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Yorodumi- PDB-6usf: CryoEM structure of human alpha4beta2 nicotinic acetylcholine rec... -
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-Basic information
Entry | Database: PDB / ID: 6usf | |||||||||
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Title | CryoEM structure of human alpha4beta2 nicotinic acetylcholine receptor with varenicline in complex with anti-BRIL synthetic antibody BAK5 | |||||||||
Components |
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Keywords | MEMBRANE 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 / 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 / visual perception / sensory perception of pain / regulation of membrane potential / learning / response to cocaine / cognition / locomotory behavior / response to nicotine / sensory perception of sound / electron transport chain / visual learning / memory / 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 | |||||||||
Biological species | Homo sapiens (human) Escherichia coli (E. coli) | |||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.87 Å | |||||||||
Authors | Alvarez, F.J.D. / Mukherjee, S. / Han, S. / Ammirati, M. / Kossiakoff, A.A. | |||||||||
Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 6usf.cif.gz | 527.8 KB | Display | PDBx/mmCIF format |
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PDB format | pdb6usf.ent.gz | 435.4 KB | Display | PDB format |
PDBx/mmJSON format | 6usf.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 6usf_validation.pdf.gz | 1.1 MB | Display | wwPDB validaton report |
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Full document | 6usf_full_validation.pdf.gz | 1.1 MB | Display | |
Data in XML | 6usf_validation.xml.gz | 89 KB | Display | |
Data in CIF | 6usf_validation.cif.gz | 134.8 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/us/6usf ftp://data.pdbj.org/pub/pdb/validation_reports/us/6usf | HTTPS FTP |
-Related structure data
Related structure data | 20863MC 6cbvC 6ur8C C: citing same article (ref.) M: map data used to model this data |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
-Protein , 2 types, 5 molecules ADBCE
#1: Protein | 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 | 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 | 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 | 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 | Source method: isolated from a genetically manipulated source #6: Polysaccharide | 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose | #7: Sugar | ChemComp-NAG / #9: Sugar | ChemComp-BMA / | |
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-Non-polymers , 1 types, 2 molecules
#8: Chemical |
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-Details
Has ligand of interest | Y |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: human alpha4beta2 nicotinic acetylcholine receptor in complex fab fragments and bound to varenicline Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT | |||||||||||||||||||||||||||||||||||
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Molecular weight | Experimental value: NO | |||||||||||||||||||||||||||||||||||
Source (natural) | Organism: Homo sapiens (human) | |||||||||||||||||||||||||||||||||||
Source (recombinant) | Organism: Homo sapiens (human) | |||||||||||||||||||||||||||||||||||
Buffer solution | pH: 7.5 | |||||||||||||||||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | |||||||||||||||||||||||||||||||||||
Specimen support | Details: -30 mA / Grid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3 | |||||||||||||||||||||||||||||||||||
Vitrification | Instrument: 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 |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal defocus max: 2400 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm |
Specimen holder | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average 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 scans | Movie frames/image: 35 |
-Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 1867100 | ||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.87 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 285852 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||
Atomic model building | Protocol: RIGID BODY FIT / Space: REAL | ||||||||||||||||||||||||||||||||||||
Atomic model building |
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