2N3O

Structure of PTB RRM1(41-163) bound to an RNA stemloop containing a structured loop derived from viral internal ribosomal entry site RNA


SOLUTION NMR
NMR Experiment
ExperimentTypeSample ContentsSolventIonic StrengthpHPressureTemperature (K)Spectrometer
13D HNCA1 mM [U-99% 13C; U-99% 15N] PTBRRM1, 1 mM RNA, 10 mM sodium phosphate, 20 mM sodium chloride90% H2O/10% D2O0.0206.5ambient313
23D HN(CO)CA1 mM [U-99% 13C; U-99% 15N] PTBRRM1, 1 mM RNA, 10 mM sodium phosphate, 20 mM sodium chloride90% H2O/10% D2O0.0206.5ambient313
33D HNCACB1 mM [U-99% 13C; U-99% 15N] PTBRRM1, 1 mM RNA, 10 mM sodium phosphate, 20 mM sodium chloride90% H2O/10% D2O0.0206.5ambient313
43D HNCO1 mM [U-99% 13C; U-99% 15N] PTBRRM1, 1 mM RNA, 10 mM sodium phosphate, 20 mM sodium chloride90% H2O/10% D2O0.0206.5ambient313
53D HCCH-TOCSY1 mM [U-99% 13C; U-99% 15N] PTBRRM1, 1 mM RNA, 10 mM sodium phosphate, 20 mM sodium chloride90% H2O/10% D2O0.0206.5ambient313
63D HCCH-TOCSY1 mM [U-99% 13C; U-99% 15N] PTBRRM1, 1 mM RNA, 10 mM sodium phosphate, 20 mM sodium chloride90% H2O/10% D2O0.0206.5ambient313
73D 1H-15N NOESY-HSQC1 mM [U-99% 15N] PTBRRM1, 1 mM RNA, 10 mM sodium phosphate, 20 mM sodium chloride90% H2O/10% D2O0.0206.5ambient313
83D 1H-13C NOESY aliphatic-HSQC1 mM [U-99% 13C; U-99% 15N] PTBRRM1, 1 mM RNA, 10 mM sodium phosphate, 20 mM sodium chloride90% H2O/10% D2O0.0206.5ambient313
93D 1H-13C NOESY aromatic-HSQC1 mM [U-99% 13C; U-99% 15N] PTBRRM1, 1 mM RNA, 10 mM sodium phosphate, 20 mM sodium chloride90% H2O/10% D2O0.0206.5ambient313
102D 1H-1H TOCSY1 mM [U-99% 15N] PTBRRM1, 1 mM RNA, 10 mM sodium phosphate, 20 mM sodium chloride100% D2O0.0206.5ambient313
112D 1H-1H NOESY1 mM [U-99% 15N] PTBRRM1, 1 mM RNA, 10 mM sodium phosphate, 20 mM sodium chloride100% D2O0.0206.5ambient313
123D f1-13Cfiltered,f2-13C-edited NOESY aliphatic-HSQC1 mM [U-99% 15N] PTBRRM1, 1 mM [U-99% 13C; U-99% 15N] RNA, 10 mM sodium phosphate, 20 mM sodium chloride100% D2O0.0206.5ambient313
133D f1-13Cfiltered,f2-13C-edited NOESY aliphatic-HSQC1 mM [U-99% 15N] PTBRRM1, 1 mM [U-13C; U-15N]-ribose-Ura10,Ura12,Ura14 RNA, 10 mM sodium phosphate, 20 mM sodium chloride100% D2O0.0206.5ambient313
142D f2-13C-filtered NOESY1 mM [U-99% 15N] PTBRRM1, 1 mM [U-99% 13C; U-99% 15N] RNA, 10 mM sodium phosphate, 20 mM sodium chloride100% D2O0.0206.5ambient313
153D aliphatic-HSQC-NOESY1 mM [U-99% 13C; U-99% 15N] PTBRRM1, 1 mM RNA, 10 mM sodium phosphate, 20 mM sodium chloride90% H2O/10% D2O0.0206.5ambient278
162D H2C2 TROSY1 mM [U-99% 15N] PTBRRM1, 1 mM [U-99% 13C; U-99% 15N] RNA, 10 mM sodium phosphate, 20 mM sodium chloride100% D2O0.0206.5ambient313
172D H6C6 TROSY1 mM [U-99% 15N] PTBRRM1, 1 mM [U-99% 13C; U-99% 15N] RNA, 10 mM sodium phosphate, 20 mM sodium chloride100% D2O0.0206.5ambient313
182D H8C8 TROSY1 mM [U-99% 15N] PTBRRM1, 1 mM [U-99% 13C; U-99% 15N] RNA, 10 mM sodium phosphate, 20 mM sodium chloride100% D2O0.0206.5ambient313
192D H2C2 TROSY1 mM [U-99% 15N] PTBRRM1, 1 mM [U-99% 13C; U-99% 15N] RNA, 10 mM sodium phosphate, 20 mM sodium chloride, 13 mg/mL Pf1 phage100% D2O0.0206.5ambient313
202D H6C6 TROSY1 mM [U-99% 15N] PTBRRM1, 1 mM [U-99% 13C; U-99% 15N] RNA, 10 mM sodium phosphate, 20 mM sodium chloride, 13 mg/mL Pf1 phage100% D2O0.0206.5ambient313
212D H8C8 TROSY1 mM [U-99% 15N] PTBRRM1, 1 mM [U-99% 13C; U-99% 15N] RNA, 10 mM sodium phosphate, 20 mM sodium chloride, 13 mg/mL Pf1 phage100% D2O0.0206.5ambient313
222D H5C5 TROSY1 mM [U-99% 15N] PTBRRM1, 1 mM [U-99% 13C; U-99% 15N] RNA, 10 mM sodium phosphate, 20 mM sodium chloride100% D2O0.0206.5ambient313
232D H1'C1' TROSY1 mM [U-99% 15N] PTBRRM1, 1 mM [U-99% 13C; U-99% 15N] RNA, 10 mM sodium phosphate, 20 mM sodium chloride100% D2O0.0206.5ambient313
242D H5C5 TROSY1 mM [U-99% 15N] PTBRRM1, 1 mM [U-99% 13C; U-99% 15N] RNA, 10 mM sodium phosphate, 20 mM sodium chloride, 13 mg/mL Pf1 phage100% D2O0.0206.5ambient313
252D H1'C1' TROSY1 mM [U-99% 15N] PTBRRM1, 1 mM [U-99% 13C; U-99% 15N] RNA, 10 mM sodium phosphate, 20 mM sodium chloride, 13 mg/mL Pf1 phage100% D2O0.0206.5ambient313
262D HN TROSY1 mM [U-99% 15N] PTBRRM1, 1 mM RNA, 10 mM sodium phosphate, 20 mM sodium chloride90% H2O/10% D2O0.0206.5ambient313
272D HN TROSY1 mM [U-99% 15N] PTBRRM1, 1 mM RNA, 10 mM sodium phosphate, 20 mM sodium chloride, 13 mg/mL Pf1 phage90% H2O/10% D2O0.0206.5ambient313
282D HN TROSY1 mM [U-99% 15N] PTBRRM1, 1 mM [U-99% 13C; U-99% 15N] RNA, 10 mM sodium phosphate, 20 mM sodium chloride, 13 mg/mL Pf1 phage90% H2O/10% D2O0.0206.5ambient278
292D HN TROSY1 mM [U-99% 15N] PTBRRM1, 1 mM [U-99% 13C; U-99% 15N] RNA, 10 mM sodium phosphate, 20 mM sodium chloride, 13 mg/mL Pf1 phage90% H2O/10% D2O0.0206.5ambient278
302D {1H}15N-NOE HSQC1 mM [U-99% 15N] PTBRRM1, 1 mM RNA, 10 mM sodium phosphate, 20 mM sodium chloride90% H2O/10% D2O0.0206.5ambient313
312D 1H-1H NOESY1 mM [U-99% 15N] PTBRRM1, 1 mM RNA, 10 mM sodium phosphate, 20 mM sodium chloride90% H2O/10% D2O0.0206.5ambient278
323D f1-13Cfiltered,f2-13C-edited NOESY aliphatic-HSQC1 mM [U-99% 15N] PTBRRM1, 1 mM [U-13C; U-15N]-Ura7,Ura10,Cyt11,Ura12,Ura13,Ura14,Cyt15,Cyt16,Ura20,Cyt21,Cyt22,Cyt23 RNA, 10 mM sodium phosphate, 20 mM sodium chloride100% D2O0.0206.5ambient313
333D 1H-13C NOESY aliphatic1 mM [U-99% 15N] PTBRRM1, 1 mM [U-13C; U-15N]-Ura7,Ura10,Cyt11,Ura12,Ura13,Ura14,Cyt15,Cyt16,Ura20,Cyt21,Cyt22,Cyt23 RNA, 10 mM sodium phosphate, 20 mM sodium chloride100% D2O0.0206.5ambient313
343D 1H-13C NOESY aliphatic1 mM [U-99% 15N] PTBRRM1, 1 mM [U-13C; U-15N]-ribose-Ura10,Ura12,Ura14 RNA, 10 mM sodium phosphate, 20 mM sodium chloride100% D2O0.0206.5ambient313
353D f1-13Cfiltered,f2-13C-edited NOESY aliphatic-HSQC1 mM [U-99% 15N] PTBRRM1, 1 mM [U-13C; U-15N]-ribose-Cyt9,Ura11,Ura13,Gua15 RNA, 10 mM sodium phosphate, 20 mM sodium chloride100% D2O0.0206.5ambient313
362D 1H-13C HSQC aliphatic1 mM [U-99% 15N] PTBRRM1, 1 mM [U-13C; U-15N]-ribose-Ura10,Ura12,Ura14 RNA, 10 mM sodium phosphate, 20 mM sodium chloride100% D2O0.0206.5ambient313
372D 1H-13C HSQC aromatic1 mM [U-99% 15N] PTBRRM1, 1 mM [U-99% 13C; U-99% 15N] RNA, 10 mM sodium phosphate, 20 mM sodium chloride100% D2O0.0206.5ambient313
383D 1H-13C NOESY aliphatic1 mM [U-99% 15N] PTBRRM1, 1 mM [U-99% 13C; U-99% 15N] RNA, 10 mM sodium phosphate, 20 mM sodium chloride100% D2O0.0206.5ambient313
393D 1H-13C NOESY aromatic1 mM [U-99% 15N] PTBRRM1, 1 mM RNA, 10 mM sodium phosphate, 20 mM sodium chloride100% D2O0.0206.5ambient313
403D 1H-13C NOESY aliphatic1 mM [U-99% 13C; U-99% 15N] RNA, 10 mM sodium phosphate, 20 mM sodium chloride100% D2O0.0206.5ambient303
413D 1H-13C NOESY aromatic1 mM [U-99% 13C; U-99% 15N] RNA, 10 mM sodium phosphate, 20 mM sodium chloride100% D2O0.0206.5ambient303
422D 1H-13C HSQC aliphatic1 mM [U-99% 15N] PTBRRM1, 1 mM [U-13C; U-15N]-ribose-Cyt9,Ura11,Ura13,Gua15 RNA, 10 mM sodium phosphate, 20 mM sodium chloride100% D2O0.0206.5ambient313
432D 1H-1H NOESY1 mM RNA, 10 mM sodium phosphate, 20 mM sodium chloride90% H2O/10% D2O0.0206.5ambient278
NMR Spectrometer Information
SpectrometerManufacturerModelField Strength
1BrukerAVANCE500
2BrukerAVANCE600
3BrukerAVANCE700
4BrukerAVANCE750
5BrukerAVANCE900
NMR Refinement
MethodDetailsSoftware
simulated annealing, simulated annealing250 CONFORMERS WERE CALCULATED WITH CYANA USING THE DEPOSITED UPL AND ACO CONSTRAINT FILES. THE 50 CONFORMERS WITH THE LOWEST CYANA TARGET FUNCTION WERE SELECTED FOR REFINEMENT WITH AMBER. 50 CYANA CONFORMERS WERE REFINED WITH AMBER USING CONSTRAINTS DEFINED IN THE DEPOSITED RST FILE. THE REFINED CONFORMERS WERE THEN SORTED BY AMBER ENERGY. THE BEST 30 WERE PRESELECTED AND FROM THESE 30 THE 20 CONFORMERS WITH LOWEST VIOLATION ENERGY WERE SORTED AND DEPOSITED.Sparky
NMR Ensemble Information
Conformer Selection Criteriastructures with the least restraint violations
Conformers Calculated Total Number250
Conformers Submitted Total Number20
Representative Model1 (lowest energy)
Additional NMR Experimental Information
DetailsStandard 3D NOESYs measured with 150 msec mixing time. 2D NOESY with 200 msec mixing time. NOESYs measured at 5C were obtained using 3919-watergate sequence before detection. RNA Constraints for all base-paired nucleotides except the closing G9-C15 basepair of the loop were derived from NOESY spectra obtained with free RNA - the NOE patterns were the same as for the RNA in the complex but signal to noise was better. 3D NOESYs of unlabeled RNA were measured with 200 msec mixing time, and 2D NOESY of unlabeled RNA was measured with 250 msec mixing time.
Computation: NMR Software
#ClassificationVersionSoftware NameAuthor
1chemical shift assignmentSparky3.114Goddard
2peak pickingSparky3.114Goddard
3peak pickingUNIO/ATNOS-CANDID10Herrmann, Guntert and Wuthrich
4noe assignmentUNIO/ATNOS-CANDID10Herrmann, Guntert and Wuthrich
5chemical shift assignmentCYANA3.0Guntert, Mumenthaler and Wuthrich
6noe assignmentCYANA3.0Guntert, Mumenthaler and Wuthrich
7structure calculationCYANA3.0Guntert, Mumenthaler and Wuthrich
8refinementAmber12Case, Darden, Cheatham, III, Simmerling, Wang, Duke, Luo, ... and Kollman
9validationProcheckNMR3.5.4Laskowski and MacArthur
10data analysisMOLMOL2K.2Koradi, Billeter and Wuthrich