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- PDB-6n86: Resistance to inhibitors of cholinesterase 8A (Ric8A) protein -

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Basic information

Entry
Database: PDB / ID: 6n86
TitleResistance to inhibitors of cholinesterase 8A (Ric8A) protein
ComponentsSynembryn-A
KeywordsCHAPERONE / Ric8a / Armadillo repeat / G alpha
Function / homology
Function and homology information


G-protein alpha-subunit binding / guanyl-nucleotide exchange factor activity / G protein-coupled receptor signaling pathway / plasma membrane / cytoplasm
Similarity search - Function
Synembryn / Guanine nucleotide exchange factor, Ric8 / Guanine nucleotide exchange factor synembryn / Armadillo-like helical / Armadillo-type fold
Similarity search - Domain/homology
Biological speciesBos taurus (cattle)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.9 Å
AuthorsSrivastava, D. / Gakhar, L. / Artemyev, N.O.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Eye Institute (NIH/NEI)NIH R01 EY12682 United States
CitationJournal: Nat Commun / Year: 2019
Title: Structural underpinnings of Ric8A function as a G-protein α-subunit chaperone and guanine-nucleotide exchange factor.
Authors: Dhiraj Srivastava / Lokesh Gakhar / Nikolai O Artemyev /
Abstract: Resistance to inhibitors of cholinesterase 8A (Ric8A) is an essential regulator of G protein α-subunits (Gα), acting as a guanine nucleotide exchange factor and a chaperone. We report two crystal ...Resistance to inhibitors of cholinesterase 8A (Ric8A) is an essential regulator of G protein α-subunits (Gα), acting as a guanine nucleotide exchange factor and a chaperone. We report two crystal structures of Ric8A, one in the apo form and the other in complex with a tagged C-terminal fragment of Gα. These structures reveal two principal domains of Ric8A: an armadillo-fold core and a flexible C-terminal tail. Additionally, they show that the Gα C-terminus binds to a highly-conserved patch on the concave surface of the Ric8A armadillo-domain, with selectivity determinants residing in the Gα sequence. Biochemical analysis shows that the Ric8A C-terminal tail is critical for its stability and function. A model of the Ric8A/Gα complex derived from crosslinking mass spectrometry and molecular dynamics simulations suggests that the Ric8A C-terminal tail helps organize the GTP-binding site of Gα. This study lays the groundwork for understanding Ric8A function at the molecular level.
History
DepositionNov 28, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 10, 2019Provider: repository / Type: Initial release
Revision 1.1Jul 24, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Oct 16, 2019Group: Data collection / Category: reflns / Item: _reflns.pdbx_CC_half
Revision 1.3Dec 11, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.4Mar 13, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
B: Synembryn-A


Theoretical massNumber of molelcules
Total (without water)57,6391
Polymers57,6391
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: SAXS
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)95.150, 134.340, 221.750
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number22
Space group name H-MF222

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Components

#1: Protein Synembryn-A / Protein Ric-8A


Mass: 57639.148 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bos taurus (cattle) / Gene: RIC8A / Plasmid: pET15b / Production host: Escherichia coli (E. coli) / Strain (production host): BL21 (DE3) / References: UniProt: Q5E9J8

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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: 3.08 Å3/Da / Density % sol: 60.1 %
Crystal growTemperature: 277 K / Method: vapor diffusion, sitting drop / pH: 6.5
Details: 0.1 M Bis-Tris propane, 0.2 M NaI, 12-20 % PEG3350, seeding
PH range: 6.0 - 7.0

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ALS / Beamline: 4.2.2 / Wavelength: 1 Å
DetectorType: RDI CMOS_8M / Detector: CMOS / Date: Feb 15, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 3.9→57.45 Å / Num. obs: 6668 / % possible obs: 99.9 % / Redundancy: 14.3 % / CC1/2: 0.999 / Rmerge(I) obs: 0.097 / Rpim(I) all: 0.028 / Rrim(I) all: 0.107 / Net I/σ(I): 18.4
Reflection shellResolution: 3.9→4.11 Å / Redundancy: 13.3 % / Rmerge(I) obs: 0.799 / Mean I/σ(I) obs: 3.3 / Num. unique obs: 954 / CC1/2: 0.955 / Rpim(I) all: 0.237 / Rrim(I) all: 0.867 / % possible all: 100

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Processing

Software
NameVersionClassification
PHENIX(1.14_3260: ???)refinement
XDSdata reduction
SCALA3.3.22data scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 3.9→57.45 Å / SU ML: 0.56 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 26.5 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2961 297 4.47 %
Rwork0.2565 --
obs0.2583 6641 99.52 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 3.9→57.45 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3157 0 0 0 3157
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0013201
X-RAY DIFFRACTIONf_angle_d0.3924325
X-RAY DIFFRACTIONf_dihedral_angle_d13.9491992
X-RAY DIFFRACTIONf_chiral_restr0.03516
X-RAY DIFFRACTIONf_plane_restr0.003557
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.9001-4.91330.35841380.27263113X-RAY DIFFRACTION99
4.9133-57.4560.27591590.24993231X-RAY DIFFRACTION100
Refinement TLS params.Method: refined / Origin x: -33.8906 Å / Origin y: 18.7197 Å / Origin z: -16.213 Å
111213212223313233
T1.0013 Å2-0.1139 Å20.1317 Å2-0.9718 Å20.1275 Å2--1.0174 Å2
L1.2082 °2-0.1989 °20.3203 °2-1.6268 °20.2166 °2--1.2475 °2
S-0.0529 Å °0.2305 Å °-0.0365 Å °0.3117 Å °0.4225 Å °-0.1164 Å °0.4815 Å °0.4819 Å °0.5924 Å °
Refinement TLS groupSelection details: all

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