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Yorodumi- EMDB-20812: Cryo-EM structure of mammalian Ric-8A:Galpha(i):nanobody complex -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-20812 | ||||||||||||
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Title | Cryo-EM structure of mammalian Ric-8A:Galpha(i):nanobody complex | ||||||||||||
Map data | mammalian Ric-8A:Galpha(i):nanobody complex | ||||||||||||
Sample |
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Function / homology | Function and homology information cell-cell adhesion involved in gastrulation / cell migration involved in gastrulation / Extra-nuclear estrogen signaling / Adenylate cyclase inhibitory pathway / basement membrane organization / vasculature development / Adrenaline,noradrenaline inhibits insulin secretion / ADP signalling through P2Y purinoceptor 12 / GTPase activating protein binding / negative regulation of synaptic transmission ...cell-cell adhesion involved in gastrulation / cell migration involved in gastrulation / Extra-nuclear estrogen signaling / Adenylate cyclase inhibitory pathway / basement membrane organization / vasculature development / Adrenaline,noradrenaline inhibits insulin secretion / ADP signalling through P2Y purinoceptor 12 / GTPase activating protein binding / negative regulation of synaptic transmission / G alpha (i) signalling events / neurotransmitter receptor localization to postsynaptic specialization membrane / G-protein alpha-subunit binding / positive regulation of protein localization to cell cortex / regulation of cAMP-mediated signaling / D2 dopamine receptor binding / G protein-coupled serotonin receptor binding / regulation of mitotic spindle organization / cellular response to forskolin / adenylate cyclase-inhibiting G protein-coupled receptor signaling pathway / guanyl-nucleotide exchange factor activity / G protein-coupled receptor binding / G-protein beta/gamma-subunit complex binding / visual learning / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / GDP binding / heterotrimeric G-protein complex / cell cortex / midbody / in utero embryonic development / postsynapse / G protein-coupled receptor signaling pathway / cell division / GTPase activity / centrosome / glutamatergic synapse / GTP binding / magnesium ion binding / protein-containing complex / nucleus / plasma membrane / cytoplasm Similarity search - Function | ||||||||||||
Biological species | Rattus norvegicus (Norway rat) / Lama glama (llama) | ||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.87 Å | ||||||||||||
Authors | Mou TC / Zhang K / Johnston JD / Chiu W / Sprang SR | ||||||||||||
Funding support | United States, 3 items
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Citation | Journal: Nat Commun / Year: 2020 Title: Structure of the G protein chaperone and guanine nucleotide exchange factor Ric-8A bound to Gαi1. Authors: Levi J McClelland / Kaiming Zhang / Tung-Chung Mou / Jake Johnston / Cindee Yates-Hansen / Shanshan Li / Celestine J Thomas / Tzanko I Doukov / Sarah Triest / Alexandre Wohlkonig / Gregory G ...Authors: Levi J McClelland / Kaiming Zhang / Tung-Chung Mou / Jake Johnston / Cindee Yates-Hansen / Shanshan Li / Celestine J Thomas / Tzanko I Doukov / Sarah Triest / Alexandre Wohlkonig / Gregory G Tall / Jan Steyaert / Wah Chiu / Stephen R Sprang / Abstract: Ric-8A is a cytosolic Guanine Nucleotide exchange Factor (GEF) that activates heterotrimeric G protein alpha subunits (Gα) and serves as an essential Gα chaperone. Mechanisms by which Ric-8A ...Ric-8A is a cytosolic Guanine Nucleotide exchange Factor (GEF) that activates heterotrimeric G protein alpha subunits (Gα) and serves as an essential Gα chaperone. Mechanisms by which Ric-8A catalyzes these activities, which are stimulated by Casein Kinase II phosphorylation, are unknown. We report the structure of the nanobody-stabilized complex of nucleotide-free Gα bound to phosphorylated Ric-8A at near atomic resolution by cryo-electron microscopy and X-ray crystallography. The mechanism of Ric-8A GEF activity differs considerably from that employed by G protein-coupled receptors at the plasma membrane. Ric-8A engages a specific conformation of Gα at multiple interfaces to form a complex that is stabilized by phosphorylation within a Ric-8A segment that connects two Gα binding sites. The C-terminus of Gα is ejected from its beta sheet core, thereby dismantling the GDP binding site. Ric-8A binds to the exposed Gα beta sheet and switch II to stabilize the nucleotide-free state of Gα. | ||||||||||||
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_20812.map.gz | 39.6 MB | EMDB map data format | |
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Header (meta data) | emd-20812-v30.xml emd-20812.xml | 21.1 KB 21.1 KB | Display Display | EMDB header |
Images | emd_20812.png | 260 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-20812 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-20812 | HTTPS FTP |
-Validation report
Summary document | emd_20812_validation.pdf.gz | 430.2 KB | Display | EMDB validaton report |
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Full document | emd_20812_full_validation.pdf.gz | 429.7 KB | Display | |
Data in XML | emd_20812_validation.xml.gz | 6.1 KB | Display | |
Data in CIF | emd_20812_validation.cif.gz | 6.9 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-20812 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-20812 | HTTPS FTP |
-Related structure data
Related structure data | 6uktMC 6tylC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_20812.map.gz / Format: CCP4 / Size: 42.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | mammalian Ric-8A:Galpha(i):nanobody complex | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.06 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : RIC-8A:Galpha(1):NB8109:NB8117:NB8119:NB9156 complex
Entire | Name: RIC-8A:Galpha(1):NB8109:NB8117:NB8119:NB9156 complex |
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Components |
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-Supramolecule #1: RIC-8A:Galpha(1):NB8109:NB8117:NB8119:NB9156 complex
Supramolecule | Name: RIC-8A:Galpha(1):NB8109:NB8117:NB8119:NB9156 complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all |
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-Supramolecule #2: RIC-8A:Galpha(1)
Supramolecule | Name: RIC-8A:Galpha(1) / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#2 |
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Source (natural) | Organism: Rattus norvegicus (Norway rat) |
Recombinant expression | Organism: Escherichia coli (E. coli) |
-Supramolecule #3: NB8109:NB8117:NB8119:NB9156
Supramolecule | Name: NB8109:NB8117:NB8119:NB9156 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3-#6 |
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Source (natural) | Organism: Lama glama (llama) |
Recombinant expression | Organism: Escherichia coli (E. coli) |
-Macromolecule #1: Resistance to inhibitors of cholinesterase 8 homolog A (C. elegans)
Macromolecule | Name: Resistance to inhibitors of cholinesterase 8 homolog A (C. elegans) type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Rattus norvegicus (Norway rat) |
Molecular weight | Theoretical: 55.836926 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: GMEPRAVADA LETGEEDAVT EALRSFNREH SQSFTFDDAQ QEDRKRLAKL LVSVLEQGLS PKHRVTWLQT IRILSRDRSC LDSFASRQS LHALACYADI AISEEPIPQP PDMDVLLESL KCLCNLVLSS PTAQMLAAEA RLVVRLAERV GLYRKRSYPH E VQFFDLRL ...String: GMEPRAVADA LETGEEDAVT EALRSFNREH SQSFTFDDAQ QEDRKRLAKL LVSVLEQGLS PKHRVTWLQT IRILSRDRSC LDSFASRQS LHALACYADI AISEEPIPQP PDMDVLLESL KCLCNLVLSS PTAQMLAAEA RLVVRLAERV GLYRKRSYPH E VQFFDLRL LFLLTALRTD VRQQLFQELH GVRLLTDALE LTLGVAPKEN PLVILPAQET ERAMEILKVL FNITFDSVKR EV DEEDAAL YRYLGTLLRH CVMADAAGDR TEEFHGHTVN LLGNLPLKCL DVLLALELHE GSLEFMGVNM DVINALLAFL EKR LHQTHR LKECVAPVLS VLTECARMHR PARKFLKAQV LPPLRDVRTR PEVGDLLRNK LVRLMTHLDT DVKRVAAEFL FVLC SESVP RFIKYTGYGN AAGLLAARGL MAGGRPEGQY (SEP)EDED(TPO)DTEE YREAKASINP VTGRVEEKPP NPMEGMT EE QKEHEAMKLV NMFDKLSR |
-Macromolecule #2: Guanine nucleotide-binding protein G(i) subunit alpha-1
Macromolecule | Name: Guanine nucleotide-binding protein G(i) subunit alpha-1 type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Rattus norvegicus (Norway rat) |
Molecular weight | Theoretical: 37.015219 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: REVKLLLLGA GESGKSTIVK QMKIIHEAGY SEEECKQYKA VVYSNTIQSI IAIIRAMGRL KIDFGDAARA DDARQLFVLA GAAEEGFMT AELAGVIKRL WKDSGVQACF NRSREYQLND SAAYYLNDLD RIAQPNYIPT QQDVLRTRVK TTGIVETHFT F KDLHFKMF ...String: REVKLLLLGA GESGKSTIVK QMKIIHEAGY SEEECKQYKA VVYSNTIQSI IAIIRAMGRL KIDFGDAARA DDARQLFVLA GAAEEGFMT AELAGVIKRL WKDSGVQACF NRSREYQLND SAAYYLNDLD RIAQPNYIPT QQDVLRTRVK TTGIVETHFT F KDLHFKMF DVGGQRSERK KWIHCFEGVT AIIFCVALSD YDLVLAEDEE MNRMHESMKL FDSICNNKWF TDTSIILFLN KK DLFEEKI KKSPLTICYP EYAGSNTYEE AAAYIQCQFE DLNKRKDTKE IYTHFTCATD TKNVQFVFDA VTDVIIKNNL KDC GLF |
-Macromolecule #3: NB8109
Macromolecule | Name: NB8109 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Lama glama (llama) |
Molecular weight | Theoretical: 13.609938 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: QVQLQESGGG LVQPGGSLRL SCAASGIIFR SNGMAWYRQA PGKEREWVAS ITSFGDAIYR DSVKGRFTIS RDNARNAVSL QTNSLKTED TAVYYCNTYP VNSAWGQGTQ VTVSSHHHHH HEPEA |
-Macromolecule #4: NB8117
Macromolecule | Name: NB8117 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Lama glama (llama) |
Molecular weight | Theoretical: 14.8944 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: QVQLQESGGG LEQAGDSLRL SCAASGLIVS NYAMGWFRQA PGKEREFVAY INWNGGVTYY TNSVKGRFTI SRDNAKNTVY LQMNSLKPE DTAVYYCART SRASVTTRVA DFGYWGQGTQ VTVSSHHHHH HEPEA |
-Macromolecule #5: NB8119
Macromolecule | Name: NB8119 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Lama glama (llama) |
Molecular weight | Theoretical: 14.247603 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: QVQLQESGGG LVQAGGSLRL SCAASGGIVH ISSMGWFRQA PGKQRELVAT SPSNGDIRYA DSVKGRFTLS RDNAKNTVSL QMNSLEPED TAVYYCHSFL RHTASASYNN YYGQGTQVTV SSHHHHHHEP EA |
-Macromolecule #6: NB9156
Macromolecule | Name: NB9156 / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Lama glama (llama) |
Molecular weight | Theoretical: 14.993406 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: QVQLQESGGG LVQAGGSLRL SCAASVRTSD TDGMAWFRQA PGKEREFVGG IRWNSATWYA DFVKGRFTIS RDNAKNTLYL QMNSLKPED TALYYCARRA YGFDTDSRES AYSNWGQGTQ VTVSSHHHHH HEPEA |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.4 mg/mL | ||||||||||||
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Buffer | pH: 8 Component:
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Grid | Model: Quantifoil R2/1 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: LACEY / Pretreatment - Type: GLOW DISCHARGE | ||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV | ||||||||||||
Details | monodisperse |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 11.5 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |