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- PDB-5lcj: In-Gel Activity-Based Protein Profiling of a Clickable Covalent E... -

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Basic information

Entry
Database: PDB / ID: 5lcj
TitleIn-Gel Activity-Based Protein Profiling of a Clickable Covalent Erk 1/2 Inhibitor
ComponentsMitogen-activated protein kinase 1
KeywordsTRANSFERASE / Erk1/2 covalent inhibitor
Function / homology
Function and homology information


phospho-PLA2 pathway / Signaling by MAPK mutants / RAF-independent MAPK1/3 activation / Suppression of apoptosis / Signaling by Activin / Gastrin-CREB signalling pathway via PKC and MAPK / cardiac neural crest cell development involved in heart development / caveolin-mediated endocytosis / ERKs are inactivated / cytosine metabolic process ...phospho-PLA2 pathway / Signaling by MAPK mutants / RAF-independent MAPK1/3 activation / Suppression of apoptosis / Signaling by Activin / Gastrin-CREB signalling pathway via PKC and MAPK / cardiac neural crest cell development involved in heart development / caveolin-mediated endocytosis / ERKs are inactivated / cytosine metabolic process / response to epidermal growth factor / Signaling by MAP2K mutants / Signaling by NODAL / RSK activation / Golgi Cisternae Pericentriolar Stack Reorganization / positive regulation of macrophage proliferation / Regulation of the apoptosome activity / outer ear morphogenesis / regulation of cellular pH / regulation of Golgi inheritance / ERBB signaling pathway / labyrinthine layer blood vessel development / mammary gland epithelial cell proliferation / trachea formation / Negative feedback regulation of MAPK pathway / regulation of early endosome to late endosome transport / regulation of stress-activated MAPK cascade / IFNG signaling activates MAPKs / Frs2-mediated activation / lung morphogenesis / positive regulation of macrophage chemotaxis / ERBB2-ERBB3 signaling pathway / response to exogenous dsRNA / regulation of cytoskeleton organization / Activation of the AP-1 family of transcription factors / face development / ERK/MAPK targets / progesterone receptor signaling pathway / androgen receptor signaling pathway / RUNX2 regulates osteoblast differentiation / pseudopodium / Recycling pathway of L1 / MAPK1 (ERK2) activation / Bergmann glial cell differentiation / negative regulation of cell differentiation / positive regulation of telomere capping / thyroid gland development / Advanced glycosylation endproduct receptor signaling / steroid hormone receptor signaling pathway / RHO GTPases Activate NADPH Oxidases / Estrogen-dependent nuclear events downstream of ESR-membrane signaling / Regulation of HSF1-mediated heat shock response / MAP kinase activity / regulation of ossification / RHO GTPases Activate WASPs and WAVEs / mitogen-activated protein kinase / phosphatase binding / Signal attenuation / Estrogen-stimulated signaling through PRKCZ / Nuclear events stimulated by ALK signaling in cancer / Schwann cell development / Growth hormone receptor signaling / stress-activated MAPK cascade / lipopolysaccharide-mediated signaling pathway / positive regulation of telomerase activity / positive regulation of telomere maintenance via telomerase / cellular response to cadmium ion / NPAS4 regulates expression of target genes / ERK1 and ERK2 cascade / cellular response to amino acid starvation / myelination / NCAM signaling for neurite out-growth / phosphotyrosine residue binding / RNA polymerase II CTD heptapeptide repeat kinase activity / ESR-mediated signaling / insulin-like growth factor receptor signaling pathway / thymus development / positive regulation of peptidyl-threonine phosphorylation / Regulation of PTEN gene transcription / Signal transduction by L1 / caveola / long-term synaptic potentiation / Negative regulation of FGFR3 signaling / Downregulation of SMAD2/3:SMAD4 transcriptional activity / Negative regulation of FGFR2 signaling / FCERI mediated MAPK activation / Negative regulation of FGFR4 signaling / FCGR3A-mediated phagocytosis / Negative regulation of FGFR1 signaling / SMAD2/SMAD3:SMAD4 heterotrimer regulates transcription / B cell receptor signaling pathway / peptidyl-threonine phosphorylation / Spry regulation of FGF signaling / Signaling by high-kinase activity BRAF mutants / response to nicotine / MAP2K and MAPK activation / regulation of protein stability / Oncogene Induced Senescence / mitotic spindle / Regulation of actin dynamics for phagocytic cup formation
Similarity search - Function
Mitogen-activated protein (MAP) kinase, ERK1/2 / Mitogen-activated protein (MAP) kinase, conserved site / MAP kinase signature. / 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 ...Mitogen-activated protein (MAP) kinase, ERK1/2 / Mitogen-activated protein (MAP) kinase, conserved site / MAP kinase signature. / 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-6TS / Mitogen-activated protein kinase 1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / FOURIER SYNTHESIS / Resolution: 1.78 Å
AuthorsO'Reilly, M. / Wright, D.
CitationJournal: Mol Biosyst / Year: 2016
Title: In-gel activity-based protein profiling of a clickable covalent ERK1/2 inhibitor.
Authors: Lebraud, H. / Wright, D.J. / East, C.E. / Holding, F.P. / O'Reilly, M. / Heightman, T.D.
History
DepositionJun 22, 2016Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 20, 2016Provider: repository / Type: Initial release
Revision 1.1Aug 31, 2016Group: Database references

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Mitogen-activated protein kinase 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)43,3324
Polymers42,5521
Non-polymers7803
Water9,638535
1


  • Idetical with deposited unit
  • defined by software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1220 Å2
ΔGint-34 kcal/mol
Surface area16920 Å2
MethodPISA
Unit cell
Length a, b, c (Å)48.729, 70.807, 60.208
Angle α, β, γ (deg.)90.00, 109.18, 90.00
Int Tables number4
Space group name H-MP1211

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Components

#1: Protein Mitogen-activated protein kinase 1 / MAPK 1 / ERT1 / Extracellular signal-regulated kinase 2 / ERK-2 / MAP kinase isoform p42 / p42-MAPK ...MAPK 1 / ERT1 / Extracellular signal-regulated kinase 2 / ERK-2 / MAP kinase isoform p42 / p42-MAPK / Mitogen-activated protein kinase 2 / MAPK 2


Mass: 42551.922 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: CYS 171 modified to CME by reaction. One letter code should is the same for both
Source: (gene. exp.) Homo sapiens (human) / Gene: MAPK1, ERK2, PRKM1, PRKM2 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: P28482, mitogen-activated protein kinase
#2: Chemical ChemComp-6TS / [(1~{R},4~{Z})-cyclooct-4-en-1-yl] ~{N}-[4-[4-[[5-chloranyl-4-[[2-(propanoylamino)phenyl]amino]pyrimidin-2-yl]amino]pyridin-2-yl]but-3-ynyl]carbamate


Mass: 588.100 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C31H34ClN7O3
#3: Chemical ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: SO4
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 535 / 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: 2.31 Å3/Da / Density % sol: 46.65 %
Crystal growTemperature: 297 K / Method: batch mode / pH: 7.2
Details: .2M (NH4)2SO4 32% MPEG 2000 .02M Mercaptoethanol .1M pH=7.2 HEPES/NaOH

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: ROTATING ANODE / Type: RIGAKU FR-E DW / Wavelength: 1.5417 Å
DetectorType: DECTRIS PILATUS 300K / Detector: PIXEL / Date: Sep 7, 2015
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.5417 Å / Relative weight: 1
ReflectionResolution: 1.78→30.05 Å / Num. obs: 102572 / % possible obs: 87.1 % / Observed criterion σ(F): 0 / Observed criterion σ(I): 0 / Redundancy: 3 % / Biso Wilson estimate: 27.18 Å2 / Rmerge(I) obs: 0.035 / Net I/σ(I): 17.2
Reflection shellResolution: 1.78→1.82 Å / Redundancy: 3 % / Rmerge(I) obs: 0.658 / Mean I/σ(I) obs: 1.9 / % possible all: 75.8

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Processing

Software
NameVersionClassification
BUSTER2.11.6refinement
iMOSFLMdata reduction
SCALEPACKdata scaling
REFMACphasing
RefinementMethod to determine structure: FOURIER SYNTHESIS / Resolution: 1.78→26.67 Å / Cor.coef. Fo:Fc: 0.961 / Cor.coef. Fo:Fc free: 0.942 / Rfactor Rfree error: 0.02 / SU R Cruickshank DPI: 0.131 / Cross valid method: THROUGHOUT / σ(F): 0 / SU R Blow DPI: 0.147 / SU Rfree Blow DPI: 0.137 / SU Rfree Cruickshank DPI: 0.129
Details: Cycles of Buster interspersed with rebuilds using Coot
RfactorNum. reflection% reflectionSelection details
Rfree0.212 1678 5.23 %RANDOM
Rwork0.162 ---
obs0.165 32060 86.9 %-
Displacement parametersBiso mean: 35.814 Å2
Baniso -1Baniso -2Baniso -3
1-2.7924 Å20 Å22.5459 Å2
2---1.5516 Å20 Å2
3----1.2408 Å2
Refine analyzeLuzzati coordinate error obs: 0.2 Å
Refinement stepCycle: 1 / Resolution: 1.78→26.67 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2817 0 62 535 3414
Refine LS restraints
Refine-IDTypeDev idealNumberRestraint functionWeight
X-RAY DIFFRACTIONt_bond_d0.0133030HARMONIC2
X-RAY DIFFRACTIONt_angle_deg1.084141HARMONIC2
X-RAY DIFFRACTIONt_dihedral_angle_d1075SINUSOIDAL2
X-RAY DIFFRACTIONt_incorr_chiral_ct
X-RAY DIFFRACTIONt_pseud_angle
X-RAY DIFFRACTIONt_trig_c_planes84HARMONIC2
X-RAY DIFFRACTIONt_gen_planes442HARMONIC16
X-RAY DIFFRACTIONt_it3024HARMONIC20
X-RAY DIFFRACTIONt_nbd
X-RAY DIFFRACTIONt_omega_torsion6.17
X-RAY DIFFRACTIONt_other_torsion17.41
X-RAY DIFFRACTIONt_improper_torsion
X-RAY DIFFRACTIONt_chiral_improper_torsion382SEMIHARMONIC5
X-RAY DIFFRACTIONt_sum_occupancies
X-RAY DIFFRACTIONt_utility_distance
X-RAY DIFFRACTIONt_utility_angle
X-RAY DIFFRACTIONt_utility_torsion
X-RAY DIFFRACTIONt_ideal_dist_contact4058SEMIHARMONIC4
LS refinement shellResolution: 1.78→1.84 Å / Rfactor Rfree error: 0 / Total num. of bins used: 16
RfactorNum. reflection% reflection
Rfree0.257 135 5.33 %
Rwork0.238 2398 -
all0.239 2533 -
obs--75.5 %
Refinement TLS params.Method: refined / Origin x: -1.1651 Å / Origin y: 3.3504 Å / Origin z: 38.0031 Å
111213212223313233
T-0.048 Å20.0076 Å20.0118 Å2--0.0574 Å2-0.031 Å2---0.0507 Å2
L0.9028 °2-0.2748 °20.2847 °2-0.595 °2-0.193 °2--1.0003 °2
S-0.0589 Å °-0.1357 Å °0.0942 Å °0.1053 Å °0.0611 Å °-0.0184 Å °-0.0729 Å °-0.0522 Å °-0.0022 Å °
Refinement TLS groupSelection details: { A|11 - A|357 }

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