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- PDB-6usf: CryoEM structure of human alpha4beta2 nicotinic acetylcholine rec... -

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Basic information

Entry
Database: PDB / ID: 6usf
TitleCryoEM structure of human alpha4beta2 nicotinic acetylcholine receptor with varenicline in complex with anti-BRIL synthetic antibody BAK5
Components
  • Fab heavy chain
  • Fab light chain
  • Neuronal acetylcholine receptor subunit beta-2
  • chimera of soluble cytochrome b562 (BRIL) and neuronal acetylcholine receptor subunit alpha-4
KeywordsMEMBRANE PROTEIN/IMMUNE SYSTEM / acetylcholine receptor / nicotine / varenicline / anti-BRIL synthetic antibody / MEMBRANE PROTEIN / MEMBRANE PROTEIN-IMMUNE SYSTEM complex
Function / homology
Function and homology information


vestibulocochlear nerve development / lateral geniculate nucleus development / regulation of circadian sleep/wake cycle, REM sleep / regulation of synaptic transmission, dopaminergic / quaternary ammonium group binding / synaptic transmission involved in micturition / Highly sodium permeable postsynaptic acetylcholine nicotinic receptors / Highly calcium permeable nicotinic acetylcholine receptors / optic nerve morphogenesis / response to acetylcholine ...vestibulocochlear nerve development / lateral geniculate nucleus development / regulation of circadian sleep/wake cycle, REM sleep / regulation of synaptic transmission, dopaminergic / quaternary ammonium group binding / synaptic transmission involved in micturition / Highly sodium permeable postsynaptic acetylcholine nicotinic receptors / Highly calcium permeable nicotinic acetylcholine receptors / optic nerve morphogenesis / response to acetylcholine / Highly calcium permeable postsynaptic nicotinic acetylcholine receptors / central nervous system projection neuron axonogenesis / acetylcholine-gated channel complex / cholinergic synapse / regulation of dopamine metabolic process / negative regulation of action potential / behavioral response to nicotine / positive regulation of dopamine secretion / acetylcholine receptor activity / inhibitory postsynaptic potential / synaptic transmission, cholinergic / acetylcholine binding / postsynaptic specialization membrane / nervous system process / acetylcholine receptor signaling pathway / acetylcholine-gated monoatomic cation-selective channel activity / regulation of synapse assembly / action potential / regulation of dendrite morphogenesis / heterocyclic compound binding / B cell activation / regulation of dopamine secretion / plasma membrane raft / membrane depolarization / associative learning / social behavior / ligand-gated monoatomic ion channel activity / smooth muscle contraction / monoatomic ion transport / positive regulation of B cell proliferation / sensory perception of pain / visual perception / response to cocaine / learning / locomotory behavior / regulation of membrane potential / electron transport chain / sensory perception of sound / response to nicotine / visual learning / memory / cognition / calcium ion transport / presynaptic membrane / chemical synaptic transmission / postsynaptic membrane / response to ethanol / response to oxidative stress / periplasmic space / electron transfer activity / response to hypoxia / neuron projection / iron ion binding / external side of plasma membrane / DNA repair / neuronal cell body / synapse / dendrite / heme binding / protein-containing complex binding / signal transduction / membrane / plasma membrane
Similarity search - Function
Nicotinic acetylcholine receptor / Acetylcholine Binding Protein; Chain: A, / Neurotransmitter-gated ion-channel ligand-binding domain / Neurotransmitter-gated ion-channel, conserved site / Neurotransmitter-gated ion-channels signature. / Neurotransmitter-gated ion-channel transmembrane domain / Neurotransmitter-gated ion-channel transmembrane region / Cytochrome b562 / Cytochrome b562 / Cytochrome c/b562 ...Nicotinic acetylcholine receptor / Acetylcholine Binding Protein; Chain: A, / Neurotransmitter-gated ion-channel ligand-binding domain / Neurotransmitter-gated ion-channel, conserved site / Neurotransmitter-gated ion-channels signature. / Neurotransmitter-gated ion-channel transmembrane domain / Neurotransmitter-gated ion-channel transmembrane region / Cytochrome b562 / Cytochrome b562 / Cytochrome c/b562 / 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 / Distorted Sandwich / Mainly Beta
Similarity search - Domain/homology
beta-D-mannopyranose / VARENICLINE / Soluble cytochrome b562 / Neuronal acetylcholine receptor subunit beta-2 / Neuronal acetylcholine receptor subunit alpha-4
Similarity search - Component
Biological speciesHomo sapiens (human)
Escherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.87 Å
AuthorsAlvarez, F.J.D. / Mukherjee, S. / Han, S. / Ammirati, M. / Kossiakoff, A.A.
CitationJournal: Nat Commun / Year: 2020
Title: Synthetic antibodies against BRIL as universal fiducial marks for single-particle cryoEM structure determination of membrane proteins.
Authors: Somnath Mukherjee / Satchal K Erramilli / Mark Ammirati / Frances J D Alvarez / Kimberly F Fennell / Michael D Purdy / Blazej M Skrobek / Katarzyna Radziwon / John Coukos / Yanyong Kang / ...Authors: Somnath Mukherjee / Satchal K Erramilli / Mark Ammirati / Frances J D Alvarez / Kimberly F Fennell / Michael D Purdy / Blazej M Skrobek / Katarzyna Radziwon / John Coukos / Yanyong Kang / Przemysław Dutka / Xiang Gao / Xiayang Qiu / Mark Yeager / H Eric Xu / Seungil Han / Anthony A Kossiakoff /
Abstract: We propose the concept of universal fiducials based on a set of pre-made semi-synthetic antibodies (sABs) generated by customized phage display selections against the fusion protein BRIL, an ...We propose the concept of universal fiducials based on a set of pre-made semi-synthetic antibodies (sABs) generated by customized phage display selections against the fusion protein BRIL, an engineered variant of apocytochrome b562a. These sABs can bind to BRIL fused either into the loops or termini of different GPCRs, ion channels, receptors and transporters without disrupting their structure. A crystal structure of BRIL in complex with an affinity-matured sAB (BAG2) that bound to all systems tested delineates the footprint of interaction. Negative stain and cryoEM data of several examples of BRIL-membrane protein chimera highlight the effectiveness of the sABs as universal fiducial marks. Taken together with a cryoEM structure of sAB bound human nicotinic acetylcholine receptor, this work demonstrates that these anti-BRIL sABs can greatly enhance the particle properties leading to improved cryoEM outcomes, especially for challenging membrane proteins.
History
DepositionOct 26, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 29, 2020Provider: repository / Type: Initial release
Revision 1.1May 6, 2020Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _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
Revision 2.0Jul 29, 2020Group: Advisory / Atomic model ...Advisory / Atomic model / Data collection / Derived calculations / Structure summary
Category: atom_site / chem_comp ...atom_site / chem_comp / entity / pdbx_branch_scheme / pdbx_chem_comp_identifier / pdbx_entity_branch / pdbx_entity_branch_descriptor / pdbx_entity_branch_link / pdbx_entity_branch_list / pdbx_entity_nonpoly / pdbx_nonpoly_scheme / pdbx_struct_assembly_gen / pdbx_unobs_or_zero_occ_atoms / pdbx_validate_close_contact / struct_asym / struct_conn
Item: _atom_site.B_iso_or_equiv / _atom_site.Cartn_x ..._atom_site.B_iso_or_equiv / _atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _atom_site.auth_asym_id / _atom_site.auth_atom_id / _atom_site.auth_comp_id / _atom_site.auth_seq_id / _atom_site.label_asym_id / _atom_site.label_atom_id / _atom_site.label_comp_id / _atom_site.label_entity_id / _atom_site.type_symbol / _chem_comp.name / _pdbx_entity_nonpoly.entity_id / _pdbx_entity_nonpoly.name / _pdbx_struct_assembly_gen.asym_id_list / _pdbx_unobs_or_zero_occ_atoms.auth_asym_id / _pdbx_unobs_or_zero_occ_atoms.auth_seq_id / _pdbx_unobs_or_zero_occ_atoms.label_asym_id / _pdbx_validate_close_contact.auth_asym_id_1 / _pdbx_validate_close_contact.auth_asym_id_2 / _pdbx_validate_close_contact.auth_atom_id_1 / _pdbx_validate_close_contact.auth_atom_id_2 / _pdbx_validate_close_contact.auth_seq_id_1 / _pdbx_validate_close_contact.auth_seq_id_2 / _struct_conn.pdbx_role / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id
Description: Carbohydrate remediation / Provider: repository / Type: Remediation

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Structure visualization

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Assembly

Deposited unit
A: chimera of soluble cytochrome b562 (BRIL) and neuronal acetylcholine receptor subunit alpha-4
B: Neuronal acetylcholine receptor subunit beta-2
C: Neuronal acetylcholine receptor subunit beta-2
D: chimera of soluble cytochrome b562 (BRIL) and neuronal acetylcholine receptor subunit alpha-4
E: Neuronal acetylcholine receptor subunit beta-2
H: Fab heavy chain
L: Fab light chain
K: Fab heavy chain
M: Fab light chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)347,97219
Polymers345,2949
Non-polymers2,67910
Water00
1


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

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Components

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Protein , 2 types, 5 molecules ADBCE

#1: Protein chimera of soluble cytochrome b562 (BRIL) and neuronal acetylcholine receptor subunit alpha-4 / Cytochrome b-562


Mass: 55914.691 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human), (gene. exp.) Escherichia coli (E. coli)
Gene: CHRNA4, NACRA4, cybC / Plasmid: pcDNA3.1(+) / Cell line (production host): Expi293F / Production host: Homo sapiens (human) / References: UniProt: P43681, UniProt: P0ABE7
#2: Protein Neuronal acetylcholine receptor subunit beta-2


Mass: 45931.953 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CHRNB2 / Plasmid: pcDNA3.1(+) / Cell line (production host): Expi293F / Production host: Homo sapiens (human) / References: UniProt: P17787

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Antibody , 2 types, 4 molecules HKLM

#3: Antibody Fab heavy chain


Mass: 24351.139 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Description: Ampicillin resistant, IPTG inducible / Plasmid: pRH2.2 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21 (gold)
#4: Antibody Fab light chain


Mass: 23483.062 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Description: Ampicillin resistant, IPTG inducible / Plasmid: pRH2.2 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21 (gold)

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Sugars , 4 types, 8 molecules

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


Type: oligosaccharide / Mass: 383.349 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/2,2,1/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5]/1-2/a4-b1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-Manp]{}}LINUCSPDB-CARE
#6: 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: obtained synthetically
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
#7: Sugar
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 4 / 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
#9: Sugar ChemComp-BMA / beta-D-mannopyranose


Type: D-saccharide, beta linking / Mass: 180.156 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Formula: C6H12O6
IdentifierTypeProgram
DManpbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
b-D-mannopyranoseCOMMON NAMEGMML 1.0
b-D-ManpIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
ManSNFG CARBOHYDRATE SYMBOLGMML 1.0

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Non-polymers , 1 types, 2 molecules

#8: Chemical ChemComp-QMR / VARENICLINE


Mass: 211.262 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C13H13N3 / Feature type: SUBJECT OF INVESTIGATION / Comment: medication, agonist*YM

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Details

Has ligand of interestY

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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

ComponentName: human alpha4beta2 nicotinic acetylcholine receptor in complex fab fragments and bound to varenicline
Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTrisC4H11NO31
2150 mMsodium chlorideNaCl1
30.1 mMtris(2-carboxyethyl)phosphineC9H15O6P1
4100 uMvareniclineC13H13N31
50.018 %n-dodecyl-beta-D-maltosideC24H46O11
60.006 %Cholesteryl HemisuccinateC31H50O41
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: -30 mA / Grid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 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 FIELD / Nominal defocus max: 2400 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 10.5 sec. / Electron dose: 52 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3418
Image scansMovie frames/image: 35

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Processing

EM software
IDNameVersionCategory
2SerialEMimage acquisition
4CTFFIND4CTF correction
7UCSF Chimera1.13model fitting
9PHENIX1.16-3549model refinement
10RELION3.0-betainitial Euler assignment
11RELION3.0-betafinal Euler assignment
13RELION3.0-beta3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1867100
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.87 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 285852 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model building
IDPDB-IDPdb chain-ID 3D fitting-ID
16CNJ

6cnj

A1
26CNJ

6cnj

B1
36CNJ

6cnj

C1
46CNJ

6cnj

D1
56CNJ

6cnj

E1
66CBV

6cbv

B1
76CBV

6cbv

H1
86CBV

6cbv

L1

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