutyric acid receptor subunit alpha-3
    • Protein or peptide: Gamma-aminobutyric acid receptor subunit beta-2
    • Protein or peptide: Gamma-aminobutyric acid receptor subunit alpha-1
    • Protein or peptide: Gamma-aminobutyric acid receptor subunit gamma-2
    • Protein or peptide: Heavy Chain of 8E3 Fab
    • Protein or peptide: Light Chain of 8E3 Fab
  • Ligand: [(2R)-2-octanoyloxy-3-[oxidanyl-[(1R,2R,3S,4R,5R,6S)-2,3,6-tris(oxidanyl)-4,5-diphosphonooxy-cyclohexyl]oxy-phosphoryl]oxy-propyl] octanoate
  • Ligand: allopregnanolone
  • Ligand: GAMMA-AMINO-BUTANOIC ACID

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Supramolecule #1: Native GABAA Receptor isolated from mouse brain containing one al...

SupramoleculeName: Native GABAA Receptor isolated from mouse brain containing one alpha1 subunit, in complex with 8E3-Fab, GABA, and allopregnanolone
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#6
Source (natural)Organism: Mus musculus (house mouse) / Strain: C57BL/6 / Organ: Brain
Molecular weightTheoretical: 350 KDa

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Macromolecule #1: Gamma-aminobutyric acid receptor subunit alpha-3

MacromoleculeName: Gamma-aminobutyric acid receptor subunit alpha-3 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse) / Strain: C57BL/6
Molecular weightTheoretical: 55.461684 KDa
SequenceString: MIITQMWHFY VTRVVLLLLI SILPGTTSQG ESRRQEPGDF VKQDIGGLSP KHAPDIPDDS TDNITIFTRI LDRLLDGYDN RLRPGLGDA VTEVKTDIYV TSFGPVSDTD MEYTIDVFFR QTWHDERLKF DGPMKILPLN NLLASKIWTP DTFFHNGKKS V AHNMTTPN ...String:
MIITQMWHFY VTRVVLLLLI SILPGTTSQG ESRRQEPGDF VKQDIGGLSP KHAPDIPDDS TDNITIFTRI LDRLLDGYDN RLRPGLGDA VTEVKTDIYV TSFGPVSDTD MEYTIDVFFR QTWHDERLKF DGPMKILPLN NLLASKIWTP DTFFHNGKKS V AHNMTTPN KLLRLVDNGT LLYTMRLTIH AECPMHLEDF PMDVHACPLK FGSYAYTKAE VIYSWTLGKN KSVEVAQDGS RL NQYDLLG HVVGTEIIRS STGEYVVMTT HFHLKRKIGY FVIQTYLPCI MTVILSQVSF WLNRESVPAR TVFGVTTVLT MTT LSISAR NSLPKVAYAT AMDWFIAVCY AFVFSALIEF ATVNYFTKRS WAWEGKKVPE ALEMKKKTPA APTKKNTTFN IVGT TYPIN LAKDTEFSTI SKSAAAPSAS STPTAIASPK ATYVQDSPAE TKTYNSVSKV DKISRIIFPV LFAIFNLVYW ATYVN RESA IKGMIRKQ

UniProtKB: Gamma-aminobutyric acid receptor subunit alpha-3

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Macromolecule #2: Gamma-aminobutyric acid receptor subunit beta-2

MacromoleculeName: Gamma-aminobutyric acid receptor subunit beta-2 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse) / Strain: C57BL/6
Molecular weightTheoretical: 59.265312 KDa
SequenceString: MWRVRKRGYF GIWSFPLIIA AVCAQSVNDP SNMSLVKETV DRLLKGYDIR LRPDFGGPPV AVGMNIDIAS IDMVSEVNMD YTLTMYFQQ AWRDKRLSYN VIPLNLTLDN RVADQLWVPD TYFLNDKKSF VHGVTVKNRM IRLHPDGTVL YGLRITTTAA C MMDLRRYP ...String:
MWRVRKRGYF GIWSFPLIIA AVCAQSVNDP SNMSLVKETV DRLLKGYDIR LRPDFGGPPV AVGMNIDIAS IDMVSEVNMD YTLTMYFQQ AWRDKRLSYN VIPLNLTLDN RVADQLWVPD TYFLNDKKSF VHGVTVKNRM IRLHPDGTVL YGLRITTTAA C MMDLRRYP LDEQNCTLEI ESYGYTTDDI EFYWRGDDNA VTGVTKIELP QFSIVDYKLI TKKVVFSTGS YPRLSLSFKL KR NIGYFIL QTYMPSILIT ILSWVSFWIN YDASAARVAL GITTVLTMTT INTHLRETLP KIPYVKAIDM YLMGCFVFVF MAL LEYALV NYIFFGRGPQ RQKKAAEKAA NANNEKMRLD VNKMFYKDIK QNGTQYRSLW DPTGDLSPTR RTTNYDFSLY TMDP HENIL LSTLEIKNEM ATSEAVMGLG DPRSTMLAYD ASSIQYRKAG LPRHSFGRNA LERHVAQKKS RLRRRASQLK ITIPD LTDV NAIDRWSRIF FPVVFSFFNI VYWLYYVN

UniProtKB: Gamma-aminobutyric acid receptor subunit beta-2

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Macromolecule #3: Gamma-aminobutyric acid receptor subunit alpha-1

MacromoleculeName: Gamma-aminobutyric acid receptor subunit alpha-1 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse) / Strain: C57BL/6
Molecular weightTheoretical: 51.818516 KDa
SequenceString: MKKSRGLSDY LWAWTLILST LSGRSYGQPS QDELKDNTTV FTRILDRLLD GYDNRLRPGL GERVTEVKTD IFVTSFGPVS DHDMEYTID VFFRQSWKDE RLKFKGPMTV LRLNNLMASK IWTPDTFFHN GKKSVAHNMT MPNKLLRITE DGTLLYTMRL T VRAECPMH ...String:
MKKSRGLSDY LWAWTLILST LSGRSYGQPS QDELKDNTTV FTRILDRLLD GYDNRLRPGL GERVTEVKTD IFVTSFGPVS DHDMEYTID VFFRQSWKDE RLKFKGPMTV LRLNNLMASK IWTPDTFFHN GKKSVAHNMT MPNKLLRITE DGTLLYTMRL T VRAECPMH LEDFPMDAHA CPLKFGSYAY TRAEVVYEWT REPARSVVVA EDGSRLNQYD LLGQTVDSGI VQSSTGEYVV MT THFHLKR KIGYFVIQTY LPCIMTVILS QVSFWLNRES VPARTVFGVT TVLTMTTLSI SARNSLPKVA YATAMDWFIA VCY AFVFSA LIEFATVNYF TKRGYAWDGK SVVPEKPKKV KDPLIKKNNT YAPTATSYTP NLARGDPGLA TIAKSATIEP KEVK PETKP PEPKKTFNSV SKIDRLSRIA FPLLFGIFNL VYWATYLNRE PQLKAPTPHQ

UniProtKB: Gamma-aminobutyric acid receptor subunit alpha-1

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Macromolecule #4: Gamma-aminobutyric acid receptor subunit gamma-2

MacromoleculeName: Gamma-aminobutyric acid receptor subunit gamma-2 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse) / Strain: C57BL/6
Molecular weightTheoretical: 55.160281 KDa
SequenceString: MSSPNTWSIG SSVYSPVFSQ KMTLWILLLL SLYPGFTSQK SDDDYEDYAS NKTWVLTPKV PEGDVTVILN NLLEGYDNKL RPDIGVKPT LIHTDMYVNS IGPVNAINME YTIDIFFAQT WYDRRLKFNS TIKVLRLNSN MVGKIWIPDT FFRNSKKADA H WITTPNRM ...String:
MSSPNTWSIG SSVYSPVFSQ KMTLWILLLL SLYPGFTSQK SDDDYEDYAS NKTWVLTPKV PEGDVTVILN NLLEGYDNKL RPDIGVKPT LIHTDMYVNS IGPVNAINME YTIDIFFAQT WYDRRLKFNS TIKVLRLNSN MVGKIWIPDT FFRNSKKADA H WITTPNRM LRIWNDGRVL YTLRLTIDAE CQLQLHNFPM DEHSCPLEFS SYGYPREEIV YQWKRSSVEV GDTRSWRLYQ FS FVGLRNT TEVVKTTSGD YVVMSVYFDL SRRMGYFTIQ TYIPCTLIVV LSWVSFWINK DAVPARTSLG ITTVLTMTTL STI ARKSLP KVSYVTAMDL FVSVCFIFVF SALVEYGTLH YFVSNRKPSK DKDKKKKNPL LRMFSFKAPT IDIRPRSATI QMNN ATHLQ ERDEEYGYEC LDGKDCASFF CCFEDCRTGA WRHGRIHIRI AKMDSYARIF FPTAFCLFNL VYWVSYLYL

UniProtKB: Gamma-aminobutyric acid receptor subunit gamma-2

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Macromolecule #5: Heavy Chain of 8E3 Fab

MacromoleculeName: Heavy Chain of 8E3 Fab / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 24.159895 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: EIQLQQSGPE LVKPGTSVKV SCKASGYSFT DYNMYWVKQS HGKSLEWIGY IDPYNADTTY NREFKGKATL TVDKSSSTAF MHLNSLTSE DSAVYYCARK RNNFYFDYWG QGTPLTVSSA KTTPPSVYPL APGCGDTTGS SVTLGCLVKG YFPESVTVTW N SGSLSSSV ...String:
EIQLQQSGPE LVKPGTSVKV SCKASGYSFT DYNMYWVKQS HGKSLEWIGY IDPYNADTTY NREFKGKATL TVDKSSSTAF MHLNSLTSE DSAVYYCARK RNNFYFDYWG QGTPLTVSSA KTTPPSVYPL APGCGDTTGS SVTLGCLVKG YFPESVTVTW N SGSLSSSV HTFPALLQSG LYTMSSSVTV PSSTWPSQTV TCSVAHPASS TTVDKKSAAL EVLFQ

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Macromolecule #6: Light Chain of 8E3 Fab

MacromoleculeName: Light Chain of 8E3 Fab / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 23.62408 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: YIVMTQSPKS MSMSLGERVT LSCRASEYVG SYVSWYQQKP EQSPKLLIYG ASNRYTGVPD RFAGSGSATD FTLTITSVQA EDLADYHCG QTYNYPTFGG GTKLEIKRAD AAPTVSIFPP SSEQLTSGGA SVVCFLNNFY PKDINVKWKI DGSERQNGVL N SWTDQDSK ...String:
YIVMTQSPKS MSMSLGERVT LSCRASEYVG SYVSWYQQKP EQSPKLLIYG ASNRYTGVPD RFAGSGSATD FTLTITSVQA EDLADYHCG QTYNYPTFGG GTKLEIKRAD AAPTVSIFPP SSEQLTSGGA SVVCFLNNFY PKDINVKWKI DGSERQNGVL N SWTDQDSK DSTYSMSSTL TLTKDEYERH NSYTCEATHK TSTSPIVKSF NRNEC

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Macromolecule #10: [(2R)-2-octanoyloxy-3-[oxidanyl-[(1R,2R,3S,4R,5R,6S)-2,3,6-tris(o...

MacromoleculeName: [(2R)-2-octanoyloxy-3-[oxidanyl-[(1R,2R,3S,4R,5R,6S)-2,3,6-tris(oxidanyl)-4,5-diphosphonooxy-cyclohexyl]oxy-phosphoryl]oxy-propyl] octanoate
type: ligand / ID: 10 / Number of copies: 2 / Formula: PIO
Molecular weightTheoretical: 746.566 Da
Chemical component information

ChemComp-PIO:
[(2R)-2-octanoyloxy-3-[oxidanyl-[(1R,2R,3S,4R,5R,6S)-2,3,6-tris(oxidanyl)-4,5-diphosphonooxy-cyclohexyl]oxy-phosphoryl]oxy-propyl] octanoate

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Macromolecule #11: allopregnanolone

MacromoleculeName: allopregnanolone / type: ligand / ID: 11 / Number of copies: 2 / Formula: Y4B
Molecular weightTheoretical: 318.493 Da
Chemical component information

ChemComp-Y4B:
allopregnanolone

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Macromolecule #12: GAMMA-AMINO-BUTANOIC ACID

MacromoleculeName: GAMMA-AMINO-BUTANOIC ACID / type: ligand / ID: 12 / Number of copies: 2 / Formula: ABU
Molecular weightTheoretical: 103.12 Da
Chemical component information

ChemComp-ABU:
GAMMA-AMINO-BUTANOIC ACID

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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

Concentration0.1 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
150.0 mMNaClSodium Chloride
20.0 mMTris-HClTris
GridModel: Quantifoil R2/1 / Material: GOLD / Mesh: 200 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 2 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK IV
DetailsThis sample was monodisperse.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.1 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 2.64 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.2) / Number images used: 137566
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-8g5f:
Native GABA-A receptor from the mouse brain, ortho-alpha1-alpha3-beta2-gamma2 subtype, in complex with GABA and allopregnanolone

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