[English] 日本語
Yorodumi
- PDB-6ur8: CryoEM structure of human alpha4beta2 nicotinic acetylcholine rec... -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: PDB / ID: 6ur8
TitleCryoEM structure of human alpha4beta2 nicotinic acetylcholine receptor in complex with varenicline
Components
  • Chimera of soluble cytochrome b562 (BRIL) and neuronal acetylcholine receptor subunit alpha-4
  • Neuronal acetylcholine receptor subunit beta-2
KeywordsMEMBRANE PROTEIN / acetylcholine receptor / nicotine / varenicline
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 / optic nerve morphogenesis / Highly sodium permeable postsynaptic acetylcholine nicotinic receptors / Highly calcium permeable nicotinic acetylcholine receptors / central nervous system projection neuron axonogenesis / synaptic transmission involved in micturition / response to acetylcholine ...vestibulocochlear nerve development / lateral geniculate nucleus development / regulation of circadian sleep/wake cycle, REM sleep / regulation of synaptic transmission, dopaminergic / optic nerve morphogenesis / Highly sodium permeable postsynaptic acetylcholine nicotinic receptors / Highly calcium permeable nicotinic acetylcholine receptors / central nervous system projection neuron axonogenesis / synaptic transmission involved in micturition / response to acetylcholine / negative regulation of action potential / Highly calcium permeable postsynaptic nicotinic acetylcholine receptors / acetylcholine receptor activity / regulation of dopamine metabolic process / positive regulation of dopamine secretion / acetylcholine-gated channel complex / behavioral response to nicotine / neuromuscular synaptic transmission / acetylcholine-gated monoatomic cation-selective channel activity / cation channel complex / acetylcholine binding / synaptic transmission, cholinergic / nervous system process / acetylcholine receptor signaling pathway / neurotransmitter receptor complex / ligand-gated monoatomic ion channel activity / regulation of dendrite morphogenesis / inhibitory postsynaptic potential / regulation of synapse assembly / regulation of dopamine secretion / B cell activation / associative learning / action potential / smooth muscle contraction / plasma membrane raft / social behavior / membrane depolarization / positive regulation of B cell proliferation / monoatomic ion transport / sensory perception of pain / visual perception / learning / response to nicotine / regulation of membrane potential / response to cocaine / locomotory behavior / sensory perception of sound / electron transport chain / visual learning / cognition / memory / calcium ion transport / presynaptic membrane / response to oxidative stress / response to ethanol / monoatomic ion transmembrane transport / chemical synaptic transmission / postsynaptic membrane / periplasmic space / response to hypoxia / electron transfer activity / neuron projection / iron ion binding / external side of plasma membrane / DNA repair / neuronal cell body / heme binding / dendrite / synapse / 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: 3.71 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 22, 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 1.2Jul 29, 2020Group: Data collection / Derived calculations / Structure summary
Category: chem_comp / entity ...chem_comp / entity / pdbx_chem_comp_identifier / pdbx_entity_nonpoly / struct_site / struct_site_gen
Item: _chem_comp.name / _entity.pdbx_description / _pdbx_entity_nonpoly.name
Description: Carbohydrate remediation / Provider: repository / Type: Remediation
Revision 1.3Nov 13, 2024Group: Data collection / Database references ...Data collection / Database references / Refinement description / Structure summary
Category: chem_comp / chem_comp_atom ...chem_comp / chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / em_admin / pdbx_entry_details / pdbx_initial_refinement_model / pdbx_modification_feature
Item: _chem_comp.pdbx_synonyms / _database_2.pdbx_DOI ..._chem_comp.pdbx_synonyms / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_admin.last_update / _pdbx_entry_details.has_protein_modification

-
Structure visualization

Movie
  • Deposited structure unit
  • Imaged by Jmol
  • Download
  • Superimposition on EM map
  • EMDB-20857
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

-
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
hetero molecules


Theoretical massNumber of molelcules
Total (without water)252,58418
Polymers249,8525
Non-polymers2,73313
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, Shift to larger molecular weight was observed by SEC after fab addition. SDS-PAGE indicates that receptor and Fab co-elute., microscopy, Negative stain TEM shows Fab bound to the receptor.
TypeNameSymmetry operationNumber
identity operation1_5551

-
Components

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


Mass: 56027.848 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
#3: 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.: 8 / 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
#4: Chemical ChemComp-QMR / VARENICLINE


Mass: 211.262 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C13H13N3 / Feature type: SUBJECT OF INVESTIGATION
#5: Sugar ChemComp-BMA / beta-D-mannopyranose / beta-D-mannose / D-mannose / mannose


Type: D-saccharide, beta linking / Mass: 180.156 Da / Num. of mol.: 3 / Source method: obtained synthetically / 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
Has ligand of interestY
Has protein modificationY

-
Experimental details

-
Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

-
Sample preparation

ComponentName: human alpha4beta2 nicotinic acetylcholine receptor in complex fab fragments and bound to varenicline
Type: COMPLEX / Entity ID: #1-#2 / 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 supportGrid material: GOLD / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

-
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
Image scansMovie frames/image: 35

-
Processing

EM software
IDNameVersionCategory
2SerialEMimage acquisition
4CTFFIND4CTF correction
7UCSF Chimera1.13model fitting
9RELION3.0-betainitial Euler assignment
10RELION3.0-betafinal Euler assignment
12RELION3.0-beta3D reconstruction
13PHENIX1.16-3549model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1867100
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.71 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 226962 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model building

3D fitting-ID: 1 / Accession code: 6CNJ / Initial refinement model-ID: 1 / PDB-ID: 6CNJ

6cnj

/ Source name: PDB / Type: experimental model

IDPdb chain-ID
1A
2B
3C
4D
5E

+
About Yorodumi

-
News

-
Feb 9, 2022. New format data for meta-information of EMDB entries

New format data for meta-information of EMDB entries

  • Version 3 of the EMDB header file is now the official format.
  • The previous official version 1.9 will be removed from the archive.

Related info.:EMDB header

External links:wwPDB to switch to version 3 of the EMDB data model

-
Aug 12, 2020. Covid-19 info

Covid-19 info

URL: https://pdbj.org/emnavi/covid19.php

New page: Covid-19 featured information page in EM Navigator.

Related info.:Covid-19 info / Mar 5, 2020. Novel coronavirus structure data

+
Mar 5, 2020. Novel coronavirus structure data

Novel coronavirus structure data

Related info.:Yorodumi Speices / Aug 12, 2020. Covid-19 info

External links:COVID-19 featured content - PDBj / Molecule of the Month (242)：Coronavirus Proteases

+
Jan 31, 2019. EMDB accession codes are about to change! (news from PDBe EMDB page)

EMDB accession codes are about to change! (news from PDBe EMDB page)

  • The allocation of 4 digits for EMDB accession codes will soon come to an end. Whilst these codes will remain in use, new EMDB accession codes will include an additional digit and will expand incrementally as the available range of codes is exhausted. The current 4-digit format prefixed with “EMD-” (i.e. EMD-XXXX) will advance to a 5-digit format (i.e. EMD-XXXXX), and so on. It is currently estimated that the 4-digit codes will be depleted around Spring 2019, at which point the 5-digit format will come into force.
  • The EM Navigator/Yorodumi systems omit the EMD- prefix.

Related info.:Q: What is EMD? / ID/Accession-code notation in Yorodumi/EM Navigator

External links:EMDB Accession Codes are Changing Soon! / Contact to PDBj

+
Jul 12, 2017. Major update of PDB

Major update of PDB

  • wwPDB released updated PDB data conforming to the new PDBx/mmCIF dictionary.
  • This is a major update changing the version number from 4 to 5, and with Remediation, in which all the entries are updated.
  • In this update, many items about electron microscopy experimental information are reorganized (e.g. em_software).
  • Now, EM Navigator and Yorodumi are based on the updated data.

External links:wwPDB Remediation / Enriched Model Files Conforming to OneDep Data Standards Now Available in the PDB FTP Archive

-
Yorodumi

Thousand views of thousand structures

  • Yorodumi is a browser for structure data from EMDB, PDB, SASBDB, etc.
  • This page is also the successor to EM Navigator detail page, and also detail information page/front-end page for Omokage search.
  • The word "yorodu" (or yorozu) is an old Japanese word meaning "ten thousand". "mi" (miru) is to see.

Related info.:EMDB / PDB / SASBDB / Comparison of 3 databanks / Yorodumi Search / Aug 31, 2016. New EM Navigator & Yorodumi / Yorodumi Papers / Jmol/JSmol / Function and homology information / Changes in new EM Navigator and Yorodumi

Read more