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- EMDB-20863: CryoEM structure of human alpha4beta2 nicotinic acetylcholine rec... -
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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-20863 | |||||||||
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Title | CryoEM structure of human alpha4beta2 nicotinic acetylcholine receptor with varenicline in complex with anti-BRIL synthetic antibody BAK5 | |||||||||
![]() | cytochrome B562-fused human alpha4beta2 nicotinic acetylcholine receptor in complex with fab fragments and bound to varenicline (focused on another alpha subunit complexed to a fab) | |||||||||
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Function / homology | ![]() 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 / regulation of dopamine metabolic process / cholinergic synapse / 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 / regulation of dopamine secretion / B cell activation / plasma membrane raft / 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 / 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 | |||||||||
Biological species | ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.87 Å | |||||||||
![]() | Alvarez FJD / Mukherjee S / Han S / Ammirati M / Kossiakoff AA | |||||||||
![]() | ![]() 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 |
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Structure visualization
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 13.4 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 26.3 KB 26.3 KB | Display Display | ![]() |
FSC (resolution estimation) | ![]() | 14.2 KB | Display | ![]() |
Images | ![]() | 83.2 KB | ||
Others | ![]() ![]() | 12.6 MB 12.3 MB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Validation report
Summary document | ![]() | 336.9 KB | Display | ![]() |
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Full document | ![]() | 336.5 KB | Display | |
Data in XML | ![]() | 13.8 KB | Display | |
Data in CIF | ![]() | 18.8 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 6usfMC ![]() 6cbvC ![]() 6ur8C M: atomic model generated by this map C: citing same article ( |
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Similar structure data |
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
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Annotation | cytochrome B562-fused human alpha4beta2 nicotinic acetylcholine receptor in complex with fab fragments and bound to varenicline (focused on another alpha subunit complexed to a fab) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.1024 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Additional map: cytochrome B562-fused human alpha4beta2 nicotinic acetylcholine receptor in...
File | emd_20863_additional_1.map | ||||||||||||
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Annotation | cytochrome B562-fused human alpha4beta2 nicotinic acetylcholine receptor in complex with fab fragments and bound to varenicline (focused on an alpha subunit complexed to a fab) | ||||||||||||
Projections & Slices |
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Density Histograms |
-Additional map: cytochrome B562-fused human alpha4beta2 nicotinic acetylcholine receptor in...
File | emd_20863_additional_2.map | ||||||||||||
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Annotation | cytochrome B562-fused human alpha4beta2 nicotinic acetylcholine receptor in complex with fab fragments and bound to varenicline (focused on an alpha subunit complexed to a fab) | ||||||||||||
Projections & Slices |
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Density Histograms |
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Sample components
-Entire : human alpha4beta2 nicotinic acetylcholine receptor in complex fab...
Entire | Name: human alpha4beta2 nicotinic acetylcholine receptor in complex fab fragments and bound to varenicline |
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Components |
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-Supramolecule #1: human alpha4beta2 nicotinic acetylcholine receptor in complex fab...
Supramolecule | Name: human alpha4beta2 nicotinic acetylcholine receptor in complex fab fragments and bound to varenicline type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4 |
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Source (natural) | Organism: ![]() |
Recombinant expression | Organism: ![]() |
-Macromolecule #1: chimera of soluble cytochrome b562 (BRIL) and neuronal acetylchol...
Macromolecule | Name: 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 |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 55.914691 KDa |
Recombinant expression | Organism: ![]() |
Sequence | String: 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 KYEDWKYVAM VIDRIFLWMF IIVCLLGTVG LFLPP WLAG MI |
-Macromolecule #2: Neuronal acetylcholine receptor subunit beta-2
Macromolecule | Name: Neuronal acetylcholine receptor subunit beta-2 / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 45.931953 KDa |
Recombinant expression | Organism: ![]() |
Sequence | String: 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 |
-Macromolecule #3: Fab heavy chain
Macromolecule | Name: Fab heavy chain / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 24.351139 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: EISEVQLVES GGGLVQPGGS LRLSCAASGF NVRSFSIHWV RQAPGKGLEW VAYISSSSGS TSYADSVKGR FTISADTSKN TAYLQMNSL RAEDTAVYYC ARWGYWPGEP WWKAFDYWGQ GTLVTVSSAS TKGPSVFPLA PSSKSTSGGT AALGCLVKDY F PEPVTVSW ...String: EISEVQLVES GGGLVQPGGS LRLSCAASGF NVRSFSIHWV RQAPGKGLEW VAYISSSSGS TSYADSVKGR FTISADTSKN TAYLQMNSL RAEDTAVYYC ARWGYWPGEP WWKAFDYWGQ GTLVTVSSAS TKGPSVFPLA PSSKSTSGGT AALGCLVKDY F PEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSSVVTV PSSSLGTQTY ICNVNHKPSN TKVDKKVEPK S |
-Macromolecule #4: Fab light chain
Macromolecule | Name: Fab light chain / type: protein_or_peptide / ID: 4 / Number of copies: 2 / Enantiomer: LEVO |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 23.483062 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: SDIQMTQSPS SLSASVGDRV TITCRASQSV SSAVAWYQQK PGKAPKLLIY SASSLYSGVP SRFSGSRSGT DFTLTISSLQ PEDFATYYC QQYLYYSLVT FGQGTKVEIK RTVAAPSVFI FPPSDSQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG N SQESVTEQ ...String: SDIQMTQSPS SLSASVGDRV TITCRASQSV SSAVAWYQQK PGKAPKLLIY SASSLYSGVP SRFSGSRSGT DFTLTISSLQ PEDFATYYC QQYLYYSLVT FGQGTKVEIK RTVAAPSVFI FPPSDSQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG N SQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGE |
-Macromolecule #7: 2-acetamido-2-deoxy-beta-D-glucopyranose
Macromolecule | Name: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 7 / Number of copies: 4 / Formula: NAG |
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Molecular weight | Theoretical: 221.208 Da |
Chemical component information | ![]() ChemComp-NAG: |
-Macromolecule #8: VARENICLINE
Macromolecule | Name: VARENICLINE / type: ligand / ID: 8 / Number of copies: 2 / Formula: QMR |
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Molecular weight | Theoretical: 211.262 Da |
Chemical component information | ![]() ChemComp-QMR: |
-Macromolecule #9: beta-D-mannopyranose
Macromolecule | Name: beta-D-mannopyranose / type: ligand / ID: 9 / Number of copies: 1 / Formula: BMA |
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Molecular weight | Theoretical: 180.156 Da |
Chemical component information | ![]() ChemComp-BMA: |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Concentration | 3 mg/mL | |||||||||||||||||||||
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Buffer | pH: 7.5 Component:
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Grid | Model: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.039 kPa / Details: -30 mA | |||||||||||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV |
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Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Number grids imaged: 1 / Number real images: 3418 / Average exposure time: 10.5 sec. / Average electron dose: 52.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | C2 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 stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Image processing
-Atomic model buiding 1
Initial model |
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Refinement | Space: REAL / Protocol: RIGID BODY FIT | ||||||||||||||||||
Output model | ![]() PDB-6usf: |