6NX4

Structure of the C-terminal Helical Repeat Domain of Eukaryotic Elongation Factor 2 Kinase (eEF-2K)

SOLUTION NMR

NMR Experiment
Experiment	Type	Sample Contents	Solvent	Ionic Strength	pH	Pressure	Temperature (K)	Spectrometer
1	3D 1H-15N NOESY	200 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% D2O	20 mM Phosphate 100 mM NaCl	6.5	1 atm	298.15	Bruker AVANCE 900
2	4D HC-HSQC-NOESY-HN-HSQC	360 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% D2O	20 mM Phosphate 100 mM NaCl	6.5	1 atm	298.15	Bruker AVANCE 900
3	3D 3D NNH (15N-HSQC-NOESY-15N-HSQC	400 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% D2O	20 mM Phosphate 100 mM NaCl	6.5	1 atm	298.15	Bruker AVANCE III 800
4	3D 1H-13C NOESY aliphatic	360 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% D2O	20 mM Phosphate 100 mM NaCl	6.5	1 atm	298.15	Bruker AVANCE 800
5	3D H-13C NOESY aromatic	360 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% D2O	20 mM Phosphate 100 mM NaCl	6.5	1 atm	298.15	Bruker AVANCE 800
6	3D HNCO	400 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% D2O	20 mM Phosphate 100 mM NaCl	6.5	1 atm	298.15	Bruker AVANCE III 700
7	3D HNCA	400 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% D2O	20 mM Phosphate 100 mM NaCl	6.5	1 atm	298.15	Bruker AVANCE III 700
8	3D HN(CO)CA	400 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% D2O	20 mM Phosphate 100 mM NaCl	6.5	1 atm	298.15	Bruker AVANCE III 600
9	3D HNCACB	400 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% D2O	20 mM Phosphate 100 mM NaCl	6.5	1 atm	298.15	Bruker AVANCE III 700
10	3D CBCA(CO)NH	400 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% D2O	20 mM Phosphate 100 mM NaCl	6.5	1 atm	298.15	Bruker AVANCE III 700
11	3D HN(CA)CO	400 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% D2O	20 mM Phosphate 100 mM NaCl	6.5	1 atm	298.15	Bruker AVANCE III 700
12	3D Hcc(co)NH	400 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% D2O	20 mM Phosphate 100 mM NaCl	6.5	1 atm	298.15	Bruker AVANCE III 600
13	3D hCc(co)NH	400 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% D2O	20 mM Phosphate 100 mM NaCl	6.5	1 atm	298.15	Bruker AVANCE III 600
14	3D HCCH-TOCSY	400 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% D2O	20 mM Phosphate 100 mM NaCl	6.5	1 atm	298.15	Bruker AVANCE III 600
15	3D HCCH-TOCSY	400 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% D2O	20 mM Phosphate 100 mM NaCl	6.5	1 atm	298.15	Bruker AVANCE III 600
16	2D 15N HSQC-IPAP	340 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% D2O	20 mM Phosphate 100 mM NaCl	6.5	1 atm	298.15	Bruker AVANCE III 700
17	3D IPAP-J-HNCO	340 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% D2O	20 mM Phosphate 100 mM NaCl	6.5	1 atm	298.15	Bruker AVANCE III 700

NMR Spectrometer Information
Spectrometer	Manufacturer	Model	Field Strength
1	Bruker	AVANCE	900
2	Bruker	AVANCE	800
3	Bruker	AVANCE III	800
4	Bruker	AVANCE III	700
5	Bruker	AVANCE III	600

NMR Refinement
Method	Details	Software
molecular dynamics	explicit water	ARIA

NMR Ensemble Information
Conformer Selection Criteria	structures with the lowest energy
Conformers Calculated Total Number	1000
Conformers Submitted Total Number	20
Representative Model	1 (medoid)

Computation: NMR Software
#	Classification	Version	Software Name	Author
1	processing	NMRPipe		Delaglio, Grzesiek, Vuister, Zhu, Pfeifer and Bax
6	processing	SMILE		Ying, J., Delaglio, F., Torchia, D. A. & Bax, A.
5	data analysis	NMRView		Johnson, One Moon Scientific
2	data analysis	TALOSN		Shen, Y. & Bax, A.
3	data analysis	PALES		Zweckstetter, M
4	peak picking	NMRView		Johnson, One Moon Scientific
7	structure calculation	ARIA		Linge, O'Donoghue and Nilges

RCSB PDB Core Operations are funded by the U.S. National Science Foundation (DBI-2321666), the US Department of Energy (DE-SC0019749), and the National Cancer Institute, National Institute of Allergy and Infectious Diseases, and National Institute of General Medical Sciences of the National Institutes of Health under grant R01GM133198.