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- PDB-3kkv: Structure of PKA with a protein Kinase B-selective inhibitor. -

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Basic information

Entry
Database: PDB / ID: 3kkv
TitleStructure of PKA with a protein Kinase B-selective inhibitor.
Components
  • PKI kinase inhibitor
  • cAMP-dependent protein kinase catalytic subunit alphaCAMP-dependent pathway
KeywordsTRANSFERASE/TRANSFERASE INHIBITOR / protein kinase / TRANSFERASE-TRANSFERASE INHIBITOR complex
Function / homology
Function and homology information


CD209 (DC-SIGN) signaling / HDL assembly / Regulation of insulin secretion / Rap1 signalling / Ion homeostasis / PKA activation in glucagon signalling / DARPP-32 events / CREB1 phosphorylation through the activation of Adenylate Cyclase / GPER1 signaling / Factors involved in megakaryocyte development and platelet production ...CD209 (DC-SIGN) signaling / HDL assembly / Regulation of insulin secretion / Rap1 signalling / Ion homeostasis / PKA activation in glucagon signalling / DARPP-32 events / CREB1 phosphorylation through the activation of Adenylate Cyclase / GPER1 signaling / Factors involved in megakaryocyte development and platelet production / Loss of Nlp from mitotic centrosomes / Recruitment of mitotic centrosome proteins and complexes / Loss of proteins required for interphase microtubule organization from the centrosome / Anchoring of the basal body to the plasma membrane / AURKA Activation by TPX2 / RET signaling / Interleukin-3, Interleukin-5 and GM-CSF signaling / Recruitment of NuMA to mitotic centrosomes / VEGFA-VEGFR2 Pathway / PKA activation / GLI3 is processed to GLI3R by the proteasome / MAPK6/MAPK4 signaling / Regulation of PLK1 Activity at G2/M Transition / Hedgehog 'off' state / cAMP-dependent protein kinase / cAMP-dependent protein kinase activity / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / cAMP-dependent protein kinase complex / AMP-activated protein kinase activity / Vasopressin regulates renal water homeostasis via Aquaporins / protein kinase A regulatory subunit binding / mesoderm formation / sperm flagellum / protein kinase A signaling / negative regulation of TORC1 signaling / acrosomal vesicle / neuromuscular junction / cellular response to heat / peptidyl-serine phosphorylation / protein kinase activity / protein domain specific binding / protein phosphorylation / protein serine kinase activity / protein serine/threonine kinase activity / perinuclear region of cytoplasm / mitochondrion / ATP binding / nucleus / plasma membrane / cytosol / cytoplasm
Similarity search - Function
cAMP-dependent protein kinase catalytic subunit / Extension to Ser/Thr-type protein kinases / AGC-kinase, C-terminal / AGC-kinase C-terminal domain profile. / Transferase(Phosphotransferase) domain 1 / Transferase(Phosphotransferase); domain 1 / Phosphorylase Kinase; domain 1 / Phosphorylase Kinase; domain 1 / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. ...cAMP-dependent protein kinase catalytic subunit / Extension to Ser/Thr-type protein kinases / AGC-kinase, C-terminal / AGC-kinase C-terminal domain profile. / Transferase(Phosphotransferase) domain 1 / Transferase(Phosphotransferase); domain 1 / Phosphorylase Kinase; domain 1 / Phosphorylase Kinase; domain 1 / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / Protein kinase domain / Serine/Threonine protein kinases, catalytic domain / Protein kinase, ATP binding site / Protein kinases ATP-binding region signature. / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily / 2-Layer Sandwich / Orthogonal Bundle / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
Chem-B99 / cAMP-dependent protein kinase catalytic subunit alpha
Similarity search - Component
Biological speciesBos taurus (cattle)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.8 Å
AuthorsElkins, P.A. / Concha, N.O.
CitationJournal: To be Published
Title: Structure of PKA with a protein Kinase B-selective inhibitor.
Authors: Lin, H. / Elkins, P.A. / Concha, N.O. / Heerding, D. / Wang, W. / Nidarmarthy, S. / Luengo, J.I. / Nelson, R. / Knick, V.B. / Choudhry, A.E.
History
DepositionNov 6, 2009Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 22, 2010Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Version format compliance
Revision 1.2Dec 10, 2014Group: Other

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: cAMP-dependent protein kinase catalytic subunit alpha
I: PKI kinase inhibitor
hetero molecules


Theoretical massNumber of molelcules
Total (without water)43,4763
Polymers43,0132
Non-polymers4641
Water4,522251
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2050 Å2
ΔGint-2 kcal/mol
Surface area16890 Å2
MethodPISA
Unit cell
Length a, b, c (Å)72.411, 75.996, 80.390
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121

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Components

#1: Protein cAMP-dependent protein kinase catalytic subunit alpha / CAMP-dependent pathway / PKA C-alpha


Mass: 40786.395 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bos taurus (cattle) / Plasmid: pDEST14 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: P00517, cAMP-dependent protein kinase
#2: Protein/peptide PKI kinase inhibitor


Mass: 2226.411 Da / Num. of mol.: 1 / Source method: obtained synthetically
Details: PKI was purchased from commercially available source
#3: Chemical ChemComp-B99 / (2S)-1-{[6-furan-3-yl-5-(3-methyl-2H-indazol-5-yl)pyridin-3-yl]oxy}-3-(1H-indol-3-yl)propan-2-amine


Mass: 463.530 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C28H25N5O2
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 251 / 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: 2.57 Å3/Da / Density % sol: 52.16 %
Crystal growTemperature: 294 K / Method: vapor diffusion, sitting drop / pH: 6.4
Details: Protein stock was at 20mg/ml in 25mM Mes-Bis Tris, pH 6.4, 75mM LiCl, 0.1M EDTA, 1mM DTT. The final concentration of inhibitor in the protein solution was 1mM from a 100mM stock solution of ...Details: Protein stock was at 20mg/ml in 25mM Mes-Bis Tris, pH 6.4, 75mM LiCl, 0.1M EDTA, 1mM DTT. The final concentration of inhibitor in the protein solution was 1mM from a 100mM stock solution of compound dissolved in DMSO.The protein was reacted with inhibitor on ice for 6 hours prior to crystallization.The final concentration of PKI in the protein solution was 2mM from stock 100mM dissolved in water. The final concentration of MEGA8 in the protein solution was 1.5mM from 225mM stock diluted from 790mM in the Hampton additive kit with water. Two microliters of protein were added to 0.35 microliters of 10% glycerol in buffer and put in sitting drops over 15% methanol diluted with water at 4C. Cryo was 100mM MES pH6.4, 100mM LiCl, 25% MeOH, 20% MPD., VAPOR DIFFUSION, SITTING DROP, temperature 294K

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Data collection

Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 17-ID / Wavelength: 1 Å
DetectorType: ADSC QUANTUM 210 / Detector: CCD / Date: Jul 1, 2005
RadiationMonochromator: downward deflecting liquid nitrogen cooled Si(111) double-crystal system monochromator designed and developed by Daresbury Laboratory (UK).
Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 1.8→50 Å / Num. all: 41841 / Num. obs: 41799 / % possible obs: 99.9 % / Observed criterion σ(F): 2 / Observed criterion σ(I): 2 / Biso Wilson estimate: 24 Å2 / Net I/σ(I): 37
Reflection shellResolution: 1.8→1.86 Å / % possible all: 99.6

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Processing

Software
NameVersionClassification
ADSCQuantumdata collection
AMoREphasing
PHENIX(phenix.refine: 1.5_2)refinement
HKL-2000data reduction
HKL-2000data scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.8→36.206 Å / SU ML: 0.22 / σ(F): 1.9 / Stereochemistry target values: ML
RfactorNum. reflection% reflectionSelection details
Rfree0.2252 2108 5.05 %Random
Rwork0.2017 ---
all0.2028 41840 --
obs0.2028 41735 99.88 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL / Bsol: 40.067 Å2 / ksol: 0.328 e/Å3
Refinement stepCycle: LAST / Resolution: 1.8→36.206 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2956 0 35 251 3242
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0013071
X-RAY DIFFRACTIONf_angle_d0.594150
X-RAY DIFFRACTIONf_dihedral_angle_d13.0791122
X-RAY DIFFRACTIONf_chiral_restr0.034429
X-RAY DIFFRACTIONf_plane_restr0.002540
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.8-1.84210.25731550.23322567X-RAY DIFFRACTION99
1.8421-1.88810.26031340.22232589X-RAY DIFFRACTION100
1.8881-1.93920.22111320.21492635X-RAY DIFFRACTION100
1.9392-1.99620.24471370.212599X-RAY DIFFRACTION100
1.9962-2.06070.20971350.20332621X-RAY DIFFRACTION100
2.0607-2.13430.23651540.19452601X-RAY DIFFRACTION100
2.1343-2.21970.26311360.20222613X-RAY DIFFRACTION100
2.2197-2.32080.26231340.20282637X-RAY DIFFRACTION100
2.3208-2.44310.21891410.2072637X-RAY DIFFRACTION100
2.4431-2.59610.26051390.20472637X-RAY DIFFRACTION100
2.5961-2.79650.25421390.22882634X-RAY DIFFRACTION100
2.7965-3.07780.2351370.22022667X-RAY DIFFRACTION100
3.0778-3.52280.21241400.20742670X-RAY DIFFRACTION100
3.5228-4.43710.20451600.17042698X-RAY DIFFRACTION100
4.4371-36.21280.17891350.17182822X-RAY DIFFRACTION99
Refinement TLS params.

Refine-ID: X-RAY DIFFRACTION

IDMethodL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
1refined0.82990.10490.00370.9053-0.411.46690.00240.0573-0.00420.0370.02780.0781-0.1238-0.1396-0.02470.10950.02350.00380.1558-0.01880.1357-13.63060.9107-8.5711
20.3703-0.1070.09650.3907-0.48320.8505-0.10050.0782-0.04890.1829-0.0804-0.0902-0.06250.1620.1060.24520.01970.00150.2167-0.0020.1795
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1chain A and resid 8:350A8 - 350
2X-RAY DIFFRACTION2chain I and resid 5:24I5 - 24

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