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- EMDB-20812: Cryo-EM structure of mammalian Ric-8A:Galpha(i):nanobody complex -

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Basic information

Entry
Database: EMDB / ID: EMD-20812
TitleCryo-EM structure of mammalian Ric-8A:Galpha(i):nanobody complex
Map datamammalian Ric-8A:Galpha(i):nanobody complex
Sample
  • Complex: RIC-8A:Galpha(1):NB8109:NB8117:NB8119:NB9156 complex
    • Complex: RIC-8A:Galpha(1)
      • Protein or peptide: Resistance to inhibitors of cholinesterase 8 homolog A (C. elegans)
      • Protein or peptide: Guanine nucleotide-binding protein G(i) subunit alpha-1
    • Complex: NB8109:NB8117:NB8119:NB9156
      • Protein or peptide: NB8109
      • Protein or peptide: NB8117
      • Protein or peptide: NB8119
      • Protein or peptide: NB9156
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 / G alpha (i) signalling events / 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 / G alpha (i) signalling events / negative regulation of synaptic transmission / GTPase activating protein binding / 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 / cell cycle / G protein-coupled receptor signaling pathway / cell division / GTPase activity / centrosome / GTP binding / magnesium ion binding / protein-containing complex / nucleus / plasma membrane / cytoplasm
Similarity search - Function
Synembryn / Guanine nucleotide exchange factor, Ric8 / Guanine nucleotide exchange factor synembryn / G-protein alpha subunit, group I / G-alpha domain profile. / Guanine nucleotide binding protein (G-protein), alpha subunit / G protein alpha subunit, helical insertion / G-protein alpha subunit / G protein alpha subunit / Armadillo-like helical ...Synembryn / Guanine nucleotide exchange factor, Ric8 / Guanine nucleotide exchange factor synembryn / G-protein alpha subunit, group I / G-alpha domain profile. / Guanine nucleotide binding protein (G-protein), alpha subunit / G protein alpha subunit, helical insertion / G-protein alpha subunit / G protein alpha subunit / Armadillo-like helical / Armadillo-type fold / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Synembryn / Guanine nucleotide-binding protein G(i) subunit alpha-1
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat) / Lama glama (llama)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.87 Å
AuthorsMou TC / Zhang K / Johnston JD / Chiu W / Sprang SR
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P20GM103546 United States
National Science Foundation (NSF, United States)1738547 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM105993 United States
CitationJournal: 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
DepositionOct 5, 2019-
Header (metadata) releaseNov 13, 2019-
Map releaseMar 11, 2020-
UpdateMar 11, 2020-
Current statusMar 11, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 4.07
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 4.07
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6ukt
  • Surface level: 4.07
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20812.png

Downloads & links

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Map

FileDownload / File: emd_20812.map.gz / Format: CCP4 / Size: 42.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationmammalian Ric-8A:Galpha(i):nanobody complex
Voxel sizeX=Y=Z: 1.06 Å
Density
Contour LevelBy AUTHOR: 4.07 / Movie #1: 4.07
Minimum - Maximum-3.7670937 - 14.8003235
Average (Standard dev.)-0.288465700 (±0.6152012)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions224224224
Spacing224224224
CellA=B=C: 237.43999 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.061.061.06
M x/y/z224224224
origin x/y/z0.0000.0000.000
length x/y/z237.440237.440237.440
α/β/γ90.00090.00090.000
start NX/NY/NZ-38-19-20
NX/NY/NZ858082
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS224224224
D min/max/mean-3.76714.800-0.000

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Supplemental data

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Sample components

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Entire : RIC-8A:Galpha(1):NB8109:NB8117:NB8119:NB9156 complex

EntireName: RIC-8A:Galpha(1):NB8109:NB8117:NB8119:NB9156 complex
Components
  • Complex: RIC-8A:Galpha(1):NB8109:NB8117:NB8119:NB9156 complex
    • Complex: RIC-8A:Galpha(1)
      • Protein or peptide: Resistance to inhibitors of cholinesterase 8 homolog A (C. elegans)
      • Protein or peptide: Guanine nucleotide-binding protein G(i) subunit alpha-1
    • Complex: NB8109:NB8117:NB8119:NB9156
      • Protein or peptide: NB8109
      • Protein or peptide: NB8117
      • Protein or peptide: NB8119
      • Protein or peptide: NB9156

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Supramolecule #1: RIC-8A:Galpha(1):NB8109:NB8117:NB8119:NB9156 complex

SupramoleculeName: 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)

SupramoleculeName: RIC-8A:Galpha(1) / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#2
Source (natural)Organism: Rattus norvegicus (Norway rat)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Supramolecule #3: NB8109:NB8117:NB8119:NB9156

SupramoleculeName: NB8109:NB8117:NB8119:NB9156 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3-#6
Source (natural)Organism: Lama glama (llama)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #1: Resistance to inhibitors of cholinesterase 8 homolog A (C. elegans)

MacromoleculeName: Resistance to inhibitors of cholinesterase 8 homolog A (C. elegans)
type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Rattus norvegicus (Norway rat)
Molecular weightTheoretical: 55.836926 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: 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

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Macromolecule #2: Guanine nucleotide-binding protein G(i) subunit alpha-1

MacromoleculeName: Guanine nucleotide-binding protein G(i) subunit alpha-1
type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Rattus norvegicus (Norway rat)
Molecular weightTheoretical: 37.015219 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: 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

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Macromolecule #3: NB8109

MacromoleculeName: NB8109 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Lama glama (llama)
Molecular weightTheoretical: 13.609938 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
QVQLQESGGG LVQPGGSLRL SCAASGIIFR SNGMAWYRQA PGKEREWVAS ITSFGDAIYR DSVKGRFTIS RDNARNAVSL QTNSLKTED TAVYYCNTYP VNSAWGQGTQ VTVSSHHHHH HEPEA

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Macromolecule #4: NB8117

MacromoleculeName: NB8117 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Lama glama (llama)
Molecular weightTheoretical: 14.8944 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
QVQLQESGGG LEQAGDSLRL SCAASGLIVS NYAMGWFRQA PGKEREFVAY INWNGGVTYY TNSVKGRFTI SRDNAKNTVY LQMNSLKPE DTAVYYCART SRASVTTRVA DFGYWGQGTQ VTVSSHHHHH HEPEA

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Macromolecule #5: NB8119

MacromoleculeName: NB8119 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Lama glama (llama)
Molecular weightTheoretical: 14.247603 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
QVQLQESGGG LVQAGGSLRL SCAASGGIVH ISSMGWFRQA PGKQRELVAT SPSNGDIRYA DSVKGRFTLS RDNAKNTVSL QMNSLEPED TAVYYCHSFL RHTASASYNN YYGQGTQVTV SSHHHHHHEP EA

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Macromolecule #6: NB9156

MacromoleculeName: NB9156 / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Lama glama (llama)
Molecular weightTheoretical: 14.993406 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
QVQLQESGGG LVQAGGSLRL SCAASVRTSD TDGMAWFRQA PGKEREFVGG IRWNSATWYA DFVKGRFTIS RDNAKNTLYL QMNSLKPED TALYYCARRA YGFDTDSRES AYSNWGQGTQ VTVSSHHHHH HEPEA

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.4 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHEPES
150.0 mMNaClSodium chloridesodium chloride
1.0 mMTCEP
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: LACEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV
Detailsmonodisperse

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 11.5 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: CTFFIND
Startup modelType of model: PDB ENTRY / Details: 6TYL
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION
Final 3D classificationNumber classes: 5 / Software - Name: cryoSPARC
Final angle assignmentType: ANGULAR RECONSTITUTION
Final reconstructionNumber classes used: 1 / Resolution.type: BY AUTHOR / Resolution: 3.87 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 338118

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Atomic model buiding 1

Initial modelPDB ID:

6nmg

RefinementProtocol: RIGID BODY FIT
Output model

PDB-6ukt:
Cryo-EM structure of mammalian Ric-8A:Galpha(i):nanobody complex

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