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- EMDB-20857: CryoEM structure of human alpha4beta2 nicotinic acetylcholine rec... -

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Basic information

Entry
Database: EMDB / ID: EMD-20857
TitleCryoEM structure of human alpha4beta2 nicotinic acetylcholine receptor in complex with varenicline
Map datacytochrome B562-fused human alpha4beta2 nicotinic acetylcholine receptor in complex with fab fragments and bound to varenicline
Sample
  • Complex: human alpha4beta2 nicotinic acetylcholine receptor in complex fab fragments and bound to varenicline
    • Protein or peptide: Chimera of soluble cytochrome b562 (BRIL) and neuronal acetylcholine receptor subunit alpha-4
    • Protein or peptide: Neuronal acetylcholine receptor subunit beta-2
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: VARENICLINE
  • Ligand: beta-D-mannopyranose
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 / synaptic transmission involved in micturition / quaternary ammonium group binding / Highly sodium permeable postsynaptic acetylcholine nicotinic receptors / Highly calcium permeable nicotinic acetylcholine receptors / optic nerve morphogenesis / central nervous system projection neuron axonogenesis ...vestibulocochlear nerve development / lateral geniculate nucleus development / regulation of circadian sleep/wake cycle, REM sleep / regulation of synaptic transmission, dopaminergic / synaptic transmission involved in micturition / quaternary ammonium group binding / Highly sodium permeable postsynaptic acetylcholine nicotinic receptors / Highly calcium permeable nicotinic acetylcholine receptors / optic nerve morphogenesis / central nervous system projection neuron axonogenesis / Highly calcium permeable postsynaptic nicotinic acetylcholine receptors / acetylcholine receptor activity / response to acetylcholine / cholinergic synapse / acetylcholine-gated channel complex / negative regulation of action potential / positive regulation of dopamine secretion / regulation of dopamine metabolic process / behavioral response to nicotine / acetylcholine-gated monoatomic cation-selective channel activity / inhibitory postsynaptic potential / nervous system process / synaptic transmission, cholinergic / acetylcholine binding / acetylcholine receptor signaling pathway / postsynaptic specialization membrane / heterocyclic compound binding / regulation of synapse assembly / regulation of dendrite morphogenesis / action potential / regulation of dopamine secretion / plasma membrane raft / B cell activation / associative learning / membrane depolarization / 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 / regulation of membrane potential / learning / response to cocaine / locomotory behavior / response to nicotine / sensory perception of sound / electron transport chain / 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 / dendrite / heme binding / synapse / protein-containing complex binding / signal transduction / membrane / plasma membrane
Similarity search - Function
Nicotinic acetylcholine receptor / 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 ...Nicotinic acetylcholine receptor / 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
Similarity search - Domain/homology
Soluble cytochrome b562 / Neuronal acetylcholine receptor subunit beta-2 / Neuronal acetylcholine receptor subunit alpha-4
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.71 Å
AuthorsAlvarez FJD / Mukherjee S / Han S / Ammirati M / Kossiakoff AA
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, 2019-
Header (metadata) releaseMar 11, 2020-
Map releaseApr 29, 2020-
UpdateJul 29, 2020-
Current statusJul 29, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.022
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.022
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6ur8
  • Surface level: 0.022
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20857.png

Downloads & links

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Map

FileDownload / File: emd_20857.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationcytochrome B562-fused human alpha4beta2 nicotinic acetylcholine receptor in complex with fab fragments and bound to varenicline
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.1 Å/pix.
x 320 pix.
= 352.768 Å		1.1 Å/pix.
x 320 pix.
= 352.768 Å		1.1 Å/pix.
x 320 pix.
= 352.768 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.1024 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.022 / Movie #1: 0.022
Minimum - Maximum-0.11499985 - 0.19475323
Average (Standard dev.)0.00011223424 (±0.0027620022)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 352.76797 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.10241.10241.1024
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z352.768352.768352.768
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ350350350
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.1150.1950.000

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Supplemental data

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Sample components

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Entire : human alpha4beta2 nicotinic acetylcholine receptor in complex fab...

EntireName: human alpha4beta2 nicotinic acetylcholine receptor in complex fab fragments and bound to varenicline
Components
  • Complex: human alpha4beta2 nicotinic acetylcholine receptor in complex fab fragments and bound to varenicline
    • Protein or peptide: Chimera of soluble cytochrome b562 (BRIL) and neuronal acetylcholine receptor subunit alpha-4
    • Protein or peptide: Neuronal acetylcholine receptor subunit beta-2
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: VARENICLINE
  • Ligand: beta-D-mannopyranose

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Supramolecule #1: human alpha4beta2 nicotinic acetylcholine receptor in complex fab...

SupramoleculeName: human alpha4beta2 nicotinic acetylcholine receptor in complex fab fragments and bound to varenicline
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)

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Macromolecule #1: Chimera of soluble cytochrome b562 (BRIL) and neuronal acetylchol...

MacromoleculeName: Chimera of soluble cytochrome b562 (BRIL) and neuronal acetylcholine receptor subunit alpha-4
type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 56.027848 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: SSHVETRAHA EERLLKKLFS GYNKWSRPVA NISDVVLVRF GLSIAQLIDV DEKNQMMTTN VWVKQEWHDY KLRWDPADYE NVTSIRIPS ELIWRPDIVL YNNADGDFAV THLTKAHLFH DGRVQWTPPA IYKSSCSIDV TFFPFDQQNC TMKFGSWTYD K AKIDLVNM ...String:
SSHVETRAHA EERLLKKLFS GYNKWSRPVA NISDVVLVRF GLSIAQLIDV DEKNQMMTTN VWVKQEWHDY KLRWDPADYE NVTSIRIPS ELIWRPDIVL YNNADGDFAV THLTKAHLFH DGRVQWTPPA IYKSSCSIDV TFFPFDQQNC TMKFGSWTYD K AKIDLVNM HSRVDQLDFW ESGEWVIVDA VGTYNTRKYE CCAEIYPDIT YAFVIRRLPL FYTINLIIPC LLISCLTVLV FY LPSECGE KITLCISVLL SLTVFLLLIT EIIPSTSLVI PLIGEYLLFT MIFVTLSIVI TVFVLNVHHR SPRTHTMPTW VRR VFLDIV PRLLLMKRPS VVDTDFADLE DNWETLNDNL KVIEKADNAA QVKDALTKMR AAALDAQKAT PPKLEDKSPD SPEM KDFRH GFDILVGQID DALKLANEGK VKEAQAAAEQ LKTTRNAYIQ KYLEDWKYVA MVIDRIFLWM FIIVCLLGTV GLFLP PWLA GMI

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Macromolecule #2: Neuronal acetylcholine receptor subunit beta-2

MacromoleculeName: Neuronal acetylcholine receptor subunit beta-2 / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 45.931953 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: TDTEERLVEH LLDPSRYNKL IRPATNGSEL VTVQLMVSLA QLISVHEREQ IMTTNVWLTQ EWEDYRLTWK PEEFDNMKKV RLPSKHIWL PDVVLYNNAD GMYEVSFYSN AVVSYDGSIF WLPPAIYKSA CKIEVKHFPF DQQNCTMKFR SWTYDRTEID L VLKSEVAS ...String:
TDTEERLVEH LLDPSRYNKL IRPATNGSEL VTVQLMVSLA QLISVHEREQ IMTTNVWLTQ EWEDYRLTWK PEEFDNMKKV RLPSKHIWL PDVVLYNNAD GMYEVSFYSN AVVSYDGSIF WLPPAIYKSA CKIEVKHFPF DQQNCTMKFR SWTYDRTEID L VLKSEVAS LDDFTPSGEW DIVALPGRRN ENPDDSTYVD ITYDFIIRRK PLFYTINLII PCVLITSLAI LVFYLPSDCG EK MTLCISV LLALTVFLLL ISKIVPPTSL DVPLVGKYLM FTMVLVTFSI VTSVCVLNVH HRSPTTHTMA PWVKVVFLEK LPA LLFMQQ DDDQSVSEDW KYVAMVIDRL FLWIFVFVCV FGTIGMFLQP LFQNYTTTTF LHSDHSAPSS KSAWSHPQFE K

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Macromolecule #3: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 3 / Number of copies: 8 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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Macromolecule #4: VARENICLINE

MacromoleculeName: VARENICLINE / type: ligand / ID: 4 / Number of copies: 2 / Formula: QMR
Molecular weightTheoretical: 211.262 Da
Chemical component information

ChemComp-QMR:
VARENICLINE / medication, agonist*YM

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Macromolecule #5: beta-D-mannopyranose

MacromoleculeName: beta-D-mannopyranose / type: ligand / ID: 5 / Number of copies: 3 / Formula: BMA
Molecular weightTheoretical: 180.156 Da
Chemical component information

ChemComp-BMA:
beta-D-mannopyranose

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration3 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMC4H11NO3Tris
150.0 mMNaClsodium chloride
0.1 mMC9H15O6Ptris(2-carboxyethyl)phosphine
100.0 uMC13H13N3varenicline
0.018 %C24H46O1n-dodecyl-beta-D-maltoside
0.006 %C31H50O4Cholesteryl Hemisuccinate
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.019 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Number grids imaged: 1 / Average exposure time: 10.5 sec. / Average electron dose: 52.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.4 µm / Nominal defocus min: 1.2 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1867100
CTF correctionSoftware - Name: CTFFIND (ver. 4.0)
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6cnj

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.71 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0-beta) / Number images used: 226962
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0-beta)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0-beta)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

6cnj

chain_id: A

6cnj

chain_id: B

6cnj

chain_id: C

6cnj

chain_id: D

6cnj

chain_id: E
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-6ur8:
CryoEM structure of human alpha4beta2 nicotinic acetylcholine receptor in complex with varenicline

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