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- PDB-6nx4: Structure of the C-terminal Helical Repeat Domain of Eukaryotic E... -

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Basic information

Entry
Database: PDB / ID: 6nx4
TitleStructure of the C-terminal Helical Repeat Domain of Eukaryotic Elongation Factor 2 Kinase (eEF-2K)
ComponentsEukaryotic elongation factor 2 kinase
KeywordsTRANSLATION / eEF2K / eEF2 / binding domain / kinase / transferase / SEL1 / elongation / TPR
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 / translational elongation / positive regulation of endocytosis / mTORC1-mediated signalling / cellular response to cAMP / cellular response to brain-derived neurotrophic factor stimulus / cellular response to calcium ion / response to ischemia / cellular response to insulin stimulus / dendritic spine / postsynaptic density / protein autophosphorylation / calmodulin binding / protein kinase activity / glutamatergic synapse / calcium ion binding / negative regulation of apoptotic process / ATP binding / cytosol / cytoplasm
Similarity search - Function
Eukaryotic elongation factor 2 kinase / : / Sel1 repeat / MHCK/EF2 kinase / Alpha-kinase family / Alpha-type protein kinase domain profile. / Alpha-kinase family / Tetratricopeptide-like helical domain superfamily / Protein kinase-like domain superfamily
Similarity search - Domain/homology
Eukaryotic elongation factor 2 kinase
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodSOLUTION NMR / molecular dynamics
AuthorsPiserchio, A. / Will, N. / Giles, D.H. / Hajredini, F. / Dalby, K.N. / Ghose, R.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)GM123252 United States
CitationJournal: J.Mol.Biol. / Year: 2019
Title: Solution Structure of the Carboxy-Terminal Tandem Repeat Domain of Eukaryotic Elongation Factor 2 Kinase and Its Role in Substrate Recognition.
Authors: Piserchio, A. / Will, N. / Giles, D.H. / Hajredini, F. / Dalby, K.N. / Ghose, R.
History
DepositionFeb 8, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 29, 2019Provider: repository / Type: Initial release
Revision 1.1Jul 10, 2019Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.title
Revision 1.2Dec 18, 2019Group: Author supporting evidence / Data collection / Category: pdbx_audit_support / pdbx_nmr_spectrometer
Item: _pdbx_audit_support.funding_organization / _pdbx_nmr_spectrometer.model
Remark 650 HELIX DETERMINATION METHOD: AUTHOR PROVIDED.

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Eukaryotic elongation factor 2 kinase


Theoretical massNumber of molelcules
Total (without water)18,8931
Polymers18,8931
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)20 / 1000structures with the lowest energy
RepresentativeModel #1medoid

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Components

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


Mass: 18893.021 Da / Num. of mol.: 1 / Fragment: 562-725 C-terminal fragment
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: EEF2K / Plasmid: pET-26b / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: O00418, elongation factor 2 kinase

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Experimental details

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Experiment

ExperimentMethod: SOLUTION NMR
NMR experiment
Conditions-IDExperiment-IDSolution-IDSample stateSpectrometer-IDType
111isotropic13D 1H-15N NOESY
124isotropic14D HC-HSQC-NOESY-HN-HSQC
132isotropic33D 3D NNH (15N-HSQC-NOESY-15N-HSQC
144isotropic23D 1H-13C NOESY aliphatic
154isotropic23D H-13C NOESY aromatic
162isotropic43D HNCO
172isotropic43D HNCA
182isotropic53D HN(CO)CA
192isotropic43D HN(CA)CB
1102isotropic43D CBCA(CO)NH
1112isotropic43D HN(CA)CO
1122isotropic53D Hcc(co)NH
1132anisotropic53D hCc(co)NH
1142isotropic53D (H)CCH-TOCSY
1152isotropic53D (H)CCH-TOCSY
1163anisotropic42D 15N HSQC-IPAP
1173anisotropic43D IPAP-J-HNCO

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Sample preparation

Details
TypeSolution-IDContentsLabelSolvent system
solution1200 uM [U-99% 13C; U-99% 15N] eEF2K (562-725), 20 mM sodium phosphate, 100 mM sodium chloride, 4 mM DTT, 0.2 mM EDTA, 2 mM AEBSF protease inhibitor, 95% H2O/5% D2O15N13C95% H2O/5% D2O
solution4360 uM [U-99% 13C; U-99% 15N] eEF2K (562-725), 20 mM sodium phosphate, 100 mM sodium chloride, 4 mM DTT, 0.2 mM EDTA, 2 mM AEBSF protease inhibitor, 95% H2O/5% D2O15N13C_395% H2O/5% D2O
solution2400 uM [U-99% 13C; U-99% 15N] eEF2K (562-725), 20 mM sodium phosphate, 100 mM sodium chloride, 4 mM DTT, 0.2 mM EDTA, 2 mM AEBSF protease inhibitor, 95% H2O/5% D2O15N13C_295% H2O/5% D2O
filamentous virus3340 uM [U-99% 13C; U-99% 15N] eEF2K (562-725), 14 mg/mL Pf1 phage, 14 mM sodium phosphate, 100 mM sodium chloride, 4 mM DTT, 0.2 mM EDTA, 2 mM AEBSF protease inhibitor, 95% H2O/5% D2OPf195% H2O/5% D2O
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
200 uMeEF2K (562-725)[U-99% 13C; U-99% 15N]1
20 mMsodium phosphatenatural abundance1
100 mMsodium chloridenatural abundance1
4 mMDTTnatural abundance1
0.2 mMEDTAnatural abundance1
2 mMAEBSF protease inhibitornatural abundance1
360 uMeEF2K (562-725)[U-99% 13C; U-99% 15N]4
20 mMsodium phosphatenatural abundance4
100 mMsodium chloridenatural abundance4
4 mMDTTnatural abundance4
0.2 mMEDTAnatural abundance4
2 mMAEBSF protease inhibitornatural abundance4
400 uMeEF2K (562-725)[U-99% 13C; U-99% 15N]2
20 mMsodium phosphatenatural abundance2
100 mMsodium chloridenatural abundance2
4 mMDTTnatural abundance2
0.2 mMEDTAnatural abundance2
2 mMAEBSF protease inhibitornatural abundance2
340 uMeEF2K (562-725)[U-99% 13C; U-99% 15N]3
14 mg/mLPf1 phagenatural abundance3
14 mMsodium phosphatenatural abundance3
100 mMsodium chloridenatural abundance3
4 mMDTTnatural abundance3
0.2 mMEDTAnatural abundance3
2 mMAEBSF protease inhibitornatural abundance3
Sample conditionsIonic strength: 20 mM Phosphate 100 mM NaCl Not defined / Label: conditions_1 / pH: 6.5 / PH err: 0.1 / Pressure: 1 atm / Temperature: 298.15 K

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NMR measurement

NMR spectrometer
TypeManufacturerModelField strength (MHz)Spectrometer-ID
Bruker AVANCEBrukerAVANCE9001
Bruker AVANCEBrukerAVANCE8002
Bruker AVANCE IIIBrukerAVANCE III8003
Bruker AVANCE IIIBrukerAVANCE III7004
Bruker AVANCE IIIBrukerAVANCE III6005

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Processing

NMR software
NameDeveloperClassification
NMRPipeDelaglio, Grzesiek, Vuister, Zhu, Pfeifer and Baxprocessing
SMILEYing, J., Delaglio, F., Torchia, D. A. & Bax, A.processing
NMRViewJohnson, One Moon Scientificdata analysis
TALOSNShen, Y. & Bax, A.data analysis
PALESZweckstetter, Mdata analysis
NMRViewJohnson, One Moon Scientificpeak picking
ARIALinge, O'Donoghue and Nilgesstructure calculation
RefinementMethod: molecular dynamics / Software ordinal: 7 / Details: explicit water
NMR representativeSelection criteria: medoid
NMR ensembleConformer selection criteria: structures with the lowest energy
Conformers calculated total number: 1000 / Conformers submitted total number: 20

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