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

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Basic information

Entry
Database: PDB / ID: 6ukt
TitleCryo-EM structure of mammalian Ric-8A:Galpha(i):nanobody complex
Components
  • Guanine nucleotide-binding protein G(i) subunit alpha-1
  • NB8109
  • NB8117
  • NB8119
  • NB9156
  • Resistance to inhibitors of cholinesterase 8 homolog A (C. elegans)
KeywordsSIGNALING PROTEIN / Ric-8A / G protein / GEF
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 / positive regulation of protein localization to cell cortex / G-protein alpha-subunit binding / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.87 Å
AuthorsMou, T.C. / Zhang, K. / Johnston, J.D. / Chiu, W. / Sprang, S.R.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P20GM103546 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM105993 United States
National Science Foundation (NSF, United States)1738547 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, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 11, 2020Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
A: Resistance to inhibitors of cholinesterase 8 homolog A (C. elegans)
B: Guanine nucleotide-binding protein G(i) subunit alpha-1
C: NB8109
D: NB8117
E: NB8119
F: NB9156


Theoretical massNumber of molelcules
Total (without water)150,5976
Polymers150,5976
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 2 types, 2 molecules AB

#1: Protein Resistance to inhibitors of cholinesterase 8 homolog A (C. elegans) / Ric8a protein / Synembryn-A


Mass: 55836.926 Da / Num. of mol.: 1 / Mutation: Y232F
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Ric8a, rCG_48458 / Production host: Escherichia coli (E. coli) / References: UniProt: B1H241
#2: Protein Guanine nucleotide-binding protein G(i) subunit alpha-1 / Adenylate cyclase-inhibiting G alpha protein


Mass: 37015.219 Da / Num. of mol.: 1 / Fragment: UNP residues 32-354
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Gnai1, Gnai-1 / Production host: Escherichia coli (E. coli) / References: UniProt: P10824

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Antibody , 4 types, 4 molecules CDEF

#3: Antibody NB8109


Mass: 13609.938 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Production host: Escherichia coli (E. coli)
#4: Antibody NB8117


Mass: 14894.400 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Production host: Escherichia coli (E. coli)
#5: Antibody NB8119


Mass: 14247.603 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Production host: Escherichia coli (E. coli)
#6: Antibody NB9156


Mass: 14993.406 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Production host: Escherichia coli (E. coli)

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Details

Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1RIC-8A:Galpha(1):NB8109:NB8117:NB8119:NB9156 complexCOMPLEXall0MULTIPLE SOURCES
2RIC-8A:Galpha(1)COMPLEX#1-#21RECOMBINANT
3NB8109:NB8117:NB8119:NB9156COMPLEX#3-#61RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Rattus norvegicus (Norway rat)10116
23Lama glama (llama)9844
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli (E. coli)562
23Escherichia coli (E. coli)562
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESC8H18N2O4S1
2150 mMsodium chlorideNaClSodium chloride1
31 mMTCEP1
SpecimenConc.: 0.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: monodisperse
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 11.5 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameCategory
2EPUimage acquisition
4CTFFINDCTF correction
7PHENIXmodel fitting
9PHENIXmodel refinement
10RELIONinitial Euler assignment
12cryoSPARCclassification
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.87 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 338118 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Atomic model buildingPDB-ID: 6NMG

6nmg

RefinementStereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 84.58 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00669126
ELECTRON MICROSCOPYf_angle_d1.013212369
ELECTRON MICROSCOPYf_chiral_restr0.0581406
ELECTRON MICROSCOPYf_plane_restr0.0071623
ELECTRON MICROSCOPYf_dihedral_angle_d19.17225485

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