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- PDB-2n3o: Structure of PTB RRM1(41-163) bound to an RNA stemloop containing... -

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Basic information

Entry
Database: PDB / ID: 2n3o
TitleStructure of PTB RRM1(41-163) bound to an RNA stemloop containing a structured loop derived from viral internal ribosomal entry site RNA
Components
  • Polypyrimidine tract-binding protein 1
  • RNA (5'-R(*GP*GP*GP*AP*CP*CP*UP*GP*GP*UP*CP*UP*UP*UP*CP*CP*AP*GP*GP*UP*CP*CP*C)-3')
KeywordsRNA BINDING PROTEIN/RNA / Polypyirimine tract binding protein / IRES / PTB / C-terminal helix formation / RNA BINDING PROTEIN-RNA complex
Function / homology
Function and homology information


negative regulation of muscle cell differentiation / poly-pyrimidine tract binding / IRES-dependent viral translational initiation / positive regulation of calcineurin-NFAT signaling cascade / pre-mRNA binding / negative regulation of RNA splicing / FGFR2 alternative splicing / negative regulation of neuron differentiation / regulation of alternative mRNA splicing, via spliceosome / regulation of RNA splicing ...negative regulation of muscle cell differentiation / poly-pyrimidine tract binding / IRES-dependent viral translational initiation / positive regulation of calcineurin-NFAT signaling cascade / pre-mRNA binding / negative regulation of RNA splicing / FGFR2 alternative splicing / negative regulation of neuron differentiation / regulation of alternative mRNA splicing, via spliceosome / regulation of RNA splicing / negative regulation of mRNA splicing, via spliceosome / regulation of cell differentiation / Processing of Capped Intron-Containing Pre-mRNA / neurogenesis / mRNA Splicing - Major Pathway / RNA splicing / mRNA processing / mRNA binding / nucleolus / positive regulation of transcription by RNA polymerase II / RNA binding / extracellular exosome / nucleoplasm / nucleus / membrane
Similarity search - Function
PTBP1, RNA recognition motif 1 / PTBP1, RNA recognition motif 3 / HnRNP-L/PTB / PTBP1-like, RNA recognition motif 2 / RRM-like domain / RNA recognition motif. (a.k.a. RRM, RBD, or RNP domain) / RRM (RNA recognition motif) domain / RNA recognition motif / RNA recognition motif / Eukaryotic RNA Recognition Motif (RRM) profile. ...PTBP1, RNA recognition motif 1 / PTBP1, RNA recognition motif 3 / HnRNP-L/PTB / PTBP1-like, RNA recognition motif 2 / RRM-like domain / RNA recognition motif. (a.k.a. RRM, RBD, or RNP domain) / RRM (RNA recognition motif) domain / RNA recognition motif / RNA recognition motif / Eukaryotic RNA Recognition Motif (RRM) profile. / RNA recognition motif domain / RNA-binding domain superfamily / Nucleotide-binding alpha-beta plait domain superfamily / Alpha-Beta Plaits / 2-Layer Sandwich / Alpha Beta
Similarity search - Domain/homology
RNA / RNA (> 10) / Polypyrimidine tract-binding protein 1
Similarity search - Component
Biological speciesHomo sapiens (human)
synthetic construct (others)
MethodSOLUTION NMR / simulated annealing, simulated annealing
AuthorsMaris, C. / Jayne, S.F. / Damberger, F.F. / Ravindranathan, S. / Allain, F.H.-T.
CitationJournal: To be Published
Title: C-terminal helix folding upon pyrimidine-rich hairpin binding to PTB RRM1. Implications for PTB function in Encephalomyocarditis virus IRES activity.
Authors: Maris, C. / Jayne, S.F. / Damberger, F.F. / Ravindranathan, S. / Allain, F.H.-T.
History
DepositionJun 8, 2015Deposition site: BMRB / Processing site: RCSB
Revision 1.0Aug 10, 2016Provider: repository / Type: Initial release
Revision 1.1May 1, 2024Group: Data collection / Database references
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_nmr_software / pdbx_nmr_spectrometer
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_nmr_software.name / _pdbx_nmr_spectrometer.model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Polypyrimidine tract-binding protein 1
B: RNA (5'-R(*GP*GP*GP*AP*CP*CP*UP*GP*GP*UP*CP*UP*UP*UP*CP*CP*AP*GP*GP*UP*CP*CP*C)-3')


Theoretical massNumber of molelcules
Total (without water)20,6842
Polymers20,6842
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1610 Å2
ΔGint-14 kcal/mol
Surface area10580 Å2
MethodPISA
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)20 / 250structures with the least restraint violations
RepresentativeModel #1lowest energy

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Components

#1: Protein Polypyrimidine tract-binding protein 1 / PTB / 57 kDa RNA-binding protein PPTB-1 / Heterogeneous nuclear ribonucleoprotein I / hnRNP I


Mass: 13375.167 Da / Num. of mol.: 1 / Fragment: UNP residues 41-163
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PTB, PTBP1 / Plasmid: pTYB11 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: P26599
#2: RNA chain RNA (5'-R(*GP*GP*GP*AP*CP*CP*UP*GP*GP*UP*CP*UP*UP*UP*CP*CP*AP*GP*GP*UP*CP*CP*C)-3')


Mass: 7308.343 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Experimental details

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Experiment

ExperimentMethod: SOLUTION NMR
NMR experiment
Conditions-IDExperiment-IDSolution-IDType
1123D HNCA
1223D HN(CO)CA
1323D HN(CA)CB
1423D HNCO
1523D (H)CCH-TOCSY
1623D (H)CCH-TOCSY
1713D 1H-15N NOESY-HSQC
1823D 1H-13C NOESY aliphatic-HSQC
1923D 1H-13C NOESY aromatic-HSQC
11052D 1H-1H TOCSY
11152D 1H-1H NOESY
11233D f1-13Cfiltered,f2-13C-edited NOESY aliphatic-HSQC
11343D f1-13Cfiltered,f2-13C-edited NOESY aliphatic-HSQC
11432D f2-13C-filtered NOESY
31523D aliphatic-HSQC-NOESY
11632D H2C2 TROSY
11732D H6C6 TROSY
11832D H8C8 TROSY
11982D H2C2 TROSY
12082D H6C6 TROSY
12182D H8C8 TROSY
12232D H5C5 TROSY
12332D H1'C1' TROSY
12482D H5C5 TROSY
12582D H1'C1' TROSY
12612D HN TROSY
12772D HN TROSY
32892D HN TROSY
32992D HN TROSY
13012D {1H}15N-NOE HSQC
33112D 1H-1H NOESY
132123D f1-13Cfiltered,f2-13C-edited NOESY aliphatic-HSQC
133123D 1H-13C NOESY aliphatic
13443D 1H-13C NOESY aliphatic
135113D f1-13Cfiltered,f2-13C-edited NOESY aliphatic-HSQC
13642D 1H-13C HSQC aliphatic
13732D 1H-13C HSQC aromatic
13833D 1H-13C NOESY aliphatic
13953D 1H-13C NOESY aromatic
440133D 1H-13C NOESY aliphatic
441133D 1H-13C NOESY aromatic
142112D 1H-13C HSQC aliphatic
343142D 1H-1H NOESY
NMR detailsText: Standard 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 ...Text: Standard 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.

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Sample preparation

Details
Solution-IDContentsSolvent system
11 mM [U-99% 15N] PTBRRM1, 1 mM RNA, 10 mM sodium phosphate, 20 mM sodium chloride, 90% H2O/10% D2O90% H2O/10% D2O
21 mM [U-99% 13C; U-99% 15N] PTBRRM1, 1 mM RNA, 10 mM sodium phosphate, 20 mM sodium chloride, 90% H2O/10% D2O90% H2O/10% D2O
31 mM [U-99% 15N] PTBRRM1, 1 mM [U-99% 13C; U-99% 15N] RNA, 10 mM sodium phosphate, 20 mM sodium chloride, 100% D2O100% D2O
41 mM [U-99% 15N] PTBRRM1, 1 mM [U-13C; U-15N]-ribose-Ura10,Ura12,Ura14 RNA, 10 mM sodium phosphate, 20 mM sodium chloride, 100% D2O100% D2O
51 mM [U-99% 15N] PTBRRM1, 1 mM RNA, 10 mM sodium phosphate, 20 mM sodium chloride, 100% D2O100% D2O
61 mM [U-99% 13C; U-99% 15N] PTBRRM1, 1 mM RNA, 10 mM sodium phosphate, 20 mM sodium chloride, 100% D2O100% D2O
71 mM [U-99% 15N] PTBRRM1, 1 mM RNA, 10 mM sodium phosphate, 20 mM sodium chloride, 13 mg/mL Pf1 phage, 90% H2O/10% D2O90% H2O/10% D2O
81 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 phage, 100% D2O100% D2O
91 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 phage, 90% H2O/10% D2O90% H2O/10% D2O
101 mM [U-99% 15N] PTBRRM1, 1 mM [U-99% 13C; U-99% 15N] RNA, 10 mM sodium phosphate, 20 mM sodium chloride, 90% H2O/10% D2O90% H2O/10% D2O
111 mM [U-99% 15N] PTBRRM1, 1 mM [U-13C; U-15N]-ribose-Cyt9,Ura11,Ura13,Gua15 RNA, 10 mM sodium phosphate, 20 mM sodium chloride, 100% D2O100% D2O
121 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 chloride, 100% D2O100% D2O
131 mM [U-99% 13C; U-99% 15N] RNA, 10 mM sodium phosphate, 20 mM sodium chloride, 100% D2O100% D2O
141 mM RNA, 10 mM sodium phosphate, 20 mM sodium chloride, 90% H2O/10% D2O90% H2O/10% D2O
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
1 mMPTBRRM1-1[U-99% 15N]1
1 mMRNA1
10 mMsodium phosphate-31
20 mMsodium chloride-41
1 mMPTBRRM1-5[U-99% 13C; U-99% 15N]2
1 mMRNA2
10 mMsodium phosphate-72
20 mMsodium chloride-82
1 mMPTBRRM1-9[U-99% 15N]3
1 mMRNA[U-99% 13C; U-99% 15N]3
10 mMsodium phosphate-113
20 mMsodium chloride-123
1 mMPTBRRM1-13[U-99% 15N]4
1 mMRNA[U-13C; U-15N]-ribose-Ura10,Ura12,Ura144
10 mMsodium phosphate-154
20 mMsodium chloride-164
1 mMPTBRRM1-17[U-99% 15N]5
1 mMRNA5
10 mMsodium phosphate-195
20 mMsodium chloride-205
1 mMPTBRRM1-21[U-99% 13C; U-99% 15N]6
1 mMRNA6
10 mMsodium phosphate-236
20 mMsodium chloride-246
1 mMPTBRRM1-25[U-99% 15N]7
1 mMRNA7
10 mMsodium phosphate-277
20 mMsodium chloride-287
13 mg/mLPf1 phage-297
1 mMPTBRRM1-30[U-99% 15N]8
1 mMRNA[U-99% 13C; U-99% 15N]8
10 mMsodium phosphate-328
20 mMsodium chloride-338
13 mg/mLPf1 phage-348
1 mMPTBRRM1-35[U-99% 15N]9
1 mMRNA[U-99% 13C; U-99% 15N]9
10 mMsodium phosphate-379
20 mMsodium chloride-389
13 mg/mLPf1 phage-399
1 mMPTBRRM1-40[U-99% 15N]10
1 mMRNA[U-99% 13C; U-99% 15N]10
10 mMsodium phosphate-4210
20 mMsodium chloride-4310
1 mMPTBRRM1-44[U-99% 15N]11
1 mMRNA[U-13C; U-15N]-ribose-Cyt9,Ura11,Ura13,Gua1511
10 mMsodium phosphate-4611
20 mMsodium chloride-4711
1 mMPTBRRM1-48[U-99% 15N]12
1 mMRNA[U-13C; U-15N]-Ura7,Ura10,Cyt11,Ura12,Ura13,Ura14,Cyt15,Cyt16,Ura20,Cyt21,Cyt22,Cyt2312
10 mMsodium phosphate-5012
20 mMsodium chloride-5112
1 mMRNA[U-99% 13C; U-99% 15N]13
10 mMsodium phosphate-5313
20 mMsodium chloride-5413
1 mMRNA14
10 mMsodium phosphate-5614
20 mMsodium chloride-5714
Sample conditions
Conditions-IDIonic strengthpHPressure (kPa)Temperature (K)
10.026.5ambient 313 K
20.026.5ambient 298 K
30.026.5ambient 278 K
40.026.5ambient 303 K

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NMR measurement

NMR spectrometer
TypeManufacturerModelField strength (MHz)Spectrometer-ID
Bruker AvanceBrukerAVANCE5001
Bruker AvanceBrukerAVANCE6002
Bruker AvanceBrukerAVANCE7003
Bruker AvanceBrukerAVANCE7504
Bruker AvanceBrukerAVANCE9005

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Processing

NMR software
NameVersionDeveloperClassification
Sparky3.114Goddardchemical shift assignment
Sparky3.114Goddardpeak picking
UNIO/ATNOS-CANDID10Herrmann, Guntert and Wuthrichpeak picking
UNIO/ATNOS-CANDID10Herrmann, Guntert and Wuthrichnoe assignment
CYANA3Guntert, Mumenthaler and Wuthrichchemical shift assignment
CYANA3Guntert, Mumenthaler and Wuthrichnoe assignment
CYANA3Guntert, Mumenthaler and Wuthrichstructure calculation
Amber12Case, Darden, Cheatham, III, Simmerling, Wang, Duke, Luo, ... and Kollmanrefinement
ProcheckNMR3.5.4Laskowski and MacArthurvalidation
MOLMOL2K.2Koradi, Billeter and Wuthrichdata analysis
RefinementMethod: simulated annealing, simulated annealing / Software ordinal: 1
Details: 250 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. ...Details: 250 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.
NMR constraintsNA alpha-angle constraints total count: 14 / NA beta-angle constraints total count: 14 / NA chi-angle constraints total count: 14 / NA delta-angle constraints total count: 92 / NA epsilon-angle constraints total count: 0 / NA gamma-angle constraints total count: 83 / NA other-angle constraints total count: 84 / NA sugar pucker constraints total count: 23 / NOE constraints total: 2284 / NOE intraresidue total count: 614 / NOE long range total count: 623 / NOE medium range total count: 381 / NOE sequential total count: 574 / Hydrogen bond constraints total count: 27
NMR representativeSelection criteria: lowest energy
NMR ensembleAverage torsion angle constraint violation: 0 °
Conformer selection criteria: structures with the least restraint violations
Conformers calculated total number: 250 / Conformers submitted total number: 20 / Maximum lower distance constraint violation: 0 Å / Maximum torsion angle constraint violation: 4.72 ° / Maximum upper distance constraint violation: 0.29 Å
NMR ensemble rmsDistance rms dev: 0 Å / Distance rms dev error: 0 Å

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