+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-24551 | |||||||||||||||
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Title | MicroED structure of the human adenosine receptor at 2.8A | |||||||||||||||
Map data | 2mFo-dFc map of A2A AR by MicroED | |||||||||||||||
Sample |
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Keywords | MEMBRANE PROTEIN | |||||||||||||||
Function / homology | Function and homology information positive regulation of acetylcholine secretion, neurotransmission / positive regulation of circadian sleep/wake cycle, sleep / regulation of norepinephrine secretion / negative regulation of alpha-beta T cell activation / Adenosine P1 receptors / G protein-coupled adenosine receptor activity / G protein-coupled adenosine receptor signaling pathway / response to purine-containing compound / sensory perception / NGF-independant TRKA activation ...positive regulation of acetylcholine secretion, neurotransmission / positive regulation of circadian sleep/wake cycle, sleep / regulation of norepinephrine secretion / negative regulation of alpha-beta T cell activation / Adenosine P1 receptors / G protein-coupled adenosine receptor activity / G protein-coupled adenosine receptor signaling pathway / response to purine-containing compound / sensory perception / NGF-independant TRKA activation / Surfactant metabolism / positive regulation of urine volume / alpha-actinin binding / synaptic transmission, dopaminergic / : / inhibitory postsynaptic potential / negative regulation of vascular permeability / type 5 metabotropic glutamate receptor binding / synaptic transmission, cholinergic / blood circulation / positive regulation of glutamate secretion / response to caffeine / intermediate filament / eating behavior / presynaptic active zone / regulation of calcium ion transport / membrane depolarization / asymmetric synapse / axolemma / : / cellular defense response / positive regulation of synaptic transmission, glutamatergic / prepulse inhibition / phagocytosis / neuron projection morphogenesis / response to amphetamine / presynaptic modulation of chemical synaptic transmission / astrocyte activation / excitatory postsynaptic potential / positive regulation of apoptotic signaling pathway / regulation of mitochondrial membrane potential / synaptic transmission, glutamatergic / positive regulation of long-term synaptic potentiation / central nervous system development / locomotory behavior / positive regulation of synaptic transmission, GABAergic / positive regulation of protein secretion / apoptotic signaling pathway / electron transport chain / negative regulation of inflammatory response / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / vasodilation / adenylate cyclase-activating G protein-coupled receptor signaling pathway / blood coagulation / cell-cell signaling / presynaptic membrane / G alpha (s) signalling events / postsynaptic membrane / negative regulation of neuron apoptotic process / periplasmic space / calmodulin binding / electron transfer activity / response to xenobiotic stimulus / inflammatory response / iron ion binding / negative regulation of cell population proliferation / neuronal cell body / lipid binding / glutamatergic synapse / dendrite / heme binding / regulation of DNA-templated transcription / protein-containing complex binding / apoptotic process / enzyme binding / identical protein binding / membrane / plasma membrane Similarity search - Function | |||||||||||||||
Biological species | Homo sapiens (human) | |||||||||||||||
Method | electron crystallography / cryo EM / Resolution: 2.79 Å | |||||||||||||||
Authors | Martynowycz MW / Shiriaeva A | |||||||||||||||
Funding support | United States, 4 items
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Citation | Journal: Proc Natl Acad Sci U S A / Year: 2021 Title: MicroED structure of the human adenosine receptor determined from a single nanocrystal in LCP. Authors: Michael W Martynowycz / Anna Shiriaeva / Xuanrui Ge / Johan Hattne / Brent L Nannenga / Vadim Cherezov / Tamir Gonen / Abstract: G protein-coupled receptors (GPCRs), or seven-transmembrane receptors, are a superfamily of membrane proteins that are critically important to physiological processes in the human body. Determining ...G protein-coupled receptors (GPCRs), or seven-transmembrane receptors, are a superfamily of membrane proteins that are critically important to physiological processes in the human body. Determining high-resolution structures of GPCRs without bound cognate signaling partners, such as a G protein, requires crystallization in lipidic cubic phase (LCP). GPCR crystals grown in LCP are often too small for traditional X-ray crystallography. These microcrystals are ideal for investigation by microcrystal electron diffraction (MicroED), but the gel-like nature of LCP makes traditional approaches to MicroED sample preparation insurmountable. Here, we show that the structure of a human A adenosine receptor can be determined by MicroED after converting the LCP into the sponge phase followed by focused ion-beam milling. We determined the structure of the A adenosine receptor to 2.8-Å resolution and resolved an antagonist in its orthosteric ligand-binding site, as well as four cholesterol molecules bound around the receptor. This study lays the groundwork for future structural studies of lipid-embedded membrane proteins by MicroED using single microcrystals that would be impossible with other crystallographic methods. | |||||||||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_24551.map.gz | 4.3 MB | EMDB map data format | |
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Header (meta data) | emd-24551-v30.xml emd-24551.xml | 16.9 KB 16.9 KB | Display Display | EMDB header |
Images | emd_24551.png | 35.2 KB | ||
Filedesc metadata | emd-24551.cif.gz | 6.4 KB | ||
Filedesc structureFactors | emd_24551_sf.cif.gz | 294.8 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-24551 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-24551 | HTTPS FTP |
-Validation report
Summary document | emd_24551_validation.pdf.gz | 498.8 KB | Display | EMDB validaton report |
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Full document | emd_24551_full_validation.pdf.gz | 498.4 KB | Display | |
Data in XML | emd_24551_validation.xml.gz | 4.6 KB | Display | |
Data in CIF | emd_24551_validation.cif.gz | 5.1 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-24551 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-24551 | HTTPS FTP |
-Related structure data
Related structure data | 7rm5MC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_24551.map.gz / Format: CCP4 / Size: 4.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | 2mFo-dFc map of A2A AR by MicroED | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X: 0.667 Å / Y: 0.669 Å / Z: 0.698 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 20 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : Adenosine receptor A2a/Soluble cytochrome b562 chimera
Entire | Name: Adenosine receptor A2a/Soluble cytochrome b562 chimera |
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Components |
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-Supramolecule #1: Adenosine receptor A2a/Soluble cytochrome b562 chimera
Supramolecule | Name: Adenosine receptor A2a/Soluble cytochrome b562 chimera type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1 |
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Source (natural) | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 58.72 KDa |
-Macromolecule #1: Adenosine receptor A2a/Soluble cytochrome b562 chimera
Macromolecule | Name: Adenosine receptor A2a/Soluble cytochrome b562 chimera type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 49.974281 KDa |
Recombinant expression | Organism: Spodoptera frugiperda (fall armyworm) |
Sequence | String: MKTIIALSYI FCLVFADYKD DDDGAPPIMG SSVYITVELA IAVLAILGNV LVCWAVWLNS NLQNVTNYFV VSLAAADIAV GVLAIPFAI TISTGFCAAC HGCLFIACFV LVLTQSSIFS LLAIAIDRYI AIRIPLRYNG LVTGTRAKGI IAICWVLSFA I GLTPMLGW ...String: MKTIIALSYI FCLVFADYKD DDDGAPPIMG SSVYITVELA IAVLAILGNV LVCWAVWLNS NLQNVTNYFV VSLAAADIAV GVLAIPFAI TISTGFCAAC HGCLFIACFV LVLTQSSIFS LLAIAIDRYI AIRIPLRYNG LVTGTRAKGI IAICWVLSFA I GLTPMLGW NNCGQPKEGK NHSQGCGEGQ VACLFEDVVP MNYMVYFNFF ACVLVPLLLM LGVYLRIFLA ARRQLADLED NW ETLNDNL KVIEKADNAA QVKDALTKMR AAALDAQKAT PPKLEDKSPD SPEMKDFRHG FDILVGQIDD ALKLANEGKV KEA QAAAEQ LKTTRNAYIQ KYLERARSTL QKEVHAAKSL AIIVGLFALC WLPLHIINCF TFFCPDCSHA PLWLMYLAIV LSHT NSVVN PFIYAYRIRE FRQTFRKIIR SHVLRQQEPF KAHHHHHHHH HH UniProtKB: Adenosine receptor A2a, Soluble cytochrome b562, Adenosine receptor A2a |
-Macromolecule #2: 4-{2-[(7-amino-2-furan-2-yl[1,2,4]triazolo[1,5-a][1,3,5]triazin-5...
Macromolecule | Name: 4-{2-[(7-amino-2-furan-2-yl[1,2,4]triazolo[1,5-a][1,3,5]triazin-5-yl)amino]ethyl}phenol type: ligand / ID: 2 / Number of copies: 1 / Formula: ZMA |
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Molecular weight | Theoretical: 337.336 Da |
Chemical component information | ChemComp-ZMA: |
-Macromolecule #3: CHOLESTEROL
Macromolecule | Name: CHOLESTEROL / type: ligand / ID: 3 / Number of copies: 4 / Formula: CLR |
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Molecular weight | Theoretical: 386.654 Da |
Chemical component information | ChemComp-CLR: |
-Macromolecule #4: SODIUM ION
Macromolecule | Name: SODIUM ION / type: ligand / ID: 4 / Number of copies: 1 |
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Molecular weight | Theoretical: 22.99 Da |
-Macromolecule #5: water
Macromolecule | Name: water / type: ligand / ID: 5 / Number of copies: 6 / Formula: HOH |
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Molecular weight | Theoretical: 18.015 Da |
Chemical component information | ChemComp-HOH: |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | electron crystallography |
Aggregation state | 3D array |
-Sample preparation
Concentration | 10 mg/mL |
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Buffer | pH: 5 Details: 25-28% (v/v) PEG 400, 0.04-0.06M sodium thiocyanate, 2% (v/v) 2,5-hexanediol, 100mM sodium citrate, pH 5.0, Lipid Cubic Phase (LCP), temperature 293K |
Grid | Model: Quantifoil R2/2 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 298 K / Instrument: LEICA EM GP |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Temperature | Min: 77.0 K / Max: 83.0 K |
Image recording | Film or detector model: FEI CETA (4k x 4k) / Digitization - Dimensions - Width: 2048 pixel / Digitization - Dimensions - Height: 2048 pixel / Number grids imaged: 1 / Number diffraction images: 134 / Average exposure time: 3.0 sec. / Average electron dose: 0.01 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: DIFFRACTION / Cs: 2.7 mm / Nominal defocus max: 0.0 µm / Nominal defocus min: 0.0 µm / Camera length: 1900 mm |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN / Tilt angle: -40.0, 40.0, 0.6 |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
+Image processing
-Atomic model buiding 1
Initial model | PDB ID: Chain - Chain ID: A / Chain - Source name: PDB / Chain - Initial model type: experimental model |
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Refinement | Space: RECIPROCAL / Protocol: RIGID BODY FIT / Overall B value: 43 / Target criteria: Maximum likelihood |
Output model | PDB-7rm5: |