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- PDB-6bu0: Crystal structure of the PI3KC2alpha C2 domain in complex with IP6 -

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Basic information

Entry
Database: PDB / ID: 6bu0
TitleCrystal structure of the PI3KC2alpha C2 domain in complex with IP6
ComponentsPhosphatidylinositol 4-phosphate 3-kinase C2 domain-containing subunit alpha
KeywordsTRANSFERASE / C2 domain / lipid binding / phosphoinositide / PI3-kinase
Function / homology
Function and homology information


vascular associated smooth muscle contraction / Synthesis of PIPs at the late endosome membrane / Synthesis of PIPs at the early endosome membrane / Synthesis of PIPs at the Golgi membrane / phosphatidylinositol-4-phosphate 3-kinase / clathrin coat assembly / membrane organization / phosphatidylinositol biosynthetic process / phosphatidylinositol 3-kinase complex / 1-phosphatidylinositol-4-phosphate 3-kinase activity ...vascular associated smooth muscle contraction / Synthesis of PIPs at the late endosome membrane / Synthesis of PIPs at the early endosome membrane / Synthesis of PIPs at the Golgi membrane / phosphatidylinositol-4-phosphate 3-kinase / clathrin coat assembly / membrane organization / phosphatidylinositol biosynthetic process / phosphatidylinositol 3-kinase complex / 1-phosphatidylinositol-4-phosphate 3-kinase activity / 1-phosphatidylinositol-4,5-bisphosphate 3-kinase activity / phosphatidylinositol-4,5-bisphosphate 3-kinase / phosphatidylinositol 3-kinase / clathrin-coated vesicle / phosphatidylinositol-3-phosphate biosynthetic process / 1-phosphatidylinositol-3-kinase activity / clathrin binding / positive regulation of cell migration involved in sprouting angiogenesis / Golgi Associated Vesicle Biogenesis / exocytosis / Synthesis of PIPs at the plasma membrane / platelet-derived growth factor receptor signaling pathway / positive regulation of autophagy / phosphatidylinositol binding / phosphatidylinositol 3-kinase/protein kinase B signal transduction / epidermal growth factor receptor signaling pathway / trans-Golgi network / endocytosis / insulin receptor signaling pathway / Clathrin-mediated endocytosis / vesicle / phosphorylation / intracellular membrane-bounded organelle / extracellular exosome / nucleoplasm / ATP binding / membrane / plasma membrane / cytosol / cytoplasm
Similarity search - Function
PI3K-C2-alpha, catalytic domain / Phosphatidylinositol 4-phosphate 3-kinase C2 domain-containing subunit alpha, PX domain / PhoX homologous domain, present in p47phox and p40phox. / PX domain profile. / PX domain / Phox homology / PX domain superfamily / C2 domain / PI3-kinase family, Ras-binding domain / Phosphatidylinositol 3-kinase Ras-binding (PI3K RBD) domain ...PI3K-C2-alpha, catalytic domain / Phosphatidylinositol 4-phosphate 3-kinase C2 domain-containing subunit alpha, PX domain / PhoX homologous domain, present in p47phox and p40phox. / PX domain profile. / PX domain / Phox homology / PX domain superfamily / C2 domain / PI3-kinase family, Ras-binding domain / Phosphatidylinositol 3-kinase Ras-binding (PI3K RBD) domain / PI3-kinase family, ras-binding domain / Phosphatidylinositol 3-kinase Ras-binding (PI3K RBD) domain profile. / Phosphoinositide 3-kinase C2 / Phosphoinositide 3-kinase, region postulated to contain C2 domain / C2 phosphatidylinositol 3-kinase-type domain / C2 phosphatidylinositol 3-kinase (PI3K)-type domain profile. / Phosphoinositide 3-kinase, accessory (PIK) domain superfamily / Phosphoinositide 3-kinase family, accessory domain (PIK domain) / Phosphoinositide 3-kinase family, accessory domain (PIK domain) / Phosphoinositide 3-kinase, accessory (PIK) domain / Phosphatidylinositol kinase / PIK helical domain profile. / C2 domain / Protein kinase C conserved region 2 (CalB) / C2 domain / C2 domain profile. / Phosphatidylinositol 3- and 4-kinases signature 1. / Phosphatidylinositol 3/4-kinase, conserved site / Phosphatidylinositol 3- and 4-kinases signature 2. / Phosphatidylinositol 3-/4-kinase, catalytic domain superfamily / Phosphoinositide 3-kinase, catalytic domain / Phosphatidylinositol 3- and 4-kinase / Phosphatidylinositol 3- and 4-kinases catalytic domain profile. / Phosphatidylinositol 3-/4-kinase, catalytic domain / C2 domain superfamily / Armadillo-type fold / Ubiquitin-like domain superfamily / Protein kinase-like domain superfamily / Immunoglobulin-like / Sandwich / Mainly Beta
Similarity search - Domain/homology
FORMIC ACID / INOSITOL HEXAKISPHOSPHATE / 1,4,7,10,13,16-HEXAOXACYCLOOCTADECANE / Phosphatidylinositol 4-phosphate 3-kinase C2 domain-containing subunit alpha
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 2.427 Å
AuthorsChen, K.-E. / Collins, B.M.
CitationJournal: Structure / Year: 2018
Title: Molecular Basis for Membrane Recruitment by the PX and C2 Domains of Class II Phosphoinositide 3-Kinase-C2α.
Authors: Kai-En Chen / Vikas A Tillu / Mintu Chandra / Brett M Collins /
Abstract: Phosphorylation of phosphoinositides by the class II phosphatidylinositol 3-kinase (PI3K) PI3K-C2α is essential for many processes, including neuroexocytosis and formation of clathrin-coated ...Phosphorylation of phosphoinositides by the class II phosphatidylinositol 3-kinase (PI3K) PI3K-C2α is essential for many processes, including neuroexocytosis and formation of clathrin-coated vesicles. A defining feature of the class II PI3Ks is a C-terminal module composed of phox-homology (PX) and C2 membrane interacting domains; however, the mechanisms that control their specific cellular localization remain poorly understood. Here we report the crystal structure of the C2 domain of PI3K-C2α in complex with the phosphoinositide head-group mimic inositol hexaphosphate, revealing two distinct pockets for membrane binding. The C2 domain preferentially binds to phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol (3,4,5)-trisphosphate, and low-resolution structures of the combined PX-C2 module by small-angle X-ray scattering reveal a compact conformation in which cooperative lipid binding by each domain binding can occur. Finally, we demonstrate an unexpected role for calcium in perturbing the membrane interactions of the PX-C2 module, which we speculate may be important for regulating the activity of PI3K-C2α.
History
DepositionDec 8, 2017Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 17, 2018Provider: repository / Type: Initial release
Revision 1.1Dec 19, 2018Group: Data collection / Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first
Revision 1.2Oct 14, 2020Group: Structure summary / Category: chem_comp / Item: _chem_comp.pdbx_synonyms
Revision 1.3Oct 4, 2023Group: Advisory / Data collection ...Advisory / Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / pdbx_unobs_or_zero_occ_atoms
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
A: Phosphatidylinositol 4-phosphate 3-kinase C2 domain-containing subunit alpha
B: Phosphatidylinositol 4-phosphate 3-kinase C2 domain-containing subunit alpha
C: Phosphatidylinositol 4-phosphate 3-kinase C2 domain-containing subunit alpha
hetero molecules


Theoretical massNumber of molelcules
Total (without water)47,75113
Polymers44,6163
Non-polymers3,13510
Water41423
1
A: Phosphatidylinositol 4-phosphate 3-kinase C2 domain-containing subunit alpha
hetero molecules


Theoretical massNumber of molelcules
Total (without water)15,0565
Polymers14,8721
Non-polymers1844
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
2
B: Phosphatidylinositol 4-phosphate 3-kinase C2 domain-containing subunit alpha
hetero molecules


Theoretical massNumber of molelcules
Total (without water)16,2384
Polymers14,8721
Non-polymers1,3663
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
3
C: Phosphatidylinositol 4-phosphate 3-kinase C2 domain-containing subunit alpha
hetero molecules


Theoretical massNumber of molelcules
Total (without water)16,4574
Polymers14,8721
Non-polymers1,5843
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)91.652, 126.715, 38.655
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number18
Space group name H-MP21212

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Components

#1: Protein Phosphatidylinositol 4-phosphate 3-kinase C2 domain-containing subunit alpha / PtdIns-3-kinase C2 subunit alpha / Phosphoinositide 3-kinase-C2-alpha


Mass: 14872.116 Da / Num. of mol.: 3 / Fragment: C2 domain / Mutation: E1654G
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PIK3C2A / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3)
References: UniProt: O00443, phosphatidylinositol-4-phosphate 3-kinase
#2: Chemical
ChemComp-FMT / FORMIC ACID / Formic acid


Mass: 46.025 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: CH2O2
#3: Chemical
ChemComp-IHP / INOSITOL HEXAKISPHOSPHATE / MYO-INOSITOL HEXAKISPHOSPHATE / INOSITOL 1,2,3,4,5,6-HEXAKISPHOSPHATE / Phytic acid


Mass: 660.035 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C6H18O24P6
#4: Chemical ChemComp-O4B / 1,4,7,10,13,16-HEXAOXACYCLOOCTADECANE / 18-Crown-6


Mass: 264.315 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C12H24O6
#5: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 23 / 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: 2.52 Å3/Da / Density % sol: 51.1 % / Mosaicity: 0.11 °
Crystal growTemperature: 295 K / Method: vapor diffusion, hanging drop / pH: 7.5 / Details: PEG 8000

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: Australian Synchrotron / Beamline: MX2 / Wavelength: 1 Å
DetectorType: ADSC QUANTUM 315r / Detector: CCD / Date: Apr 19, 2017
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 2.43→45.83 Å / Num. obs: 17713 / % possible obs: 99.6 % / Redundancy: 6.5 % / Biso Wilson estimate: 56.8 Å2 / CC1/2: 0.999 / Rmerge(I) obs: 0.046 / Rpim(I) all: 0.02 / Rrim(I) all: 0.051 / Net I/σ(I): 16.8 / Num. measured all: 114498
Reflection shellResolution: 2.43→2.52 Å / Redundancy: 6.6 % / Rmerge(I) obs: 0.496 / Num. measured all: 11479 / Num. unique obs: 1747 / CC1/2: 0.915 / Rpim(I) all: 0.208 / Rrim(I) all: 0.538 / Net I/σ(I) obs: 2.7 / % possible all: 97

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Phasing

PhasingMethod: molecular replacement
Phasing MRModel details: Phaser MODE: MR_AUTO
Highest resolutionLowest resolution
Rotation2.5 Å43.09 Å
Translation2.5 Å43.09 Å

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Processing

Software
NameVersionClassificationNB
PHENIXrefinement
XDSdata reduction
Aimless0.5.29data scaling
PHASER2.3.0phasing
PDB_EXTRACT3.24data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 2B3R

2b3r


Resolution: 2.427→43.094 Å / SU ML: 0.32 / Cross valid method: THROUGHOUT / σ(F): 1.38 / Phase error: 27.99 / Stereochemistry target values: MLHL
RfactorNum. reflection% reflection
Rfree0.2501 897 5.08 %
Rwork0.2094 16759 -
obs0.2115 17656 99.54 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 152.43 Å2 / Biso mean: 58.7087 Å2 / Biso min: 30 Å2
Refinement stepCycle: final / Resolution: 2.427→43.094 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3012 0 177 23 3212
Biso mean--98.45 63.49 -
Num. residues----377
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0093277
X-RAY DIFFRACTIONf_angle_d1.4144455
X-RAY DIFFRACTIONf_chiral_restr0.088501
X-RAY DIFFRACTIONf_plane_restr0.007524
X-RAY DIFFRACTIONf_dihedral_angle_d16.4661939
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 6

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
2.4275-2.57960.38081450.28972684282998
2.5796-2.77870.31821540.262227622916100
2.7787-3.05830.28311510.246627442895100
3.0583-3.50060.23681490.221427972946100
3.5006-4.40970.24791550.188127942949100
4.4097-43.10120.22181430.192929783121100

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