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- PDB-6tyl: Crystal structure of mammalian Ric-8A:Galpha(i):nanobody complex -

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Basic information

Entry
Database: PDB / ID: 6tyl
TitleCrystal structure of mammalian Ric-8A:Galpha(i):nanobody complex
Components
  • Guanine nucleotide-binding protein G(i) subunit alpha-1
  • Nanobody A
  • Nanobody B
  • Nanobody C
  • 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)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.3 Å
AuthorsMou, T.C. / McClelland, L. / Yates-Hansen, C. / 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 (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
DepositionAug 9, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 11, 2020Provider: repository / Type: Initial release
Revision 1.1Mar 16, 2022Group: Author supporting evidence / Database references / Category: database_2 / pdbx_audit_support
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_audit_support.funding_organization
Revision 1.2Oct 11, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Resistance to inhibitors of cholinesterase 8 homolog A (C. elegans)
C: Nanobody A
H: Nanobody A
E: Nanobody B
J: Nanobody B
D: Nanobody C
I: Nanobody C
B: Guanine nucleotide-binding protein G(i) subunit alpha-1
G: Guanine nucleotide-binding protein G(i) subunit alpha-1
F: Resistance to inhibitors of cholinesterase 8 homolog A (C. elegans)


Theoretical massNumber of molelcules
Total (without water)277,97610
Polymers277,97610
Non-polymers00
Water0
1
A: Resistance to inhibitors of cholinesterase 8 homolog A (C. elegans)
C: Nanobody A
E: Nanobody B
D: Nanobody C
B: Guanine nucleotide-binding protein G(i) subunit alpha-1


Theoretical massNumber of molelcules
Total (without water)138,9885
Polymers138,9885
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
H: Nanobody A
J: Nanobody B
I: Nanobody C
G: Guanine nucleotide-binding protein G(i) subunit alpha-1
F: Resistance to inhibitors of cholinesterase 8 homolog A (C. elegans)


Theoretical massNumber of molelcules
Total (without water)138,9885
Polymers138,9885
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)93.048, 144.726, 114.420
Angle α, β, γ (deg.)90.00, 94.66, 90.00
Int Tables number4
Space group name H-MP1211
Symmetry operation#1: x,y,z
#2: -x,y+1/2,-z

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Components

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


Mass: 55836.926 Da / Num. of mol.: 2 / 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: Antibody Nanobody A


Mass: 13609.938 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Production host: Escherichia coli (E. coli)
#3: Antibody Nanobody B


Mass: 14247.603 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Production host: Escherichia coli (E. coli)
#4: Antibody Nanobody C


Mass: 14894.400 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Production host: Escherichia coli (E. coli)
#5: Protein Guanine nucleotide-binding protein G(i) subunit alpha-1 / Adenylate cyclase-inhibiting G alpha protein


Mass: 40399.031 Da / Num. of mol.: 2
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
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.76 Å3/Da / Density % sol: 55.47 %
Crystal growTemperature: 285 K / Method: vapor diffusion, hanging drop / Details: 1.2-1.4M Sodium Malonate / PH range: 6-8

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: NSLS-II / Beamline: 17-ID-2 / Wavelength: 0.987 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Nov 9, 2018
RadiationMonochromator: Si(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.987 Å / Relative weight: 1
ReflectionResolution: 3.3→39.58 Å / Num. obs: 20707 / % possible obs: 90.1 % / Redundancy: 3.6 % / Biso Wilson estimate: 91.88 Å2 / CC1/2: 0.99 / Rpim(I) all: 0.11 / Rrim(I) all: 0.21 / Net I/σ(I): 3.7
Reflection shellResolution: 3.3→3.9 Å / Mean I/σ(I) obs: 1.7 / Num. unique obs: 1035 / CC1/2: 0.7 / Rpim(I) all: 0.38 / Rrim(I) all: 0.7

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Processing

Software
NameClassification
PHENIXrefinement
XDSdata reduction
STARANISOdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 6NMG

6nmg


Resolution: 3.3→39.53 Å / Cross valid method: FREE R-VALUE
RfactorNum. reflection% reflection
Rfree0.287 -5 %
Rwork0.248 --
obs-20707 90.1 %
Displacement parametersBiso mean: 109.9 Å2
Refinement stepCycle: LAST / Resolution: 3.3→39.53 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms15938 0 0 0 15938
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00216223
X-RAY DIFFRACTIONf_angle_d0.54821930
X-RAY DIFFRACTIONf_dihedral_angle_d17.8129825
X-RAY DIFFRACTIONf_chiral_restr0.0382461
X-RAY DIFFRACTIONf_plane_restr0.0042841
Refinement TLS params.Method: refined / Origin x: 25.8389 Å / Origin y: -18.0538 Å / Origin z: 27.0874 Å
111213212223313233
T0.5501 Å2-0.1561 Å2-0.0023 Å2-0.5406 Å2-0.0783 Å2--0.4201 Å2
L0.6963 °2-0.0485 °2-0.0697 °2-1.1735 °2-0.1776 °2--0.941 °2
S0.12 Å °-0.0093 Å °-0.0531 Å °-0.0238 Å °-0.1963 Å °0.2957 Å °0.1999 Å °0.0576 Å °0.0799 Å °
Refinement TLS groupSelection details: all

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