1OM2

SOLUTION NMR STRUCTURE OF THE MITOCHONDRIAL PROTEIN IMPORT RECEPTOR TOM20 FROM RAT IN A COMPLEX WITH A PRESEQUENCE PEPTIDE DERIVED FROM RAT ALDEHYDE DEHYDROGENASE (ALDH)

SOLUTION NMR

NMR Experiment
Experiment	Type	Sample Contents	Solvent	Ionic Strength	pH	Pressure	Temperature (K)	Spectrometer
1	HNCA	90% WATER/10% D2O, 100% D2O		0.02M	6.8	1 atm	303
2	HN(CO)CA	90% WATER/10% D2O, 100% D2O		0.02M	6.8	1 atm	303
3	CBCA(CO)NH	90% WATER/10% D2O, 100% D2O		0.02M	6.8	1 atm	303
4	HNCACB	90% WATER/10% D2O, 100% D2O		0.02M	6.8	1 atm	303
5	HBHA(CO)NH	90% WATER/10% D2O, 100% D2O		0.02M	6.8	1 atm	303
6	C(CO)NH-TOCSY HC(CO)NH-TOCSY	90% WATER/10% D2O, 100% D2O		0.02M	6.8	1 atm	303
7	HCCH-COSY	90% WATER/10% D2O, 100% D2O		0.02M	6.8	1 atm	303
8	HCCH-TOCSY	90% WATER/10% D2O, 100% D2O		0.02M	6.8	1 atm	303
9	15N-RESOLVED NOESY	90% WATER/10% D2O, 100% D2O		0.02M	6.8	1 atm	303
10	15N-RESOLVED TOCSY	90% WATER/10% D2O, 100% D2O		0.02M	6.8	1 atm	303
11	NOESY	90% WATER/10% D2O, 100% D2O		0.02M	6.8	1 atm	303
12	TOCSY	90% WATER/10% D2O, 100% D2O		0.02M	6.8	1 atm	303
13	HNHA	90% WATER/10% D2O, 100% D2O		0.02M	6.8	1 atm	303
14	{1H}-15N NOE	90% WATER/10% D2O, 100% D2O		0.02M	6.8	1 atm	303
15	3D F1 13C-FILTERED-F3 13C-RESOLVED NOESY	90% WATER/10% D2O, 100% D2O		0.02M	6.8	1 atm	303
16	2D F1/F2 13C-FILTERED NOESY	90% WATER/10% D2O, 100% D2O		0.02M	6.8	1 atm	303

NMR Spectrometer Information
Spectrometer	Manufacturer	Model	Field Strength
1	Bruker	DMX600	600
2	Bruker	DMX750	750

NMR Refinement
Method	Details	Software
TORSION ANGLE DYNAMICS FOLLOWED BY RESTRAINED ENERGY MINIMIZATION WITH AMBER FORCE FIELD	DEPOSITED COORDINATES WERE CALCULATED BASED ON 1012 NOE-DERIVED DISTANCE, 80 SLOWLY EXCHANGING AMIDE PROTON-DERIVED DISTANCE, AND 39 DIHEDRAL ANGLE RESTRAINTS. NO VIOLATIONS OF DISTANCE RESTRAINTS EXCEED 0.33 ANGSTROMS, AND NO VIOLATIONS OF ANGLE CONSTRAINTS EXCEED 2.8 DEGREES.	DYANA 1.5, EMBOSS

NMR Ensemble Information
Conformer Selection Criteria	LOWEST TARGET FUNCTION
Conformers Calculated Total Number	200
Conformers Submitted Total Number	20
Representative Model	1 (n/a)

Additional NMR Experimental Information
Details	THE INTRA-TOM20 PROTEIN NOES WERE OBTAINED AT PH5.4 AND 0.02M CHAPSO ( DETERGENT) IN THE ABSENCE OF THE PEPTIDE USING 3D 15N-RESOLVED AND 3D 13C- RESOLVED NOESY SPECTRA. THE INTRA-PEPTIDE NOES AND INTERMOLECULAR NOES WERE COLLECTED AT PH6.8 IN THE ABSENCE OF CHAPSO USING 2D AND 3D NOESY SPECTRA WITH ISOTOPE FILTERS. THE NOE ASSIGNMENT WAS COMPLETED BY USING SEQUENCE- SPECIFICALLY DEUTERATED DERIVATIVES OF THE PEPTIDE. THESE THREE NOE SETS WERE COMBINED TO CALCULATE THE COMPLEX STRUCTURE OF TOM20 AND THE PRESEQUENCE PEPTIDE.

Additional NMR Experimental Information

Details

THE INTRA-TOM20 PROTEIN NOES WERE OBTAINED AT PH5.4 AND 0.02M CHAPSO ( DETERGENT) IN THE ABSENCE OF THE PEPTIDE USING 3D 15N-RESOLVED AND 3D 13C- RESOLVED NOESY SPECTRA. THE INTRA-PEPTIDE NOES AND INTERMOLECULAR NOES WERE COLLECTED AT PH6.8 IN THE ABSENCE OF CHAPSO USING 2D AND 3D NOESY SPECTRA WITH ISOTOPE FILTERS. THE NOE ASSIGNMENT WAS COMPLETED BY USING SEQUENCE- SPECIFICALLY DEUTERATED DERIVATIVES OF THE PEPTIDE. THESE THREE NOE SETS WERE COMBINED TO CALCULATE THE COMPLEX STRUCTURE OF TOM20 AND THE PRESEQUENCE PEPTIDE.

Computation: NMR Software
#	Classification	Version	Software Name	Author
1	refinement	DYANA 1.5, EMBOSS	5.0	GUNTERT & WUTHRICH, NAKAI & NAKAMURA
2	structure solution	DYANA
3	structure solution	EMBOSS

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.

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1OM2

SOLUTION NMR STRUCTURE OF THE MITOCHONDRIAL PROTEIN IMPORT RECEPTOR TOM20 FROM RAT IN A COMPLEX WITH A PRESEQUENCE PEPTIDE DERIVED FROM RAT ALDEHYDE DEHYDROGENASE (ALDH)