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- PDB-5m6y: Cocrystal structure of cAMP-dependent Protein Kinase (PKA) in com... -

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Basic information

Entry
Database: PDB / ID: 5m6y
TitleCocrystal structure of cAMP-dependent Protein Kinase (PKA) in complex with a methylisoquinoline Fasudil-derivative
Components
  • cAMP-dependent protein kinase catalytic subunit alpha
  • cAMP-dependent protein kinase inhibitor alpha
KeywordsTRANSFERASE / PKA / Kinase / Fasudil-derivative / Inhibition / Cocrystal / 3x-Phosphorylated
Function / homology
Function and homology information


negative regulation of cAMP-dependent protein kinase activity / negative regulation of cAMP/PKA signal transduction / regulation of protein processing / protein localization to lipid droplet / regulation of bicellular tight junction assembly / cellular response to parathyroid hormone stimulus / cAMP-dependent protein kinase inhibitor activity / cellular response to cold / cAMP-dependent protein kinase / regulation of osteoblast differentiation ...negative regulation of cAMP-dependent protein kinase activity / negative regulation of cAMP/PKA signal transduction / regulation of protein processing / protein localization to lipid droplet / regulation of bicellular tight junction assembly / cellular response to parathyroid hormone stimulus / cAMP-dependent protein kinase inhibitor activity / cellular response to cold / cAMP-dependent protein kinase / regulation of osteoblast differentiation / cAMP-dependent protein kinase activity / sperm capacitation / negative regulation of glycolytic process through fructose-6-phosphate / ciliary base / cAMP-dependent protein kinase complex / AMP-activated protein kinase activity / negative regulation of protein import into nucleus / postsynaptic modulation of chemical synaptic transmission / protein kinase A regulatory subunit binding / plasma membrane raft / protein kinase A catalytic subunit binding / axoneme / mesoderm formation / sperm flagellum / regulation of proteasomal protein catabolic process / negative regulation of smoothened signaling pathway / positive regulation of gluconeogenesis / negative regulation of TORC1 signaling / regulation of G2/M transition of mitotic cell cycle / cellular response to glucagon stimulus / protein serine/threonine/tyrosine kinase activity / protein kinase A signaling / protein export from nucleus / acrosomal vesicle / positive regulation of protein export from nucleus / neural tube closure / cellular response to glucose stimulus / neuromuscular junction / positive regulation of insulin secretion / adenylate cyclase-activating G protein-coupled receptor signaling pathway / mRNA processing / manganese ion binding / cellular response to heat / dendritic spine / regulation of cell cycle / nuclear speck / protein domain specific binding / protein serine kinase activity / protein serine/threonine kinase activity / centrosome / glutamatergic synapse / ubiquitin protein ligase binding / protein kinase binding / perinuclear region of cytoplasm / negative regulation of transcription by RNA polymerase II / magnesium ion binding / mitochondrion / ATP binding / nucleus / cytosol / cytoplasm
Similarity search - Function
cAMP-dependent protein kinase inhibitor / cAMP-dependent protein kinase inhibitor / 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 ...cAMP-dependent protein kinase inhibitor / cAMP-dependent protein kinase inhibitor / 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-7KB / cAMP-dependent protein kinase catalytic subunit alpha / cAMP-dependent protein kinase inhibitor alpha
Similarity search - Component
Biological speciesCricetulus griseus (Chinese hamster)
Homo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.367 Å
AuthorsWienen-Schmidt, B. / Heine, A. / Klebe, G.
Funding support Belgium, 1items
OrganizationGrant numberCountry
European Research Council268145 - Project Acronym: DrugProfilBind Belgium
CitationJournal: To be Published
Title: Cocrystal structure of cAMP-dependent Protein Kinase (PKA) in complex with differently methylated Fasudil-derived ligands
Authors: Wienen-Schmidt, B. / Jonker, H. / Gerber, H.-D. / Saxena, K. / Kudlinzki, D. / Sreeramulu, S. / Heine, A. / Schwalbe, H. / Klebe, G.
History
DepositionOct 26, 2016Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 6, 2018Provider: repository / Type: Initial release
Revision 1.1Jan 17, 2024Group: 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
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Revision 1.2Nov 6, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature

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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
B: cAMP-dependent protein kinase inhibitor alpha
hetero molecules


Theoretical massNumber of molelcules
Total (without water)43,7644
Polymers43,3402
Non-polymers4242
Water7,566420
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2160 Å2
ΔGint-17 kcal/mol
Surface area17070 Å2
MethodPISA
Unit cell
Length a, b, c (Å)58.688, 73.142, 109.323
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 / PKA C-alpha


Mass: 41113.766 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Phosphorylated at Ser11, Thr198, Ser339 / Source: (gene. exp.) Cricetulus griseus (Chinese hamster) / Gene: PRKACA / Production host: Escherichia coli (E. coli) / References: UniProt: P25321, cAMP-dependent protein kinase
#2: Protein/peptide cAMP-dependent protein kinase inhibitor alpha / PKI-alpha / cAMP-dependent protein kinase inhibitor / muscle/brain isoform


Mass: 2226.411 Da / Num. of mol.: 1 / Fragment: UNP Residues 6-25 / Source method: obtained synthetically
Details: Amino acids 5-24 from cAMP-dependent protein kinase inhibitor alpha from Sigma (order ID: P7739)
Source: (synth.) Homo sapiens (human) / References: UniProt: P61925
#3: Chemical ChemComp-7KB / 5-(1,4-diazepan-1-ylsulfonyl)-4-methyl-isoquinoline


Mass: 305.395 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C15H19N3O2S
#4: Chemical ChemComp-MPD / (4S)-2-METHYL-2,4-PENTANEDIOL


Mass: 118.174 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H14O2 / Comment: precipitant*YM
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 420 / Source method: isolated from a natural source / Formula: H2O
Has protein modificationY

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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: 2.15 Å3/Da / Density % sol: 54.56 %
Crystal growTemperature: 277.15 K / Method: vapor diffusion, hanging drop / pH: 6.9
Details: Drop: 10 mg/ml PKA (0.240 mM) 30 mM MBT (MES/Bis-Tris Puffer pH 6.9) 1 mM DTT 0.1 mM EDTA 75 mM LiCl 0.03 mM Mega 8 0.07mM PKI (Sigma: P7739) 1.2 mM ligand solved in DMSO (50 mM Stock) ...Details: Drop: 10 mg/ml PKA (0.240 mM) 30 mM MBT (MES/Bis-Tris Puffer pH 6.9) 1 mM DTT 0.1 mM EDTA 75 mM LiCl 0.03 mM Mega 8 0.07mM PKI (Sigma: P7739) 1.2 mM ligand solved in DMSO (50 mM Stock) Reservoir: 20% Methanol 0.003 mL drop volume, 0.4 mL reservoir volume

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: BESSY / Beamline: 14.1 / Wavelength: 0.918409 Å
DetectorType: DECTRIS PILATUS3 S 6M / Detector: PIXEL / Date: Feb 7, 2015
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.918409 Å / Relative weight: 1
ReflectionResolution: 1.367→45.774 Å / Num. obs: 98699 / % possible obs: 98.7 % / Redundancy: 3.968 % / Rsym value: 0.033 / Net I/σ(I): 20.1
Reflection shellResolution: 1.37→1.45 Å / Redundancy: 3.878 % / Rmerge(I) obs: 0.484 / Mean I/σ(I) obs: 2.31 / % possible all: 96.5

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Processing

Software
NameVersionClassification
PHENIX(1.10.1_2155: ???)refinement
XDSdata reduction
XDSdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 1Q8W

1q8w


Resolution: 1.367→45.774 Å / SU ML: 0.11 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 14.82
RfactorNum. reflection% reflection
Rfree0.1663 4935 5 %
Rwork0.1441 --
obs0.1452 98694 98.68 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Refinement stepCycle: LAST / Resolution: 1.367→45.774 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3007 0 29 420 3456
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0083249
X-RAY DIFFRACTIONf_angle_d0.9894417
X-RAY DIFFRACTIONf_dihedral_angle_d21.5671214
X-RAY DIFFRACTIONf_chiral_restr0.071460
X-RAY DIFFRACTIONf_plane_restr0.007584
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.3673-1.38280.25411490.2342834X-RAY DIFFRACTION90
1.3828-1.39910.2431620.20743082X-RAY DIFFRACTION99
1.3991-1.41610.23981600.19393034X-RAY DIFFRACTION98
1.4161-1.43410.22521620.1873074X-RAY DIFFRACTION98
1.4341-1.45290.20271600.17653056X-RAY DIFFRACTION99
1.4529-1.47290.1881640.16293118X-RAY DIFFRACTION99
1.4729-1.49390.16441630.1513080X-RAY DIFFRACTION98
1.4939-1.51620.17951640.1473131X-RAY DIFFRACTION99
1.5162-1.53990.18791620.13853075X-RAY DIFFRACTION99
1.5399-1.56510.1731640.13163108X-RAY DIFFRACTION99
1.5651-1.59210.16611630.12893106X-RAY DIFFRACTION99
1.5921-1.62110.17471640.12963109X-RAY DIFFRACTION99
1.6211-1.65230.1751630.12723093X-RAY DIFFRACTION99
1.6523-1.6860.16561650.12973131X-RAY DIFFRACTION99
1.686-1.72260.17751630.12863108X-RAY DIFFRACTION99
1.7226-1.76270.1861640.12753119X-RAY DIFFRACTION99
1.7627-1.80680.15751640.13213117X-RAY DIFFRACTION99
1.8068-1.85570.18871660.1393157X-RAY DIFFRACTION99
1.8557-1.91030.16611650.14193130X-RAY DIFFRACTION99
1.9103-1.97190.18551660.14373146X-RAY DIFFRACTION99
1.9719-2.04240.18651650.14533136X-RAY DIFFRACTION99
2.0424-2.12420.17231660.14323166X-RAY DIFFRACTION100
2.1242-2.22080.16731650.13533134X-RAY DIFFRACTION99
2.2208-2.33790.15691660.13453156X-RAY DIFFRACTION99
2.3379-2.48440.14711680.13893174X-RAY DIFFRACTION100
2.4844-2.67620.1631670.14453190X-RAY DIFFRACTION99
2.6762-2.94550.15991690.15243193X-RAY DIFFRACTION99
2.9455-3.37160.16991670.15393183X-RAY DIFFRACTION99
3.3716-4.24740.14211720.1423258X-RAY DIFFRACTION99
4.2474-45.80.15591770.14083361X-RAY DIFFRACTION99

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