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- PDB-8gm5: Functional construct of the Eukaryotic elongation factor 2 kinase... -

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Basic information

Entry
Database: PDB / ID: 8gm5
TitleFunctional construct of the Eukaryotic elongation factor 2 kinase bound to Calmodulin, ADP and to the A-484954 inhibitor and showing two conformations for the 498-520 loop
Components
  • Calmodulin-1
  • Eukaryotic elongation factor 2 kinase
KeywordsTRANSLATION/Transferase / elongation factor 2 kinase / eEF2 / eEF2K / eEF-2K / Calmodulin / TRANSLATION / A-484954 / ADP / allostery / TRANSLATION-Transferase complex / alternate conformations / SIGNALING PROTEIN
Function / homology
Function and homology information


elongation factor 2 kinase / elongation factor-2 kinase activity / response to prolactin / regulation of translation at postsynapse / myosin II filament disassembly / regulation of protein autophosphorylation / cellular response to anoxia / positive regulation of dendritic spine morphogenesis / translation factor activity, RNA binding / positive regulation of synapse assembly ...elongation factor 2 kinase / elongation factor-2 kinase activity / response to prolactin / regulation of translation at postsynapse / myosin II filament disassembly / regulation of protein autophosphorylation / cellular response to anoxia / positive regulation of dendritic spine morphogenesis / translation factor activity, RNA binding / positive regulation of synapse assembly / CaM pathway / Cam-PDE 1 activation / Sodium/Calcium exchangers / Calmodulin induced events / Reduction of cytosolic Ca++ levels / CREB1 phosphorylation through the activation of CaMKII/CaMKK/CaMKIV cascasde / Activation of Ca-permeable Kainate Receptor / Loss of phosphorylation of MECP2 at T308 / CREB1 phosphorylation through the activation of Adenylate Cyclase / PKA activation / negative regulation of high voltage-gated calcium channel activity / translational elongation / CaMK IV-mediated phosphorylation of CREB / Glycogen breakdown (glycogenolysis) / organelle localization by membrane tethering / negative regulation of calcium ion export across plasma membrane / Activation of RAC1 downstream of NMDARs / mitochondrion-endoplasmic reticulum membrane tethering / CLEC7A (Dectin-1) induces NFAT activation / regulation of cardiac muscle cell action potential / autophagosome membrane docking / positive regulation of ryanodine-sensitive calcium-release channel activity / Negative regulation of NMDA receptor-mediated neuronal transmission / regulation of cell communication by electrical coupling involved in cardiac conduction / negative regulation of peptidyl-threonine phosphorylation / Unblocking of NMDA receptors, glutamate binding and activation / Synthesis of IP3 and IP4 in the cytosol / Phase 0 - rapid depolarisation / mTORC1-mediated signalling / protein phosphatase activator activity / positive regulation of endocytosis / RHO GTPases activate PAKs / positive regulation of cyclic-nucleotide phosphodiesterase activity / positive regulation of phosphoprotein phosphatase activity / Ion transport by P-type ATPases / Long-term potentiation / Uptake and function of anthrax toxins / Calcineurin activates NFAT / Regulation of MECP2 expression and activity / catalytic complex / DARPP-32 events / detection of calcium ion / negative regulation of ryanodine-sensitive calcium-release channel activity / Smooth Muscle Contraction / RHO GTPases activate IQGAPs / regulation of cardiac muscle contraction / calcium channel inhibitor activity / cellular response to interferon-beta / regulation of cardiac muscle contraction by regulation of the release of sequestered calcium ion / Protein methylation / voltage-gated potassium channel complex / eNOS activation / Activation of AMPK downstream of NMDARs / regulation of release of sequestered calcium ion into cytosol by sarcoplasmic reticulum / cellular response to cAMP / regulation of calcium-mediated signaling / positive regulation of protein dephosphorylation / Tetrahydrobiopterin (BH4) synthesis, recycling, salvage and regulation / titin binding / Ion homeostasis / regulation of ryanodine-sensitive calcium-release channel activity / cellular response to brain-derived neurotrophic factor stimulus / positive regulation of protein autophosphorylation / sperm midpiece / calcium channel complex / cellular response to calcium ion / substantia nigra development / adenylate cyclase activator activity / Ras activation upon Ca2+ influx through NMDA receptor / regulation of heart rate / protein serine/threonine kinase activator activity / sarcomere / FCERI mediated Ca+2 mobilization / FCGR3A-mediated IL10 synthesis / VEGFR2 mediated vascular permeability / positive regulation of peptidyl-threonine phosphorylation / Antigen activates B Cell Receptor (BCR) leading to generation of second messengers / VEGFR2 mediated cell proliferation / regulation of cytokinesis / response to ischemia / Translocation of SLC2A4 (GLUT4) to the plasma membrane / spindle microtubule / RAF activation / positive regulation of receptor signaling pathway via JAK-STAT / positive regulation of protein serine/threonine kinase activity / Transcriptional activation of mitochondrial biogenesis / Stimuli-sensing channels / spindle pole / cellular response to type II interferon / response to calcium ion
Similarity search - Function
Eukaryotic elongation factor 2 kinase / : / MHCK/EF2 kinase / Alpha-kinase family / Alpha-type protein kinase domain profile. / Alpha-kinase family / EF-hand domain pair / EF-hand, calcium binding motif / EF-Hand 1, calcium-binding site / EF-hand calcium-binding domain. ...Eukaryotic elongation factor 2 kinase / : / MHCK/EF2 kinase / Alpha-kinase family / Alpha-type protein kinase domain profile. / Alpha-kinase family / EF-hand domain pair / EF-hand, calcium binding motif / EF-Hand 1, calcium-binding site / EF-hand calcium-binding domain. / EF-hand calcium-binding domain profile. / EF-hand domain / EF-hand domain pair / Tetratricopeptide-like helical domain superfamily / Protein kinase-like domain superfamily
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / Chem-EKI / Eukaryotic elongation factor 2 kinase / Calmodulin-1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.12 Å
AuthorsPiserchio, A. / Isiorho, E.A. / Dalby, K.N. / Ghose, R.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM123252 United States
CitationJournal: J.Biol.Chem. / Year: 2023
Title: Structure of the complex between calmodulin and a functional construct of eukaryotic elongation factor 2 kinase bound to an ATP-competitive inhibitor.
Authors: Piserchio, A. / Isiorho, E.A. / Dalby, K.N. / Ghose, R.
History
DepositionMar 24, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 31, 2023Provider: repository / Type: Initial release
Revision 1.1Jun 21, 2023Group: Database references / Category: citation / Item: _citation.journal_volume
Revision 1.2Oct 25, 2023Group: 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
A: Eukaryotic elongation factor 2 kinase
B: Calmodulin-1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)77,9647
Polymers77,1022
Non-polymers8625
Water4,324240
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area4480 Å2
ΔGint-55 kcal/mol
Surface area25280 Å2
MethodPISA
Unit cell
Length a, b, c (Å)80.909, 61.041, 88.958
Angle α, β, γ (deg.)90.00, 111.26, 90.00
Int Tables number4
Space group name H-MP1211
Symmetry operation#1: x,y,z
#2: -x,y+1/2,-z

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Components

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Protein , 2 types, 2 molecules AB

#1: Protein Eukaryotic elongation factor 2 kinase / eEF-2 kinase / eEF-2K / Calcium/calmodulin-dependent eukaryotic elongation factor 2 kinase


Mass: 60380.727 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: EEF2K / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: O00418, elongation factor 2 kinase
#2: Protein Calmodulin-1 /


Mass: 16721.350 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CALM1, CALM, CAM, CAM1 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0DP23

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Non-polymers , 5 types, 245 molecules

#3: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
#4: Chemical ChemComp-EKI / 7-amino-1-cyclopropyl-3-ethyl-2,4-dioxo-1,2,3,4-tetrahydropyrido[2,3-d]pyrimidine-6-carboxamide


Mass: 289.290 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C13H15N5O3
#5: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
#6: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ca
#7: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 240 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY

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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: 2.68 Å3/Da / Density % sol: 53.71 %
Crystal growTemperature: 295.15 K / Method: vapor diffusion / pH: 6.5
Details: Cocktail:16.55% PEG-3350, 0.2 M NaF, 100 mM BisTris-Propane Protein solution: 10.3 mg/mL 20 mM Tris pH 7.5, 100 mM NaCl, 3 mM CaCl2, 1mM TCEP, 1.5 m Inhibitor , 3.1 % DMSO 2protein/1cocktail (0.2 ul total)
Temp details: Room temperature, not controlled

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Data collection

DiffractionMean temperature: 100 K / Ambient temp details: Cold nitrogen stream / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: NSLS-II / Beamline: 17-ID-2 / Wavelength: 0.97934 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: May 28, 2022
Details: Horizontal pre-focus bimorph mirror & KB bimorph mirrors
RadiationMonochromator: Si(111) DCM / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97934 Å / Relative weight: 1
ReflectionResolution: 2.12→82.906 Å / Num. obs: 30789 / % possible obs: 92 % / Redundancy: 4.93 % / CC1/2: 0.996 / Rmerge(I) obs: 0.1221 / Rpim(I) all: 0.0603 / Rrim(I) all: 0.1367 / Net I/σ(I): 5.89
Reflection shell
Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. measured allNum. measured obsNum. unique allNum. unique obsCC1/2CC1/2 anomalousRpim(I) allRrim(I) allAbsDiff over sigma anomalous% possible anomalous% possible ellipsoidal% possible ellipsoidal anomalous% possible spherical% possible spherical anomalousRedundancy anomalous% possible all
6.673-82.9064.940.048813.4476017601153915390.997-0.4440.02350.05440.40798.79998.79998.72.6899
5.288-6.6725.030.068511.4977477747154015400.995-0.210.03330.07640.48299.799.999.799.999.72.6399.9
4.605-5.2864.830.066211.3474397439153915390.995-0.1820.03290.07420.51698.999.898.999.898.92.5499.8
4.179-4.6054.60.067910.8770847084154015400.995-0.0380.03550.07690.52798.599.798.599.798.52.499.7
3.871-4.1794.790.08199.8573727372153915390.994-0.0450.04150.09220.56596.699.796.699.796.62.5299.7
3.643-3.8714.940.1078.6576057605154015400.993-0.0450.0530.11980.619910099100992.57100
3.458-3.6435.150.1235879237923153915390.989-0.010.06020.13790.60399.910099.910099.92.64100
3.308-3.4585.180.15347.0579727972154015400.987-0.0270.07450.17110.62299.799.999.799.999.72.6599.9
3.176-3.3075.260.18856.0380968096153915390.9850.0490.09060.20990.65199.899.999.899.999.82.799.9
3.065-3.1765.310.23395.1581698169153915390.981-0.0790.11120.25990.62699.899.899.899.899.82.7299.8
2.97-3.0655.340.29194.4382228222154015400.968-0.0050.13770.32390.63199.899.999.899.999.82.7399.9
2.884-2.975.320.36293.6981918191153915390.9530.0310.1710.40260.64799.810099.810099.82.73100
2.807-2.8845.380.4543.1382808280154015400.929-0.0050.21240.50290.66699.599.699.599.699.52.7599.6
2.734-2.8075.260.46462.9681018101153915390.9230.010.22020.5160.65296.395.796.393.494.32.6895.7
2.659-2.7345.020.55512.4277247724154015400.890.0380.27060.61990.6892.892.792.883.884.52.5592.7
2.586-2.6594.610.57282.1871007100153915390.854-0.0130.29570.64760.63788.890.988.875.574.72.3790.9
2.509-2.5864.60.56482.1670847084154015400.854-0.040.28970.63720.65583.886.183.864.163.12.3886.1
2.429-2.5094.490.6361.9269156915153915390.782-0.0050.33280.72080.66680.883.280.854.653.62.3283.2
2.338-2.4294.120.7121.6663506350154015400.68-0.0130.39610.81830.65272.776.472.741.539.92.1576.4
2.124-2.3384.450.88881.4668536853153915390.5670.030.47361.00910.66354.153.454.113.513.82.2553.4

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Processing

Software
NameVersionClassification
PHENIX1.20.1_4487refinement
autoPROC1.0.5 20221121data processing
XDSJan 10, 2022data reduction
Aimless0.7.7data scaling
PHASER2.3.90phasing
Coot0.9.6model building
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 7SHQ

7shq


Resolution: 2.12→45.94 Å / SU ML: 0.241 / Cross valid method: FREE R-VALUE / σ(F): 1.36 / Phase error: 27.122
Stereochemistry target values: GEOSTD + MONOMER LIBRARY + CDL V1.2
RfactorNum. reflection% reflection
Rfree0.225 1534 4.98 %
Rwork0.181 --
obs0.183 30782 67.1 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 42.27 Å2
Refinement stepCycle: LAST / Resolution: 2.12→45.94 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4304 0 51 240 4595
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0034502
X-RAY DIFFRACTIONf_angle_d0.5186084
X-RAY DIFFRACTIONf_dihedral_angle_d5.304613
X-RAY DIFFRACTIONf_chiral_restr0.039637
X-RAY DIFFRACTIONf_plane_restr0.004793
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.12-2.190.3201140.2591168X-RAY DIFFRACTION4
2.19-2.270.3538220.2504491X-RAY DIFFRACTION13
2.27-2.360.3113570.25831148X-RAY DIFFRACTION29
2.36-2.470.28291060.25771851X-RAY DIFFRACTION47
2.47-2.60.29071050.2382502X-RAY DIFFRACTION63
2.6-2.760.2981770.24743230X-RAY DIFFRACTION82
2.76-2.980.28442160.21683876X-RAY DIFFRACTION99
2.98-3.280.21772150.19713964X-RAY DIFFRACTION100
3.28-3.750.21112320.15993945X-RAY DIFFRACTION100
3.75-4.720.18951910.13783996X-RAY DIFFRACTION100
4.72-45.940.19871990.17214077X-RAY DIFFRACTION99

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