+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-29586 | |||||||||
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Title | MicroED structure of A2A from plasma milled lamellae | |||||||||
Map data | Structure of the human A2A adenosine receptor from plasma milled lamellae | |||||||||
Sample |
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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: 2.0 Å | |||||||||
Authors | Martynowycz MW / Shiriaeva A / Clabbers MTB / Nicolas WJ / Weaver SJ / Hattne J / Gonen T | |||||||||
Funding support | United States, 2 items
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Citation | Journal: Nat Commun / Year: 2023 Title: A robust approach for MicroED sample preparation of lipidic cubic phase embedded membrane protein crystals. Authors: Michael W Martynowycz / Anna Shiriaeva / Max T B Clabbers / William J Nicolas / Sara J Weaver / Johan Hattne / Tamir Gonen / Abstract: Crystallizing G protein-coupled receptors (GPCRs) in lipidic cubic phase (LCP) often yields crystals suited for the cryogenic electron microscopy (cryoEM) method microcrystal electron diffraction ...Crystallizing G protein-coupled receptors (GPCRs) in lipidic cubic phase (LCP) often yields crystals suited for the cryogenic electron microscopy (cryoEM) method microcrystal electron diffraction (MicroED). However, sample preparation is challenging. Embedded crystals cannot be targeted topologically. Here, we use an integrated fluorescence light microscope (iFLM) inside of a focused ion beam and scanning electron microscope (FIB-SEM) to identify fluorescently labeled GPCR crystals. Crystals are targeted using the iFLM and LCP is milled using a plasma focused ion beam (pFIB). The optimal ion source for preparing biological lamellae is identified using standard crystals of proteinase K. Lamellae prepared using either argon or xenon produced the highest quality data and structures. MicroED data are collected from the milled lamellae and the structures are determined. This study outlines a robust approach to identify and mill membrane protein crystals for MicroED and demonstrates plasma ion-beam milling is a powerful tool for preparing biological lamellae. | |||||||||
History |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_29586.map.gz | 13.1 MB | EMDB map data format | |
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Header (meta data) | emd-29586-v30.xml emd-29586.xml | 16.5 KB 16.5 KB | Display Display | EMDB header |
Images | emd_29586.png | 62.6 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-29586 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-29586 | HTTPS FTP |
-Validation report
Summary document | emd_29586_validation.pdf.gz | 454.6 KB | Display | EMDB validaton report |
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Full document | emd_29586_full_validation.pdf.gz | 454.1 KB | Display | |
Data in XML | emd_29586_validation.xml.gz | 4.5 KB | Display | |
Data in CIF | emd_29586_validation.cif.gz | 5 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-29586 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-29586 | HTTPS FTP |
-Related structure data
Related structure data | 8fynMC 8fyoC 8fypC 8fyqC 8fyrC 8fysC 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_29586.map.gz / Format: CCP4 / Size: 14.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||
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Annotation | Structure of the human A2A adenosine receptor from plasma milled lamellae | ||||||||||||||||||||
Voxel size | X: 0.4885 Å / Y: 0.4885 Å / Z: 0.48365 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 20 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Sample components
-Entire : A2A BRIL Adenosine receptor
Entire | Name: A2A BRIL Adenosine receptor |
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Components |
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-Supramolecule #1: A2A BRIL Adenosine receptor
Supramolecule | Name: A2A BRIL Adenosine receptor / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1 |
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Source (natural) | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 41 KDa |
-Macromolecule #1: Adenosine receptor A2a,Soluble cytochrome b562
Macromolecule | Name: Adenosine receptor A2a,Soluble cytochrome b562 / 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 aff. frugiperda 1 BOLD-2017 (butterflies/moths) |
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 |
-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: 3 / Formula: CLR |
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Molecular weight | Theoretical: 386.654 Da |
Chemical component information | ChemComp-CLR: |
-Macromolecule #4: OLEIC ACID
Macromolecule | Name: OLEIC ACID / type: ligand / ID: 4 / Number of copies: 15 / Formula: OLA |
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Molecular weight | Theoretical: 282.461 Da |
Chemical component information | ChemComp-OLA: |
-Macromolecule #5: (2R)-2,3-dihydroxypropyl (9Z)-octadec-9-enoate
Macromolecule | Name: (2R)-2,3-dihydroxypropyl (9Z)-octadec-9-enoate / type: ligand / ID: 5 / Number of copies: 5 / Formula: OLC |
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Molecular weight | Theoretical: 356.54 Da |
Chemical component information | ChemComp-OLC: |
-Macromolecule #6: (2S)-2,3-dihydroxypropyl (9Z)-octadec-9-enoate
Macromolecule | Name: (2S)-2,3-dihydroxypropyl (9Z)-octadec-9-enoate / type: ligand / ID: 6 / Number of copies: 1 / Formula: OLB |
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Molecular weight | Theoretical: 356.54 Da |
Chemical component information | ChemComp-OLB: |
-Macromolecule #7: SODIUM ION
Macromolecule | Name: SODIUM ION / type: ligand / ID: 7 / Number of copies: 1 |
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Molecular weight | Theoretical: 22.99 Da |
-Macromolecule #8: water
Macromolecule | Name: water / type: ligand / ID: 8 / Number of copies: 174 / 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 | 25 mg/mL |
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Buffer | pH: 4.7 |
Grid | Model: Quantifoil R2/2 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 10.0 nm |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: LEICA PLUNGER |
Details | Milled microcrystals |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Temperature | Min: 77.0 K / Max: 90.0 K |
Image recording | Film or detector model: FEI FALCON IV (4k x 4k) / Digitization - Dimensions - Width: 2048 pixel / Digitization - Dimensions - Height: 2048 pixel / Number grids imaged: 1 / Number real images: 1 / Number diffraction images: 840 / Average exposure time: 0.5 sec. / Average electron dose: 0.001 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: 1202 mm |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN / Tilt angle: -30.0, 30.0 |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
Details | Binned by 2 |
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Final reconstruction | Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.0 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES |
Merging software list | Software - Name: AIMLESS |
Crystallography statistics | Number intensities measured: 569407 / Number structure factors: 64974 / Fourier space coverage: 87.58 / R sym: 0.073 / R merge: 0.236 / Overall phase error: 30 / Overall phase residual: 0 / Phase error rejection criteria: None / High resolution: 0.87 Å / Shell - Shell ID: 1 / Shell - High resolution: 0.87 Å / Shell - Low resolution: 0.9 Å / Shell - Number structure factors: 2783 / Shell - Phase residual: 30 / Shell - Fourier space coverage: 37.64 / Shell - Multiplicity: 2.1 |
-Atomic model buiding 1
Refinement | Space: RECIPROCAL / Protocol: AB INITIO MODEL / Overall B value: 12.54 / Target criteria: Maximum likelihood |
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Output model | PDB-8fyn: |