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- PDB-6qfa: CryoEM structure of a beta3K279T GABA(A)R homomer in complex with... -

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Basic information

Entry
Database: PDB / ID: 6qfa
TitleCryoEM structure of a beta3K279T GABA(A)R homomer in complex with histamine and megabody Mb25
Components
  • Gamma-aminobutyric acid receptor subunit beta-3,Gamma-aminobutyric acid receptor subunit beta-3
  • Outer membrane protein,Outer membrane protein,Outer membrane protein,Outer membrane protein,Uncharacterized protein,Uncharacterized protein,Mb-c7HopQ-Nb25
KeywordsMEMBRANE PROTEIN / Megabody / protein engineering / cryo-EM
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
HISTAMINE / Outer membrane protein / Gamma-aminobutyric acid receptor subunit beta-3
Similarity search - Component
Biological speciesHomo sapiens (human)
Helicobacter pylori (bacteria)
Lama glama (llama)
MethodELECTRON MICROSCOPY / single 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, A.R. / Pardon, E. / Steyaert, J.
Funding support United Kingdom, Belgium, 5items
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, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 4, 2021Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
B: Gamma-aminobutyric acid receptor subunit beta-3,Gamma-aminobutyric acid receptor subunit beta-3
K: Outer membrane protein,Outer membrane protein,Outer membrane protein,Outer membrane protein,Uncharacterized protein,Uncharacterized protein,Mb-c7HopQ-Nb25
O: Outer membrane protein,Outer membrane protein,Outer membrane protein,Outer membrane protein,Uncharacterized protein,Uncharacterized protein,Mb-c7HopQ-Nb25
N: Outer membrane protein,Outer membrane protein,Outer membrane protein,Outer membrane protein,Uncharacterized protein,Uncharacterized protein,Mb-c7HopQ-Nb25
M: Outer membrane protein,Outer membrane protein,Outer membrane protein,Outer membrane protein,Uncharacterized protein,Uncharacterized protein,Mb-c7HopQ-Nb25
L: Outer membrane protein,Outer membrane protein,Outer membrane protein,Outer membrane protein,Uncharacterized protein,Uncharacterized protein,Mb-c7HopQ-Nb25
C: Gamma-aminobutyric acid receptor subunit beta-3,Gamma-aminobutyric acid receptor subunit beta-3
D: Gamma-aminobutyric acid receptor subunit beta-3,Gamma-aminobutyric acid receptor subunit beta-3
E: Gamma-aminobutyric acid receptor subunit beta-3,Gamma-aminobutyric acid receptor subunit beta-3
A: Gamma-aminobutyric acid receptor subunit beta-3,Gamma-aminobutyric acid receptor subunit beta-3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)485,23725
Polymers479,02110
Non-polymers6,21615
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Gamma-aminobutyric acid receptor subunit beta-3,Gamma-aminobutyric acid receptor subunit beta-3 / GABA(A) receptor subunit beta-3


Mass: 39503.555 Da / Num. of mol.: 5 / Mutation: K279T
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GABRB3 / Cell line (production host): HEK293S GnTI- / Production host: Homo sapiens (human) / References: UniProt: P28472
#2: Antibody
Outer membrane protein,Outer membrane protein,Outer membrane protein,Outer membrane protein,Uncharacterized protein,Uncharacterized protein,Mb-c7HopQ-Nb25


Mass: 56300.629 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Helicobacter pylori (strain G27) (bacteria), (gene. exp.) Lama glama (llama)
Strain: G27 / Gene: hopQ, HPG27_1120 / Production host: Escherichia coli (E. coli) / References: UniProt: B5Z8H1
#3: Polysaccharide
alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]beta-D-mannopyranose-(1-4)-2-acetamido-2- ...alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 910.823 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-3[DManpa1-6]DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(3+1)][a-D-Manp]{}[(6+1)][a-D-Manp]{}}}}}LINUCSPDB-CARE
#4: Sugar
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
#5: Chemical
ChemComp-HSM / HISTAMINE


Mass: 111.145 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Formula: C5H9N3 / Feature type: SUBJECT OF INVESTIGATION / Comment: neurotransmitter, hormone*YM

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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

Component
IDNameTypeEntity IDParent-IDSource
1Homomeric truncated (M3-M4 loop) GABAAR b3K279T receptor in complex with histamine and megabody Mb25COMPLEX#1-#20MULTIPLE SOURCES
2Homomeric truncated (M3-M4 loop) GABAAR b3K279T receptorCOMPLEX#11RECOMBINANT
3Megabody Mb25COMPLEX#21RECOMBINANT
Molecular weightValue: 0.5 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-IDCellular locationOrgan
32Homo sapiens (human)9606Plasma membraneBrain
43Lama glama (llama)9844
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDCellPlasmid
22Homo sapiens (human)9606HEK293S-GnTI-pHL-sec
43Escherichia coli (E. coli)562
Buffer solutionpH: 7.6
SpecimenConc.: 0.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Monodisperse sample
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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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 magnification: 75000 X / Nominal defocus max: 700 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 30 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k)
EM imaging opticsPhase plate: VOLTA PHASE PLATE

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Processing

EM software
IDNameVersionCategoryDetails
2EPUimage acquisition
4Gctf1.08CTF correction
7UCSF Chimeramodel fitting
9PHENIXmodel refinement
10RELION3initial Euler assignmentRefine3D
11RELION3final Euler assignmentRefine3D
12RELION3classificationClass3D
13RELION33D reconstructionRefine3D
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 315183
SymmetryPoint symmetry: C5 (5 fold cyclic)
3D reconstructionResolution: 2.49 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 171761 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT

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