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- PDB-7a5v: CryoEM structure of a human gamma-aminobutyric acid receptor, the... -

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Entry
Database: PDB / ID: 7a5v
TitleCryoEM structure of a human gamma-aminobutyric acid receptor, the GABA(A)R-beta3 homopentamer, in complex with histamine and megabody Mb25 in lipid nanodisc
Components
  • Gamma-aminobutyric acid receptor subunit beta-3,Gamma-aminobutyric acid receptor subunit beta-3
  • Megabody Mb25
KeywordsMEMBRANE PROTEIN / Pentameric ligand-gated ion channel / Neurotrasmitter receptor / GABA(A) receptor
Function / homology
Function and homology information


inner ear receptor cell development / GABA-A receptor activity / GABA-gated chloride ion channel activity / GABA-A receptor complex / cellular response to histamine / inhibitory synapse assembly / gamma-aminobutyric acid signaling pathway / innervation / inhibitory postsynaptic potential / neurotransmitter receptor activity ...inner ear receptor cell development / GABA-A receptor activity / GABA-gated chloride ion channel activity / GABA-A receptor complex / cellular response to histamine / inhibitory synapse assembly / gamma-aminobutyric acid signaling pathway / innervation / inhibitory postsynaptic potential / neurotransmitter receptor activity / nervous system process / GABA-ergic synapse / chloride channel complex / cochlea development / chloride transmembrane transport / roof of mouth development / transmitter-gated ion channel activity involved in regulation of postsynaptic membrane potential / ion transmembrane transport / regulation of membrane potential / sensory perception of sound / chemical synaptic transmission / cytoplasmic vesicle membrane / postsynaptic membrane / negative regulation of neuron apoptotic process / neuron projection / synapse / signal transduction / integral component of plasma membrane / identical protein binding / plasma membrane
Neurotransmitter-gated ion-channel ligand-binding domain superfamily / Neurotransmitter-gated ion-channel transmembrane domain superfamily / Neurotransmitter-gated ion-channel, conserved site / Neurotransmitter-gated ion-channel ligand-binding domain / Neurotransmitter-gated ion-channel / Neurotransmitter-gated ion-channel transmembrane domain / Gamma-aminobutyric acid A receptor/Glycine receptor alpha / Gamma-aminobutyric-acid A receptor, beta subunit
Gamma-aminobutyric acid receptor subunit beta-3 / polysac:dglcpnacb1-4dglcpnacb1-roh: / polysac:dmanpa1-3[dmanpa1-6]dmanpb1-4dglcpnacb1-4dglcpnacb1-roh:
Biological speciesHomo sapiens (human)
Lama glama (llama)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 1.7 Å
AuthorsNakane, T. / Kotecha, A. / Sente, A. / Yamashita, K. / McMullan, G. / Masiulis, S. / Brown, P.M.G.E. / Grigoras, I.T. / Malinauskaite, L. / Malinauskas, T. ...Nakane, T. / Kotecha, A. / Sente, A. / Yamashita, K. / McMullan, G. / Masiulis, S. / Brown, P.M.G.E. / Grigoras, I.T. / Malinauskaite, L. / Malinauskas, T. / Miehling, J. / Yu, L. / Karia, D. / Pechnikova, E.V. / de Jong, E. / Keizer, J. / Bischoff, M. / McCormack, J. / Tiemeijer, P. / Hardwick, S.W. / Chirgadze, D.Y. / Murshudov, G. / Aricescu, A.R. / Scheres, S.H.W.
Funding support United Kingdom, 6items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MC_UP_A025_1012 United Kingdom
Medical Research Council (MRC, United Kingdom)MR/L009609/1 United Kingdom
Medical Research Council (MRC, United Kingdom)MC_UP_1201/15 United Kingdom
Medical Research Council (MRC, United Kingdom)MC_UP_A025_1013 United Kingdom
Wellcome Trust206171/Z/17/Z United Kingdom
Wellcome Trust202905/Z/16/Z) United Kingdom
CitationJournal: Nature / Year: 2020
Title: Single-particle cryo-EM at atomic resolution.
Authors: Takanori Nakane / Abhay Kotecha / Andrija Sente / Greg McMullan / Simonas Masiulis / Patricia M G E Brown / Ioana T Grigoras / Lina Malinauskaite / Tomas Malinauskas / Jonas Miehling / ...Authors: Takanori Nakane / Abhay Kotecha / Andrija Sente / Greg McMullan / Simonas Masiulis / Patricia M G E Brown / Ioana T Grigoras / Lina Malinauskaite / Tomas Malinauskas / Jonas Miehling / Tomasz Uchański / Lingbo Yu / Dimple Karia / Evgeniya V Pechnikova / Erwin de Jong / Jeroen Keizer / Maarten Bischoff / Jamie McCormack / Peter Tiemeijer / Steven W Hardwick / Dimitri Y Chirgadze / Garib Murshudov / A Radu Aricescu / Sjors H W Scheres /
Abstract: The three-dimensional positions of atoms in protein molecules define their structure and their roles in biological processes. The more precisely atomic coordinates are determined, the more chemical ...The three-dimensional positions of atoms in protein molecules define their structure and their roles in biological processes. The more precisely atomic coordinates are determined, the more chemical information can be derived and the more mechanistic insights into protein function may be inferred. Electron cryo-microscopy (cryo-EM) single-particle analysis has yielded protein structures with increasing levels of detail in recent years. However, it has proved difficult to obtain cryo-EM reconstructions with sufficient resolution to visualize individual atoms in proteins. Here we use a new electron source, energy filter and camera to obtain a 1.7 Å resolution cryo-EM reconstruction for a human membrane protein, the β3 GABA receptor homopentamer. Such maps allow a detailed understanding of small-molecule coordination, visualization of solvent molecules and alternative conformations for multiple amino acids, and unambiguous building of ordered acidic side chains and glycans. Applied to mouse apoferritin, our strategy led to a 1.22 Å resolution reconstruction that offers a genuine atomic-resolution view of a protein molecule using single-particle cryo-EM. Moreover, the scattering potential from many hydrogen atoms can be visualized in difference maps, allowing a direct analysis of hydrogen-bonding networks. Our technological advances, combined with further approaches to accelerate data acquisition and improve sample quality, provide a route towards routine application of cryo-EM in high-throughput screening of small molecule modulators and structure-based drug discovery.
Validation Report
SummaryFull reportAbout validation report
History
DepositionAug 22, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 18, 2020Provider: repository / Type: Initial release
Revision 1.1Nov 25, 2020Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

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  • Biological unit as author_and_software_defined_assembly
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Gamma-aminobutyric acid receptor subunit beta-3,Gamma-aminobutyric acid receptor subunit beta-3
O: Megabody Mb25
hetero molecules


Theoretical massNumber of molelcules
Total (without water)104,69314
Polymers102,0452
Non-polymers2,64812
Water3,657203
1
A: Gamma-aminobutyric acid receptor subunit beta-3,Gamma-aminobutyric acid receptor subunit beta-3
O: Megabody Mb25
hetero molecules

A: Gamma-aminobutyric acid receptor subunit beta-3,Gamma-aminobutyric acid receptor subunit beta-3
O: Megabody Mb25
hetero molecules

A: Gamma-aminobutyric acid receptor subunit beta-3,Gamma-aminobutyric acid receptor subunit beta-3
O: Megabody Mb25
hetero molecules

A: Gamma-aminobutyric acid receptor subunit beta-3,Gamma-aminobutyric acid receptor subunit beta-3
O: Megabody Mb25
hetero molecules

A: Gamma-aminobutyric acid receptor subunit beta-3,Gamma-aminobutyric acid receptor subunit beta-3
O: Megabody Mb25
hetero molecules


Theoretical massNumber of molelcules
Total (without water)523,46670
Polymers510,22510
Non-polymers13,24160
Water18010
TypeNameSymmetry operationNumber
identity operation1
point symmetry operation4
Methodauthor_and_software_defined_assembly
Noncrystallographic symmetry (NCS)NCS oper:
IDCodeMatrixVector
1given(1), (1), (1)
2generate(0.309017, -0.951057), (0.951057, 0.309017), (1)239.91182, -37.9983
3generate(-0.809017, -0.587785), (0.587785, -0.809017), (1)350.18719, 178.42929
4generate(-0.809017, 0.587785), (-0.587785, -0.809017), (1)178.42929, 350.18719
5generate(0.309017, 0.951057), (-0.951057, 0.309017), (1)-37.9983, 239.91182

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Components

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Protein / Antibody , 2 types, 2 molecules AO

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


Mass: 45744.285 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GABRB3 / Cell line (production host): HEK293S / Production host: Homo sapiens (human) / References: UniProt: P28472
#2: Antibody Megabody Mb25


Mass: 56300.629 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Production host: Escherichia coli (E. coli)

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Sugars , 3 types, 3 molecules

#3: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}LINUCSPDB-CARE
#4: 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.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-3[DManpa1-6]DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/3,5,4/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1122h-1a_1-5]/1-1-2-3-3/a4-b1_b4-c1_c3-d1_c6-e1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(3+1)][a-D-Manp]{}[(6+1)][a-D-Manp]{}}}}LINUCSPDB-CARE
#7: 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 / N-Acetylglucosamine


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 1 / Source method: obtained synthetically / 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

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Non-polymers , 6 types, 212 molecules

#5: Chemical ChemComp-R16 / HEXADECANE / Hexadecane


Mass: 226.441 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C16H34
#6: Chemical
ChemComp-D10 / DECANE / Decane


Mass: 142.282 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H22
#8: Chemical ChemComp-OCT / N-OCTANE / Octane


Mass: 114.229 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C8H18
#9: Chemical ChemComp-HSM / HISTAMINE / Histamine


Mass: 111.145 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C5H9N3 / Comment: neurotransmitter*YM
#10: Chemical ChemComp-CL / CHLORIDE ION / Chloride


Mass: 35.453 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Cl
#11: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 203 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestN

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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
1Human GABA(A)R-beta3 homopentamer in complex with Megabody-25 in lipid nanodiscCOMPLEX#1-#20MULTIPLE SOURCES
2Human GABA(A)R-beta3 homopentamerCOMPLEX#11RECOMBINANT
3Megabody-25COMPLEX#21RECOMBINANT
Molecular weightValue: 0.485 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Homo sapiens (human)9606
33Lama glama (llama)9844
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrain
22Homo sapiens (human)9606HEK293S
33Escherichia coli (E. coli)562
Buffer solutionpH: 7.6
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: LEICA PLUNGER / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 287 K

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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 FIELDBright-field microscopy / Nominal magnification: 165000 X / Nominal defocus max: 1100 nm / Nominal defocus min: 300 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 40 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)
Image scansWidth: 4096 / Height: 4096

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Processing

SoftwareName: REFMAC / Version: 5.8.0270 / Classification: refinement
EM software
IDNameVersionCategory
2EPUimage acquisition
4CTFFIND4.1.13CTF correction
7REFMAC5.8.0270model fitting
9RELION3.1initial Euler assignment
10RELION3.1final Euler assignment
11RELION3.1classification
12RELION3.13D reconstruction
13REFMAC5.8.0270model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C5 (5 fold cyclic)
3D reconstructionResolution: 1.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 371693 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: RECIPROCAL
RefinementResolution: 1.7→292.21 Å / Cor.coef. Fo:Fc: 0.862 / SU B: 0.591 / SU ML: 0.017 / ESU R: 0.009
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS /
Rfactor% reflection
Rwork0.27621 -
Obs0.27621 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 50.129 Å2
Baniso -1Baniso -2Baniso -3
1-0.26 Å2-0 Å20 Å2
2--0.25 Å20 Å2
3----0.51 Å2
Refinement stepCycle: 1 / Total: 4231
Refine LS restraints
Refinement-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0090.0134156
ELECTRON MICROSCOPYr_bond_other_d00.0173930
ELECTRON MICROSCOPYr_angle_refined_deg1.31.6875678
ELECTRON MICROSCOPYr_angle_other_deg1.1611.6029052
ELECTRON MICROSCOPYr_dihedral_angle_1_deg7.1355499
ELECTRON MICROSCOPYr_dihedral_angle_2_deg30.34321.048210
ELECTRON MICROSCOPYr_dihedral_angle_3_deg13.05215643
ELECTRON MICROSCOPYr_dihedral_angle_4_deg17.1691528
ELECTRON MICROSCOPYr_chiral_restr0.0650.2556
ELECTRON MICROSCOPYr_gen_planes_refined0.0180.025073
ELECTRON MICROSCOPYr_gen_planes_other0.010.021051
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it5.654.6681912
ELECTRON MICROSCOPYr_mcbond_other5.6474.6591911
ELECTRON MICROSCOPYr_mcangle_it8.9646.9152413
ELECTRON MICROSCOPYr_mcangle_other8.9656.9242414
ELECTRON MICROSCOPYr_scbond_it6.9055.4952244
ELECTRON MICROSCOPYr_scbond_other6.9035.52245
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other11.347.8343256
ELECTRON MICROSCOPYr_long_range_B_refined15.67655.1264532
ELECTRON MICROSCOPYr_long_range_B_other15.67555.1824533
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 1.7→1.717 Å / Total num. of bins used: 50
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork0.641 317209 -
Obs--100 %

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