1K1G

STRUCTURAL BASIS FOR RECOGNITION OF THE INTRON BRANCH SITE RNA BY SPLICING FACTOR 1

SOLUTION NMR

NMR Experiment
Experiment	Type	Sample Contents	Solvent	Ionic Strength	pH	Pressure	Temperature (K)	Spectrometer
1	3D_13C-separated_NOESY	1 mM U-15N,13C SF1 KH-QUA2/unlabeled BPS in 20 mM phosphate buffer NA pH 6.5, 50 mM NaCl, 2 mM DTT	100% D2O	50 mM NaCl	6.5	ambient	295
2	3D_13C/15N_edited/filtered_NOESY	1 mM U-15N,13C SF1 KH-QUA2/unlabeled BPS in 20 mM phosphate buffer NA pH 6.5, 50 mM NaCl, 2 mM DTT	100% D2O	50 mM NaCl	6.5	ambient	295
3	3D_15N-separated_NOESY	1 mM U-15N SF1 KH-QUA2/unlabeled BPS in 20 mM phosphate buffer NA pH 6.5, 50 mM NaCl, 2 mM DTT	90% H2O/10% D2O	50 mM NaCl	6.5	ambient	295

NMR Spectrometer Information
Spectrometer	Manufacturer	Model	Field Strength
1	Bruker	DRX	500
2	Bruker	DRX	600
3	Bruker	DRX	800

NMR Refinement
Method	Details	Software
restrained molecular dynamics, simulated annealing	STRUCTURES WERE CALCULATED WITH A MIXED TORSION AND CARTESIAN ANGLE DYNAMICS PROTOCOL USING ARIA/CNS. A REDUCED RELAXATION MATRIX APPROACH WAS USED FOR THE NOE CALIBRATION. THE STRUCTURES ARE CURRENTLY BEING REFINED USING RESIDUAL DIPOLAR COUPLINGS AND WILL BE UPDATED IN THE FUTURE.	XwinNMR

NMR Ensemble Information
Conformer Selection Criteria	10 lowest energy structures consistent with experimental distance and dihedral angle restraints
Conformers Calculated Total Number	100
Conformers Submitted Total Number	10
Representative Model	1 (lowest energy)

Additional NMR Experimental Information
Details	Backbone and side chain 1H, 15N and 13C resonances were assigned using standard triple resonance experiments. Distance restraints were derived from 13C- and 15N-edited 3D NOESY experiments. Assignments for the RNA were obtained from 2D isotope-filtered experiments. Intermolecular distance restraints were measured in 3D 13C-edited/filtered experiments. Dihedral angle restraints for the backbone angle phi were derived from 3J(HN,Ha) coupling constants measured in an HNHA-J experiment, additional phi/psi restraints were derived from TALOS. Hydrogen bond restraints for secondary structure elements in the protein were defined from slowly exchanging amide protons, identified after exchange of the H2O buffer to D2O.

Additional NMR Experimental Information

Details

Backbone and side chain 1H, 15N and 13C resonances were assigned using standard triple resonance experiments. Distance restraints were derived from 13C- and 15N-edited 3D NOESY experiments. Assignments for the RNA were obtained from 2D isotope-filtered experiments. Intermolecular distance restraints were measured in 3D 13C-edited/filtered experiments. Dihedral angle restraints for the backbone angle phi were derived from 3J(HN,Ha) coupling constants measured in an HNHA-J experiment, additional phi/psi restraints were derived from TALOS. Hydrogen bond restraints for secondary structure elements in the protein were defined from slowly exchanging amide protons, identified after exchange of the H2O buffer to D2O.

Computation: NMR Software
#	Classification	Version	Software Name	Author
1	collection	XwinNMR	2.6	Bruker
2	processing	NMRPipe	1.8	F. Delaglio, S. Grzesiek, G. Vuister, G. Zhu, J. Pfeifer, & A. Bax
3	data analysis	XEASY	1.2	Ch. Bartels, T.-H. Xia, M. Billeter, P. Gntert and K. Wthrich
4	structure solution	ARIA/CNS	1.0	J. Linge, M. Nilges
5	refinement	ARIA/CNS	1.0	J. Linge, M. 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.

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1K1G

STRUCTURAL BASIS FOR RECOGNITION OF THE INTRON BRANCH SITE RNA BY SPLICING FACTOR 1