+Open data
-Basic information
Entry | Database: PDB / ID: 7rm5 | |||||||||||||||
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Title | MicroED structure of the human adenosine receptor at 2.8A | |||||||||||||||
Components | Adenosine receptor A2a/Soluble cytochrome b562 chimera | |||||||||||||||
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) Escherichia coli (E. coli) | |||||||||||||||
Method | ELECTRON CRYSTALLOGRAPHY / electron crystallography / MOLECULAR REPLACEMENT / cryo EM / Resolution: 2.79 Å | |||||||||||||||
Authors | Martynowycz, M.W. / Shiriaeva, A. / Ge, X. / Hattne, J. / Nannenga, B.L. / Cherezov, V. / Gonen, T. | |||||||||||||||
Funding support | United States, 4items
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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 | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 7rm5.cif.gz | 98.1 KB | Display | PDBx/mmCIF format |
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PDB format | pdb7rm5.ent.gz | 68.5 KB | Display | PDB format |
PDBx/mmJSON format | 7rm5.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 7rm5_validation.pdf.gz | 1.1 MB | Display | wwPDB validaton report |
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Full document | 7rm5_full_validation.pdf.gz | 1.1 MB | Display | |
Data in XML | 7rm5_validation.xml.gz | 17.1 KB | Display | |
Data in CIF | 7rm5_validation.cif.gz | 23.9 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/rm/7rm5 ftp://data.pdbj.org/pub/pdb/validation_reports/rm/7rm5 | HTTPS FTP |
-Related structure data
Related structure data | 24551MC 4eiyS |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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Unit cell |
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-Components
#1: Protein | Mass: 49974.281 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human), (gene. exp.) Escherichia coli (E. coli) Gene: ADORA2A, ADORA2, cybC / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P29274, UniProt: P0ABE7 | ||||||
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#2: Chemical | ChemComp-ZMA / | ||||||
#3: Chemical | ChemComp-CLR / #4: Chemical | ChemComp-NA / | #5: Water | ChemComp-HOH / | Has ligand of interest | N | |
-Experimental details
-Experiment
Experiment | Method: ELECTRON CRYSTALLOGRAPHY |
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EM experiment | Aggregation state: 3D ARRAY / 3D reconstruction method: electron crystallography |
-Sample preparation
Component | Name: Adenosine receptor A2a/Soluble cytochrome b562 chimera Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT | ||||||||||||
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Molecular weight | Value: 0.05872 MDa / Experimental value: NO | ||||||||||||
Source (natural) |
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Source (recombinant) |
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Buffer solution | 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 | ||||||||||||
Specimen | Conc.: 10 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | ||||||||||||
Specimen support | Grid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2 | ||||||||||||
Vitrification | Instrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 298 K |
-Data collection
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: DIFFRACTION / Nominal defocus max: 0 nm / Nominal defocus min: 0 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: BASIC |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 83 K / Temperature (min): 77 K |
Image recording | Average exposure time: 3 sec. / Electron dose: 0.01 e/Å2 / Film or detector model: FEI CETA (4k x 4k) / Num. of diffraction images: 134 / Num. of grids imaged: 1 |
Image scans | Sampling size: 28 µm / Width: 2048 / Height: 2048 |
EM diffraction | Camera length: 1900 mm / Tilt angle list: -40, +40, 0.6 |
EM diffraction shell | Resolution: 37.91→2.79 Å / Fourier space coverage: 77 % / Multiplicity: 3.7 / Num. of structure factors: 10071 / Phase residual: 30 ° |
EM diffraction stats | Fourier space coverage: 77 % / High resolution: 2.79 Å / Num. of intensities measured: 37130 / Num. of structure factors: 10071 / Phase error: 30 ° / Phase error rejection criteria: none / Rmerge: 0.299 |
Diffraction | Serial crystal experiment: N |
Detector | Detector: CMOS / Date: Sep 11, 2020 |
-Processing
Software |
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EM software |
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Image processing | Details: CetaD binned by 2 | |||||||||||||||||||||
EM 3D crystal entity | ∠α: 90 ° / ∠β: 90 ° / ∠γ: 90 ° / A: 40 Å / B: 180.5 Å / C: 139.7 Å / Space group name: C2221 / Space group num: 20 | |||||||||||||||||||||
CTF correction | Type: NONE | |||||||||||||||||||||
3D reconstruction | Resolution: 2.79 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Algorithm: FOURIER SPACE / Symmetry type: 3D CRYSTAL | |||||||||||||||||||||
Atomic model building | B value: 43 / Protocol: RIGID BODY FIT / Space: RECIPROCAL / Target criteria: Maximum likelihood | |||||||||||||||||||||
Atomic model building | PDB-ID: 4EIY Pdb chain-ID: A / Accession code: 4EIY / Source name: PDB / Type: experimental model | |||||||||||||||||||||
Refinement | Method to determine structure: MOLECULAR REPLACEMENT Starting model: 4EIY Resolution: 2.79→37.91 Å / SU B: 0.48 / Cross valid method: FREE R-VALUE / σ(F): 1.3 / Phase error: 0.3063
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Solvent computation | Shrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å | |||||||||||||||||||||
Displacement parameters | Biso mean: 43 Å2 | |||||||||||||||||||||
LS refinement shell | Resolution: 2.79→3.07 Å
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