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Yorodumi- EMDB-11610: CryoEM structure of a beta3K279T GABA(A)R homomer in complex with... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-11610 | ||||||||||||||||||
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Title | CryoEM structure of a beta3K279T GABA(A)R homomer in complex with megabody MbNbF3c7HopQ | ||||||||||||||||||
Map data | GABAAR-b3 homomer solubilised in nanodiscs and in complex with anti-MSP megabodies | ||||||||||||||||||
Sample |
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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 | ||||||||||||||||||
Biological species | Homo sapiens (human) / Lama glama (llama) | ||||||||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.0 Å | ||||||||||||||||||
Authors | Uchanski T / Masiulis S | ||||||||||||||||||
Funding support | United Kingdom, Belgium, 5 items
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Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_11610.map.gz | 16.6 MB | EMDB map data format | |
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Header (meta data) | emd-11610-v30.xml emd-11610.xml | 16.2 KB 16.2 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_11610_fsc.xml | 14.2 KB | Display | FSC data file |
Images | emd_11610.png | 137.2 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-11610 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-11610 | HTTPS FTP |
-Validation report
Summary document | emd_11610_validation.pdf.gz | 246.8 KB | Display | EMDB validaton report |
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Full document | emd_11610_full_validation.pdf.gz | 246 KB | Display | |
Data in XML | emd_11610_validation.xml.gz | 14.1 KB | Display | |
Data in CIF | emd_11610_validation.cif.gz | 18.9 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-11610 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-11610 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_11610.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | GABAAR-b3 homomer solubilised in nanodiscs and in complex with anti-MSP megabodies | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 0.705 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : Homomeric truncated (M3-M4 loop) GABAAR b3K279T receptor in compl...
Entire | Name: Homomeric truncated (M3-M4 loop) GABAAR b3K279T receptor in complex with megabody MbNbF3c7HopQ |
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Components |
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-Supramolecule #1: Homomeric truncated (M3-M4 loop) GABAAR b3K279T receptor in compl...
Supramolecule | Name: Homomeric truncated (M3-M4 loop) GABAAR b3K279T receptor in complex with megabody MbNbF3c7HopQ type: complex / ID: 1 / Parent: 0 / Macromolecule list: all |
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Molecular weight | Theoretical: 500 KDa |
-Supramolecule #2: Homomeric truncated (M3-M4 loop) GABAAR b3K279T receptor
Supramolecule | Name: Homomeric truncated (M3-M4 loop) GABAAR b3K279T receptor type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1 |
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Source (natural) | Organism: Homo sapiens (human) / Organ: Brain / Location in cell: Plasma membrane |
Recombinant expression | Organism: Homo sapiens (human) / Recombinant cell: HEK293S-GnTI- / Recombinant plasmid: pHL-sec |
-Supramolecule #3: Megabody MbNbF3c7HopQ
Supramolecule | Name: Megabody MbNbF3c7HopQ / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2 |
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Source (natural) | Organism: Lama glama (llama) |
Recombinant expression | Organism: Escherichia coli (E. coli) / Recombinant strain: WK6 su- |
-Macromolecule #1: Gamma-aminobutyric acid receptor subunit beta-3,Gamma-aminobutyri...
Macromolecule | Name: Gamma-aminobutyric acid receptor subunit beta-3,Gamma-aminobutyric acid receptor subunit beta-3 type: protein_or_peptide / ID: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Homo sapiens (human) |
Recombinant expression | Organism: Homo sapiens (human) |
Sequence | String: 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 |
-Macromolecule #2: Megabody MbNbF3c7HopQ
Macromolecule | Name: Megabody MbNbF3c7HopQ / type: protein_or_peptide / ID: 2 Details: Megabody is a chimeric antigen binding protein build of a nanobody and the adhesin domain of H. pylori (HopQ) Enantiomer: LEVO |
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Source (natural) | Organism: Lama glama (llama) |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: QVQLVESGGG LVQTKTTTSV IDTTNDAQNL LTQAQTIVNT LKDYCPILIA KSSSSNGGTN NANTPSWQTA GGGKNSCATF GAEFSAASDM INNAQKIVQE TQQLSANQPK NITQPHNLNL NSPSSLTALA QKMLKNAQSQ AEILKLANQV ESDFNKLSSG HLKDYIGKCD ...String: QVQLVESGGG LVQTKTTTSV IDTTNDAQNL LTQAQTIVNT LKDYCPILIA KSSSSNGGTN NANTPSWQTA GGGKNSCATF GAEFSAASDM INNAQKIVQE TQQLSANQPK NITQPHNLNL NSPSSLTALA QKMLKNAQSQ AEILKLANQV ESDFNKLSSG HLKDYIGKCD ASAISSANMT MQNQKNNWGN GCAGVEETQS LLKTSAADFN NQTPQINQAQ NLANTLIQEL GNNTYEQLSR LLTNDNGTNS KTSAQAINQA VNNLNERAKT LAGGTTNSPA YQATLLALRS VLGLWNSMGY AVICGGYTKS PGENNQKDFH YTDENGNGTT INCGGSTNSN GTHSYNGTNT LKADKNVSLS IEQYEKIHEA YQILSKALKQ AGLAPLNSKG EKLEAHVTTS KYGSLRLSCA ASGSGFGPNV MGWYRQAPGK RRELVATINR IGNINYGDSV KGRFTLSREI AVNTVYLQMN SLKPEDAANY YCYAAKYGVD YWGKGTQVTV SSHHHHHHEP EA |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.1 mg/mL |
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Buffer | pH: 7.6 |
Vitrification | Cryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV |
Details | Monodisperse sample |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Specialist optics | Phase plate: OTHER |
Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 65.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination 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: 165000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
+Image processing
-Atomic model buiding 1
Refinement | Protocol: FLEXIBLE FIT |
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