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- EMDB-4542: CryoEM structure of a beta3K279T GABA(A)R homomer in complex with... -

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Basic information

Entry
Database: EMDB / ID: EMD-4542
TitleCryoEM structure of a beta3K279T GABA(A)R homomer in complex with histamine and megabody Mb25
Map dataFinal EM map from RELION 3.0 Postprocess job
Sample
  • Complex: Homomeric truncated (M3-M4 loop) GABAAR b3K279T receptor in complex with histamine and megabody Mb25
    • Complex: Homomeric truncated (M3-M4 loop) GABAAR b3K279T receptor
      • Protein or peptide: Gamma-aminobutyric acid receptor subunit beta-3,Gamma-aminobutyric acid receptor subunit beta-3
    • Complex: Megabody Mb25
      • Protein or peptide: Outer membrane protein,Outer membrane protein,Outer membrane protein,Outer membrane protein,Uncharacterized protein,Uncharacterized protein,Mb-c7HopQ-Nb25
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: HISTAMINE
Function / homology
Function and homology information


cellular response to histamine / GABA receptor activation / GABA-gated chloride ion channel activity / GABA-A receptor activity / GABA-A receptor complex / inhibitory synapse assembly / synaptic transmission, GABAergic / gamma-aminobutyric acid signaling pathway / nervous system process / neurotransmitter receptor activity ...cellular response to histamine / GABA receptor activation / GABA-gated chloride ion channel activity / GABA-A receptor activity / GABA-A receptor complex / inhibitory synapse assembly / synaptic transmission, GABAergic / gamma-aminobutyric acid signaling pathway / nervous system process / neurotransmitter receptor activity / roof of mouth development / Signaling by ERBB4 / chloride channel complex / transmembrane transporter complex / chloride transmembrane transport / regulation of membrane potential / cytoplasmic vesicle membrane / postsynaptic membrane / neuron projection / synapse / signal transduction / identical protein binding / plasma membrane
Similarity search - Function
SabA, N-terminal extracellular adhesion domain / SabA N-terminal extracellular adhesion domain / Outer membrane protein, Helicobacter / Helicobacter outer membrane protein / Gamma-aminobutyric-acid A receptor, beta 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 ...SabA, N-terminal extracellular adhesion domain / SabA N-terminal extracellular adhesion domain / Outer membrane protein, Helicobacter / Helicobacter outer membrane protein / Gamma-aminobutyric-acid A receptor, beta 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
Outer membrane protein / Gamma-aminobutyric acid receptor subunit beta-3
Similarity search - Component
Biological speciesHomo sapiens (human) / Lama glama (llama)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.49 Å
AuthorsUchanski T / Masiulis S / Fischer B / Kalichuk V / Wohlkoening A / Zoegg T / Remaut H / Vranken W / Aricescu AR / Pardon E / Steyaert J
Funding support United Kingdom, Belgium, 5 items
OrganizationGrant numberCountry
Medical Research Council (United Kingdom)MC_UP_1201/15 United Kingdom
Medical Research Council (United Kingdom)L009609/1 United Kingdom
European Union731005 Belgium
Grenoble Instruct-ERIC Center Belgium
Research Foundation - Flanders Belgium
CitationJournal: Nat Methods / Year: 2021
Title: Megabodies expand the nanobody toolkit for protein structure determination by single-particle cryo-EM.
Authors: Tomasz Uchański / Simonas Masiulis / Baptiste Fischer / Valentina Kalichuk / Uriel López-Sánchez / Eleftherios Zarkadas / Miriam Weckener / Andrija Sente / Philip Ward / Alexandre ...Authors: Tomasz Uchański / Simonas Masiulis / Baptiste Fischer / Valentina Kalichuk / Uriel López-Sánchez / Eleftherios Zarkadas / Miriam Weckener / Andrija Sente / Philip Ward / Alexandre Wohlkönig / Thomas Zögg / Han Remaut / James H Naismith / Hugues Nury / Wim Vranken / A Radu Aricescu / Els Pardon / Jan Steyaert /
Abstract: Nanobodies are popular and versatile tools for structural biology. They have a compact single immunoglobulin domain organization, bind target proteins with high affinities while reducing their ...Nanobodies are popular and versatile tools for structural biology. They have a compact single immunoglobulin domain organization, bind target proteins with high affinities while reducing their conformational heterogeneity and stabilize multi-protein complexes. Here we demonstrate that engineered nanobodies can also help overcome two major obstacles that limit the resolution of single-particle cryo-electron microscopy reconstructions: particle size and preferential orientation at the water-air interfaces. We have developed and characterized constructs, termed megabodies, by grafting nanobodies onto selected protein scaffolds to increase their molecular weight while retaining the full antigen-binding specificity and affinity. We show that the megabody design principles are applicable to different scaffold proteins and recognition domains of compatible geometries and are amenable for efficient selection from yeast display libraries. Moreover, we demonstrate that megabodies can be used to obtain three-dimensional reconstructions for membrane proteins that suffer from severe preferential orientation or are otherwise too small to allow accurate particle alignment.
History
DepositionJan 9, 2019-
Header (metadata) releaseAug 4, 2021-
Map releaseAug 4, 2021-
UpdateAug 4, 2021-
Current statusAug 4, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.08
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.08
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6qfa
  • Surface level: 0.08
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4542.png

Downloads & links

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Map

FileDownload / File: emd_4542.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFinal EM map from RELION 3.0 Postprocess job
Voxel sizeX=Y=Z: 1.07 Å
Density
Contour LevelBy AUTHOR: 0.08 / Movie #1: 0.08
Minimum - Maximum-0.35078228 - 0.5620734
Average (Standard dev.)0.00046119175 (±0.01360003)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 273.92 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.071.071.07
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z273.920273.920273.920
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ200200200
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.3510.5620.000

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Supplemental data

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Mask #1

Fileemd_4542_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Combined, unfiltered/unsharpened map from Relion Refine3D

Fileemd_4542_additional_1.map
AnnotationCombined, unfiltered/unsharpened map from Relion Refine3D
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map from Relion

Fileemd_4542_half_map_1.map
AnnotationHalf-map from Relion
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map from Relion

Fileemd_4542_half_map_2.map
AnnotationHalf-map from Relion
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Homomeric truncated (M3-M4 loop) GABAAR b3K279T receptor in compl...

EntireName: Homomeric truncated (M3-M4 loop) GABAAR b3K279T receptor in complex with histamine and megabody Mb25
Components
  • Complex: Homomeric truncated (M3-M4 loop) GABAAR b3K279T receptor in complex with histamine and megabody Mb25
    • Complex: Homomeric truncated (M3-M4 loop) GABAAR b3K279T receptor
      • Protein or peptide: Gamma-aminobutyric acid receptor subunit beta-3,Gamma-aminobutyric acid receptor subunit beta-3
    • Complex: Megabody Mb25
      • Protein or peptide: Outer membrane protein,Outer membrane protein,Outer membrane protein,Outer membrane protein,Uncharacterized protein,Uncharacterized protein,Mb-c7HopQ-Nb25
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: HISTAMINE

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Supramolecule #1: Homomeric truncated (M3-M4 loop) GABAAR b3K279T receptor in compl...

SupramoleculeName: Homomeric truncated (M3-M4 loop) GABAAR b3K279T receptor in complex with histamine and megabody Mb25
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Molecular weightTheoretical: 500 KDa

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Supramolecule #2: Homomeric truncated (M3-M4 loop) GABAAR b3K279T receptor

SupramoleculeName: Homomeric truncated (M3-M4 loop) GABAAR b3K279T receptor
type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human) / Organ: Brain / Location in cell: Plasma membrane
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293S-GnTI- / Recombinant plasmid: pHL-sec

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Supramolecule #3: Megabody Mb25

SupramoleculeName: Megabody Mb25 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Lama glama (llama)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #1: Gamma-aminobutyric acid receptor subunit beta-3,Gamma-aminobutyri...

MacromoleculeName: Gamma-aminobutyric acid receptor subunit beta-3,Gamma-aminobutyric acid receptor subunit beta-3
type: protein_or_peptide / ID: 1 / Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 39.503555 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: QSVNDPGNMS FVKETVDKLL KGYDIRLRPD FGGPPVCVGM NIDIASIDMV SEVNMDYTLT MYFQQYWRDK RLAYSGIPLN LTLDNRVAD QLWVPDTYFL NDKKSFVHGV TVKNRMIRLH PDGTVLYGLR ITTTAACMMD LRRYPLDEQN CTLEIESYGY T TDDIEFYW ...String:
QSVNDPGNMS FVKETVDKLL KGYDIRLRPD FGGPPVCVGM NIDIASIDMV SEVNMDYTLT MYFQQYWRDK RLAYSGIPLN LTLDNRVAD QLWVPDTYFL NDKKSFVHGV TVKNRMIRLH PDGTVLYGLR ITTTAACMMD LRRYPLDEQN CTLEIESYGY T TDDIEFYW RGGDKAVTGV ERIELPQFSI VEHRLVSRNV VFATGAYPRL SLSFRLKRNI GYFILQTYMP SILITILSWV SF WINYDAS AARVALGITT VLTMTTINTH LRETLPKIPY VTAIDMYLMG CFVFVFLALL EYAFVNYIFF SQPARAAAID RWS RIVFPF TFSLFNLVYW LYYVN

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Macromolecule #2: Outer membrane protein,Outer membrane protein,Outer membrane prot...

MacromoleculeName: Outer membrane protein,Outer membrane protein,Outer membrane protein,Outer membrane protein,Uncharacterized protein,Uncharacterized protein,Mb-c7HopQ-Nb25
type: protein_or_peptide / ID: 2 / Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: Lama glama (llama)
Molecular weightTheoretical: 56.300629 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: QVQLVESGGG LVQTKTTTSV IDTTNDAQNL LTQAQTIVNT LKDYCPILIA KSSSSNGGTN NANTPSWQTA GGGKNSCATF GAEFSAASD MINNAQKIVQ ETQQLSANQP KNITQPHNLN LNSPSSLTAL AQKMLKNAQS QAEILKLANQ VESDFNKLSS G HLKDYIGK ...String:
QVQLVESGGG LVQTKTTTSV IDTTNDAQNL LTQAQTIVNT LKDYCPILIA KSSSSNGGTN NANTPSWQTA GGGKNSCATF GAEFSAASD MINNAQKIVQ ETQQLSANQP KNITQPHNLN LNSPSSLTAL AQKMLKNAQS QAEILKLANQ VESDFNKLSS G HLKDYIGK CDASAISSAN MTMQNQKNNW GNGCAGVEET QSLLKTSAAD FNNQTPQINQ AQNLANTLIQ ELGNNTYEQL SR LLTNDNG TNSKTSAQAI NQAVNNLNER AKTLAGGTTN SPAYQATLLA LRSVLGLWNS MGYAVICGGY TKSPGENNQK DFH YTDENG NGTTINCGGS TNSNGTHSYN GTNTLKADKN VSLSIEQYEK IHEAYQILSK ALKQAGLAPL NSKGEKLEAH VTTS KYGSL RLSCAASGHT FNYPIMGWFR QAPGKEREFV GAISWSGGST SYADSVKDRF TISRDNAKNT VYLEMNNLKP EDTAV YYCA AKGRYSGGLY YPTNYDYWGQ GTQVTVSSHH HHHHEPEA

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Macromolecule #4: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 4 / Number of copies: 5 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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Macromolecule #5: HISTAMINE

MacromoleculeName: HISTAMINE / type: ligand / ID: 5 / Number of copies: 5 / Formula: HSM
Molecular weightTheoretical: 111.145 Da
Chemical component information

ChemComp-HSM:
HISTAMINE / neurotransmitter, hormone*YM

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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: 7.6
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV
DetailsMonodisperse sample

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsPhase plate: VOLTA PHASE PLATE
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Average electron dose: 30.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 0.7000000000000001 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 75000
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: 315183
CTF correctionSoftware - Name: Gctf (ver. 1.08)
Startup modelType of model: OTHER / Details: Relion 3D ab-initio
Final reconstructionApplied symmetry - Point group: C5 (5 fold cyclic) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 2.49 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Software - details: Refine3D / Number images used: 171761
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3.0) / Software - details: Refine3D
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3.0) / Software - details: Refine3D
Final 3D classificationSoftware - Name: RELION (ver. 3.0) / Software - details: Class3D

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Atomic model buiding 1

RefinementProtocol: FLEXIBLE FIT
Output model

PDB-6qfa:
CryoEM structure of a beta3K279T GABA(A)R homomer in complex with histamine and megabody Mb25

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