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- PDB-8gm4: Functional construct of the Eukaryotic elongation factor 2 kinase... -

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Basic information

Entry
Database: PDB / ID: 8gm4
TitleFunctional construct of the Eukaryotic elongation factor 2 kinase bound to an ATP-competitive inhibitor
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 / 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 / cellular response to anoxia / positive regulation of dendritic spine morphogenesis / translation factor activity, RNA binding / positive regulation of synapse assembly / CaM pathway ...elongation factor 2 kinase / elongation factor-2 kinase activity / response to prolactin / regulation of translation at postsynapse / myosin II filament disassembly / 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 / Activation of Ca-permeable Kainate Receptor / CREB1 phosphorylation through the activation of CaMKII/CaMKK/CaMKIV cascasde / Loss of phosphorylation of MECP2 at T308 / CREB1 phosphorylation through the activation of Adenylate Cyclase / PKA activation / CaMK IV-mediated phosphorylation of CREB / negative regulation of high voltage-gated calcium channel activity / Glycogen breakdown (glycogenolysis) / CLEC7A (Dectin-1) induces NFAT activation / Activation of RAC1 downstream of NMDARs / negative regulation of calcium ion export across plasma membrane / organelle localization by membrane tethering / mitochondrion-endoplasmic reticulum membrane tethering / autophagosome membrane docking / presynaptic endocytosis / regulation of cardiac muscle cell action potential / positive regulation of ryanodine-sensitive calcium-release channel activity / Synthesis of IP3 and IP4 in the cytosol / regulation of cell communication by electrical coupling involved in cardiac conduction / Phase 0 - rapid depolarisation / Negative regulation of NMDA receptor-mediated neuronal transmission / negative regulation of ryanodine-sensitive calcium-release channel activity / Unblocking of NMDA receptors, glutamate binding and activation / RHO GTPases activate PAKs / calcineurin-mediated signaling / positive regulation of endocytosis / Ion transport by P-type ATPases / Uptake and function of anthrax toxins / mTORC1-mediated signalling / Long-term potentiation / Regulation of MECP2 expression and activity / Calcineurin activates NFAT / protein phosphatase activator activity / regulation of ryanodine-sensitive calcium-release channel activity / DARPP-32 events / translational elongation / Smooth Muscle Contraction / catalytic complex / detection of calcium ion / regulation of cardiac muscle contraction / RHO GTPases activate IQGAPs / regulation of cardiac muscle contraction by regulation of the release of sequestered calcium ion / presynaptic cytosol / calcium channel inhibitor activity / cellular response to interferon-beta / Protein methylation / Activation of AMPK downstream of NMDARs / Ion homeostasis / regulation of release of sequestered calcium ion into cytosol by sarcoplasmic reticulum / eNOS activation / regulation of calcium-mediated signaling / Tetrahydrobiopterin (BH4) synthesis, recycling, salvage and regulation / titin binding / cellular response to brain-derived neurotrophic factor stimulus / cellular response to cAMP / voltage-gated potassium channel complex / sperm midpiece / substantia nigra development / calcium channel complex / calyx of Held / FCERI mediated Ca+2 mobilization / Ras activation upon Ca2+ influx through NMDA receptor / FCGR3A-mediated IL10 synthesis / adenylate cyclase activator activity / regulation of heart rate / cellular response to calcium ion / Antigen activates B Cell Receptor (BCR) leading to generation of second messengers / protein serine/threonine kinase activator activity / VEGFR2 mediated cell proliferation / sarcomere / regulation of cytokinesis / response to ischemia / VEGFR2 mediated vascular permeability / Translocation of SLC2A4 (GLUT4) to the plasma membrane / positive regulation of receptor signaling pathway via JAK-STAT / spindle microtubule / RAF activation / Transcriptional activation of mitochondrial biogenesis / Stimuli-sensing channels / cellular response to type II interferon / long-term synaptic potentiation / cellular response to insulin stimulus / response to calcium ion / RAS processing / spindle pole / Signaling by RAF1 mutants
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 ...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
Revision 1.3Oct 23, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature / Item: _pdbx_entry_details.has_protein_modification

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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
Water6,521362
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area4410 Å2
ΔGint-52 kcal/mol
Surface area24930 Å2
MethodPISA
Unit cell
Length a, b, c (Å)80.544, 60.684, 88.704
Angle α, β, γ (deg.)90.000, 111.100, 90.000
Int Tables number4
Space group name H-MP1211
Space group name HallP2yb
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, 367 molecules

#3: Chemical ChemComp-ADP / ADENOSINE-5'-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 / Feature type: SUBJECT OF INVESTIGATION
#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


Mass: 18.015 Da / Num. of mol.: 362 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY
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: 2.64 Å3/Da / Density % sol: 53.11 %
Crystal growTemperature: 295.15 K / Method: vapor diffusion / pH: 6.2
Details: Cocktail:16.55% PEG-3350, 200 mM NaF, 100 mM BisTris-Propane Protein sol: 10.3 mg/mL 20 mM Tris pH 7.5, 100 mM NaCl, 3 mM CaCl2, 1mM TCEP, 0.3 mM ADP, 0.3 m Inhibitor , 0.7 % DMSO ...Details: Cocktail:16.55% PEG-3350, 200 mM NaF, 100 mM BisTris-Propane Protein sol: 10.3 mg/mL 20 mM Tris pH 7.5, 100 mM NaCl, 3 mM CaCl2, 1mM TCEP, 0.3 mM ADP, 0.3 m Inhibitor , 0.7 % DMSO 2protein/1cocktail (0.2 ul total)
Temp details: Room temperature, no control

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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: 2.009→75.133 Å / Num. obs: 53518 / % possible obs: 100 % / Redundancy: 5.99 %
Details: Some remarks regarding the mmCIF items written, the PDB Exchange Dictionary (PDBx/mmCIF) Version 5.0 supporting the data files in the current PDB archive (dictionary version 5.325, last ...Details: Some remarks regarding the mmCIF items written, the PDB Exchange Dictionary (PDBx/mmCIF) Version 5.0 supporting the data files in the current PDB archive (dictionary version 5.325, last updated 2020-04-13: http://mmcif.wwpdb.org/dictionaries/mmcif_pdbx_v50.dic/Index/) and the actual quantities provided by MRFANA (https://github.com/githubgphl/MRFANA) from the autoPROC package (https://www.globalphasing.com/autoproc/). In general, the mmCIF categories here should provide items that are currently used in the PDB archive. If there are alternatives, the one recommended by the PDB developers has been selected. The distinction between *_all and *_obs quantities is not always clear: often only one version is actively used within the PDB archive (or is the one recommended by PDB developers). The intention of distinguishing between classes of reflections before and after some kind of observation criterion was applied, can in principle be useful - but such criteria change in various ways throughout the data processing steps (rejection of overloaded or too partial reflections, outlier/misfit rejections during scaling etc) and there is no retrospect computation of data scaling/merging statistics for the reflections used in the final refinement (where another observation criterion might have been applied). Typical data processing will usually only provide one version of statistics at various stages and these are given in the recommended item here, irrespective of the "_all" and "_obs" connotation, see e.g. the use of _reflns.pdbx_Rmerge_I_obs, _reflns.pdbx_Rrim_I_all and _reflns.pdbx_Rpim_I_all. Please note that all statistics related to "merged intensities" (or "merging") are based on inverse-variance weighting of the individual measurements making up a symmetry-unique reflection. This is standard for several decades now, even if some of the dictionary definitions seem to suggest that a simple "mean" or "average" intensity is being used instead. R-values are always given for all symmetry-equivalent reflections following Friedel's law, i.e. Bijvoet pairs are not treated separately (since we want to describe the overall mean intensity and not the mean I(+) and I(-) here). The Rrim metric is identical to the Rmeas R-value and only differs in name. _reflns.pdbx_number_measured_all is the number of measured intensities just before the final merging step (at which point no additional rejection takes place). _reflns.number_obs is the number of symmetry-unique observations, i.e. the result of merging those measurements via inverse-variance weighting. _reflns.pdbx_netI_over_sigmaI is based on the merged intensities (_reflns.number_obs) as expected. _reflns.pdbx_redundancy is synonymous with "multiplicity". The per-shell item _reflns_shell.number_measured_all corresponds to the overall value _reflns.pdbx_number_measured_all. The per-shell item _reflns_shell.number_unique_all corresponds to the overall value _reflns.number_obs. The per-shell item _reflns_shell.percent_possible_all corresponds to the overall value _reflns.percent_possible_obs. The per-shell item _reflns_shell.meanI_over_sigI_obs corresponds to the overall value given as _reflns.pdbx_netI_over_sigmaI. But be aware of the incorrect definition of the former in the current dictionary!
CC1/2: 0.985 / CC1/2 anomalous: -0.096 / Rmerge(I) obs: 0.2073 / Rpim(I) all: 0.0924 / Rrim(I) all: 0.2274 / AbsDiff over sigma anomalous: 0.746 / Net I/σ(I): 4.97 / Num. measured all: 320683 / % possible anomalous: 99.3 / Redundancy anomalous: 3.07
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 anomalousRedundancy anomalous% possible all
5.452-75.1336.070.083813.321691316913278627860.978-0.0890.03840.09250.55199.63.2299.9
4.328-5.4525.790.093212.361570515705271127110.989-0.1840.04190.10240.60599.83100
3.781-4.3285.70.107411.361540015400270427040.983-0.1480.04950.11850.65799.82.93100
3.435-3.7816.10.133610.541636316363268426840.982-0.1930.05940.14640.7371003.12100
3.189-3.4356.220.15019.131668316683268326830.986-0.0530.06590.16410.7561003.18100
3.001-3.1896.270.1847.761676016760267126710.979-0.1290.08020.2010.751003.21100
2.851-3.0016.30.2416.221688316883268126810.972-0.0280.10460.26310.76399.83.22100
2.726-2.8516.310.29785.191685116851267226720.969-0.0630.12890.3250.76599.53.23100
2.622-2.7265.90.388841570915709266326630.947-0.0670.17480.42720.75199.13.02100
2.531-2.6225.820.45623.51534215342263626360.92900.20560.50140.7799.22.98100
2.452-2.5316.010.58162.931614116141268526850.9120.0510.25660.63670.78398.93.09100
2.382-2.4525.720.7242.411536515365268726870.8560.0430.32860.79680.78399.12.93100
2.319-2.3825.550.87352.021472814728265326530.815-0.0080.40250.9640.76298.22.86100
2.263-2.3195.851.25021.61552615526265526550.7290.0360.55931.3720.79498.92.99100
2.211-2.2635.951.7921.481586115861266526650.525-0.0420.79231.96280.91499.13.0499.9
2.164-2.21161.81361.221585015850264326430.627-0.0110.79751.98450.76798.93.07100
2.121-2.1646.021.96181.141611416114267526750.5620.0180.86012.14530.76199.13.07100
2.081-2.1216.072.5070.941607716077264826480.4650.0021.0932.7390.741993.11100
2.044-2.0816.072.87510.831624816248267826780.44-0.0321.2533.1410.74299.23.1100
2.009-2.0446.133.65110.681616416164263826380.311-0.0091.58253.98510.75699.43.12100

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Processing

Software
NameVersionClassification
PHENIX1.20.1_4487refinement
autoPROC1.0.5 20221121data processing
Aimless0.7.7data scaling
TRUNCATE7.1.018data processing
XDSJan 10, 2022 (BUILT 20220220)data reduction
PHASER1.20.1_4487phasing
Coot0.9.6model building
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 7SHQ

7shq


Resolution: 2.12→47.73 Å / SU ML: 0.2731 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 28.1087
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2376 2303 5.08 %
Rwork0.2071 43015 -
obs0.2086 45318 99.14 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 48.69 Å2
Refinement stepCycle: LAST / Resolution: 2.12→47.73 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4215 0 51 362 4628
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00274369
X-RAY DIFFRACTIONf_angle_d0.51485903
X-RAY DIFFRACTIONf_chiral_restr0.0376609
X-RAY DIFFRACTIONf_plane_restr0.0034772
X-RAY DIFFRACTIONf_dihedral_angle_d5.9337591
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.12-2.160.41711220.36262495X-RAY DIFFRACTION92.08
2.16-2.220.35021490.33092599X-RAY DIFFRACTION97.55
2.22-2.270.39031450.35612622X-RAY DIFFRACTION97.74
2.27-2.330.33291600.29112683X-RAY DIFFRACTION99.79
2.33-2.40.27441460.25852703X-RAY DIFFRACTION99.96
2.4-2.480.28071440.24082665X-RAY DIFFRACTION99.96
2.48-2.570.25191420.22012712X-RAY DIFFRACTION100
2.57-2.670.25871420.22672692X-RAY DIFFRACTION99.96
2.67-2.790.24781470.21422703X-RAY DIFFRACTION99.93
2.79-2.940.25961430.21482697X-RAY DIFFRACTION99.96
2.94-3.120.22121420.20272713X-RAY DIFFRACTION99.93
3.12-3.360.1911530.1992733X-RAY DIFFRACTION99.93
3.36-3.70.22841620.18522689X-RAY DIFFRACTION99.93
3.7-4.240.20211440.1622724X-RAY DIFFRACTION99.97
4.24-5.340.19651220.16192773X-RAY DIFFRACTION99.97
5.34-47.730.21591400.19882812X-RAY DIFFRACTION99.63

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