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- PDB-6n85: Resistance to inhibitors of cholinesterase 8A (Ric8A) protein in ... -

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Entry
Database: PDB / ID: 6n85
TitleResistance to inhibitors of cholinesterase 8A (Ric8A) protein in complex with MBP-tagged transducin-alpha residues 327-350
Components
  • Maltose/maltodextrin-binding periplasmic protein,Guanine nucleotide-binding protein G(t) subunit alpha-2
  • Synembryn-A
KeywordsCHAPERONE / Ric8a / Armadillo repeat / G alpha / MBP
Function / homology
Function and homology information


detection of light stimulus involved in visual perception / retinal cone cell development / detection of chemical stimulus involved in sensory perception of bitter taste / response to light intensity / G protein-coupled photoreceptor activity / photoreceptor outer segment membrane / phototransduction / carbohydrate transmembrane transporter activity / photoreceptor outer segment / G-protein alpha-subunit binding ...detection of light stimulus involved in visual perception / retinal cone cell development / detection of chemical stimulus involved in sensory perception of bitter taste / response to light intensity / G protein-coupled photoreceptor activity / photoreceptor outer segment membrane / phototransduction / carbohydrate transmembrane transporter activity / photoreceptor outer segment / G-protein alpha-subunit binding / photoreceptor inner segment / visual perception / guanyl-nucleotide exchange factor activity / G protein-coupled receptor binding / G-protein beta/gamma-subunit complex binding / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / heterotrimeric G-protein complex / Ca2+ pathway / outer membrane-bounded periplasmic space / positive regulation of cytosolic calcium ion concentration / G alpha (i) signalling events / G protein-coupled receptor signaling pathway / GTPase activity / GTP binding / metal ion binding / plasma membrane / cytoplasm
Similarity search - Function
Synembryn / Guanine nucleotide exchange factor, Ric8 / Guanine nucleotide exchange factor synembryn / Maltose/Cyclodextrin ABC transporter, substrate-binding protein / Solute-binding family 1, conserved site / Bacterial extracellular solute-binding proteins, family 1 signature. / Bacterial extracellular solute-binding protein / G-protein alpha subunit, group I / Bacterial extracellular solute-binding protein / G-alpha domain profile. ...Synembryn / Guanine nucleotide exchange factor, Ric8 / Guanine nucleotide exchange factor synembryn / Maltose/Cyclodextrin ABC transporter, substrate-binding protein / Solute-binding family 1, conserved site / Bacterial extracellular solute-binding proteins, family 1 signature. / Bacterial extracellular solute-binding protein / G-protein alpha subunit, group I / Bacterial extracellular solute-binding protein / 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
alpha-maltose / Maltose/maltodextrin-binding periplasmic protein / Guanine nucleotide-binding protein G(t) subunit alpha-2 / Synembryn-A
Similarity search - Component
Biological speciesBos taurus (cattle)
Escherichia coli O157:H7 (bacteria)
Homo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.5 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 1.2Dec 11, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 2.0Jul 29, 2020Group: Atomic model / Data collection ...Atomic model / Data collection / Derived calculations / Non-polymer description / Structure summary
Category: atom_site / atom_site_anisotrop ...atom_site / atom_site_anisotrop / chem_comp / entity / entity_name_com / pdbx_branch_scheme / pdbx_chem_comp_identifier / pdbx_entity_branch / pdbx_entity_branch_descriptor / pdbx_entity_branch_link / pdbx_entity_branch_list / pdbx_entity_nonpoly / pdbx_molecule_features / pdbx_nonpoly_scheme / struct_conn / struct_site / struct_site_gen
Item: _atom_site.B_iso_or_equiv / _atom_site.Cartn_x ..._atom_site.B_iso_or_equiv / _atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _atom_site.auth_asym_id / _atom_site.auth_atom_id / _atom_site.auth_comp_id / _atom_site.auth_seq_id / _atom_site.label_atom_id / _atom_site.label_comp_id / _atom_site_anisotrop.U[1][1] / _atom_site_anisotrop.U[1][2] / _atom_site_anisotrop.U[1][3] / _atom_site_anisotrop.U[2][2] / _atom_site_anisotrop.U[2][3] / _atom_site_anisotrop.U[3][3] / _atom_site_anisotrop.pdbx_auth_asym_id / _atom_site_anisotrop.pdbx_auth_atom_id / _atom_site_anisotrop.pdbx_auth_comp_id / _atom_site_anisotrop.pdbx_auth_seq_id / _atom_site_anisotrop.pdbx_label_atom_id / _atom_site_anisotrop.pdbx_label_comp_id / _chem_comp.formula / _chem_comp.formula_weight / _chem_comp.id / _chem_comp.mon_nstd_flag / _chem_comp.name / _chem_comp.type / _entity.formula_weight / _entity.pdbx_description / _entity.type
Description: Carbohydrate remediation / Provider: repository / Type: Remediation
Revision 3.0Nov 24, 2021Group: Advisory / Atomic model ...Advisory / Atomic model / Data collection / Database references / Derived calculations / Polymer sequence / Structure summary
Category: atom_site / atom_site_anisotrop ...atom_site / atom_site_anisotrop / chem_comp / database_2 / entity / entity_poly / entity_poly_seq / pdbx_nonpoly_scheme / pdbx_poly_seq_scheme / pdbx_struct_sheet_hbond / pdbx_unobs_or_zero_occ_atoms / struct_conf / struct_conn / struct_ref / struct_ref_seq / struct_ref_seq_dif / struct_sheet_range
Item: _atom_site.B_iso_or_equiv / _atom_site.Cartn_x ..._atom_site.B_iso_or_equiv / _atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _atom_site.auth_comp_id / _atom_site.label_comp_id / _atom_site_anisotrop.U[1][1] / _atom_site_anisotrop.U[1][2] / _atom_site_anisotrop.U[1][3] / _atom_site_anisotrop.U[2][2] / _atom_site_anisotrop.U[2][3] / _atom_site_anisotrop.U[3][3] / _atom_site_anisotrop.pdbx_auth_comp_id / _atom_site_anisotrop.pdbx_label_comp_id / _chem_comp.pdbx_synonyms / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _entity.formula_weight / _entity.pdbx_description / _entity_poly.pdbx_seq_one_letter_code / _entity_poly.pdbx_seq_one_letter_code_can / _entity_poly_seq.mon_id / _pdbx_nonpoly_scheme.auth_seq_num / _pdbx_poly_seq_scheme.mon_id / _pdbx_poly_seq_scheme.pdb_mon_id / _pdbx_struct_sheet_hbond.range_1_auth_comp_id / _pdbx_struct_sheet_hbond.range_1_label_comp_id / _struct_conf.beg_auth_comp_id / _struct_conf.beg_label_comp_id / _struct_conn.pdbx_value_order / _struct_ref.pdbx_seq_one_letter_code / _struct_ref_seq.db_align_end / _struct_ref_seq.pdbx_auth_seq_align_end / _struct_ref_seq.seq_align_end / _struct_sheet_range.end_auth_comp_id / _struct_sheet_range.end_label_comp_id
Revision 3.1May 22, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: Synembryn-A
M: Maltose/maltodextrin-binding periplasmic protein,Guanine nucleotide-binding protein G(t) subunit alpha-2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)103,2563
Polymers102,9132
Non-polymers3421
Water1,71195
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3410 Å2
ΔGint-14 kcal/mol
Surface area33930 Å2
MethodPISA
Unit cell
Length a, b, c (Å)65.740, 91.920, 86.230
Angle α, β, γ (deg.)90.00, 112.30, 90.00
Int Tables number4
Space group name H-MP1211

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Components

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


Mass: 57464.977 Da / Num. of mol.: 1 / Mutation: E460A, E461A, K462A
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
#2: Protein Maltose/maltodextrin-binding periplasmic protein,Guanine nucleotide-binding protein G(t) subunit alpha-2 / MMBP / Maltodextrin-binding protein / Maltose-binding protein / MBP / Transducin alpha-2 chain


Mass: 45448.391 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli O157:H7 (bacteria), (gene. exp.) Homo sapiens (human)
Gene: malE, Z5632, ECs5017, GNAT2, GNATC / Production host: Escherichia coli (E. coli) / References: UniProt: P0AEY0, UniProt: P19087
#3: Polysaccharide alpha-D-glucopyranose-(1-4)-alpha-D-glucopyranose / alpha-maltose


Type: oligosaccharide, Oligosaccharide / Class: Nutrient / Mass: 342.297 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: oligosaccharide / References: alpha-maltose
DescriptorTypeProgram
DGlcpa1-4DGlcpa1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1a_1-5]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[][a-D-Glcp]{[(4+1)][a-D-Glcp]{}}LINUCSPDB-CARE
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 95 / Source method: isolated from a natural source / Formula: H2O

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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: 2.34 Å3/Da / Density % sol: 47.46 %
Crystal growTemperature: 291 K / Method: vapor diffusion, hanging drop / pH: 8 / Details: 0.1 M MIB buffer, 25 % PEG3350pH 8.0

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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 7, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 2.5→60.18 Å / Num. obs: 32806 / % possible obs: 99.8 % / Redundancy: 7.1 % / Biso Wilson estimate: 44.67 Å2 / CC1/2: 0.999 / Rmerge(I) obs: 0.066 / Rpim(I) all: 0.03 / Rrim(I) all: 0.08 / Net I/σ(I): 20.4
Reflection shellResolution: 2.5→2.64 Å / Redundancy: 6.7 % / Rmerge(I) obs: 0.716 / Mean I/σ(I) obs: 2.6 / Num. unique obs: 4784 / CC1/2: 0.899 / Rpim(I) all: 0.332 / Rrim(I) all: 0.871 / % possible all: 99.8

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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: 2.5→41.386 Å / SU ML: 0.33 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 30.57 / Stereochemistry target values: ML
RfactorNum. reflection% reflectionSelection details
Rfree0.2489 1664 5.06 %Random selection
Rwork0.1964 ---
obs0.1991 32862 99.63 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 2.5→41.386 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms6221 0 23 95 6339
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0026364
X-RAY DIFFRACTIONf_angle_d0.4788626
X-RAY DIFFRACTIONf_dihedral_angle_d16.553868
X-RAY DIFFRACTIONf_chiral_restr0.036993
X-RAY DIFFRACTIONf_plane_restr0.0031111
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.5-2.57360.35331630.2732588X-RAY DIFFRACTION100
2.5736-2.65660.3171270.26352551X-RAY DIFFRACTION99
2.6566-2.75160.33171470.2392616X-RAY DIFFRACTION99
2.7516-2.86170.26581260.23982604X-RAY DIFFRACTION99
2.8617-2.99190.34481110.24292581X-RAY DIFFRACTION99
2.9919-3.14960.3212990.24552629X-RAY DIFFRACTION100
3.1496-3.34680.28541640.24152562X-RAY DIFFRACTION100
3.3468-3.60510.27361280.21282621X-RAY DIFFRACTION100
3.6051-3.96770.22941440.18882586X-RAY DIFFRACTION100
3.9677-4.54120.21061400.15442623X-RAY DIFFRACTION100
4.5412-5.7190.21511390.1592612X-RAY DIFFRACTION100
5.719-41.3920.2061760.1642625X-RAY DIFFRACTION100
Refinement TLS params.Method: refined / Origin x: 2.5053 Å / Origin y: 59.2762 Å / Origin z: 19.8312 Å
111213212223313233
T0.3122 Å20.0683 Å20.0141 Å2-0.2531 Å2-0.0403 Å2--0.3229 Å2
L0.7597 °20.1996 °20.6871 °2-0.0369 °2-0.0641 °2--1.0759 °2
S-0.008 Å °0.1145 Å °0.0526 Å °-0.0098 Å °0.0035 Å °-0.0046 Å °-0.0647 Å °-0.1397 Å °-0.0003 Å °
Refinement TLS groupSelection details: all

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