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- PDB-2yh1: Model of human U2AF65 tandem RRM1 and RRM2 domains with eight-sit... -

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Basic information

Entry
Database: PDB / ID: 2yh1
TitleModel of human U2AF65 tandem RRM1 and RRM2 domains with eight-site uridine binding
Components
  • 5'-R(*UP*UP*UP*UP*UP*UP*UP*UP*UP)-3'
  • SPLICING FACTOR U2AF 65 KDA SUBUNIT
KeywordsTRANSCRIPTION / PRE-MRNA SPLICING
Function / homology
Function and homology information


U2AF complex / poly-pyrimidine tract binding / pre-mRNA 3'-splice site binding / mRNA 3'-end processing / C2H2 zinc finger domain binding / commitment complex / Transport of Mature mRNA derived from an Intron-Containing Transcript / RNA Polymerase II Transcription Termination / U2-type prespliceosome / molecular function inhibitor activity ...U2AF complex / poly-pyrimidine tract binding / pre-mRNA 3'-splice site binding / mRNA 3'-end processing / C2H2 zinc finger domain binding / commitment complex / Transport of Mature mRNA derived from an Intron-Containing Transcript / RNA Polymerase II Transcription Termination / U2-type prespliceosome / molecular function inhibitor activity / spliceosomal complex assembly / Protein hydroxylation / negative regulation of mRNA splicing, via spliceosome / negative regulation of protein ubiquitination / mRNA Splicing - Major Pathway / positive regulation of RNA splicing / spliceosomal complex / mRNA splicing, via spliceosome / mRNA processing / nuclear speck / enzyme binding / RNA binding / nucleoplasm / nucleus
Similarity search - Function
U2 snRNP auxilliary factor, large subunit, splicing factor / 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 / Splicing factor U2AF 65 kDa subunit
Similarity search - Component
Biological speciesHOMO SAPIENS (human)
MethodSOLUTION NMR / MODIFIED ARIA
AuthorsMackereth, C.D. / Madl, T. / Simon, B. / Zanier, K. / Gasch, A. / Sattler, M.
Citation
Journal: Nature / Year: 2011
Title: Multi-Domain Conformational Selection Underlies Pre-Mrna Splicing Regulation by U2Af
Authors: Mackereth, C.D. / Madl, T. / Bonnal, S. / Simon, B. / Zanier, K. / Gasch, A. / Rybin, V. / Valcarcel, J. / Sattler, M.
#1: Journal: Mol.Cell / Year: 2006
Title: Structural Basis for Polypyrimidine Tract Recognition by the Essential Pre-Mrna Splicing Factor U2Af65.
Authors: Sickmier, E.A. / Frato, K.E. / Shen, H. / Paranawithana, S.R. / Green, M.R. / Kielkopf, C.L.
History
DepositionApr 26, 2011Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 20, 2011Provider: repository / Type: Initial release
Revision 1.1Jul 27, 2011Group: Atomic model / Database references / Other
Revision 1.2Sep 25, 2019Group: Data collection / Other
Category: pdbx_database_status / pdbx_nmr_software / pdbx_nmr_spectrometer
Item: _pdbx_database_status.status_code_cs / _pdbx_database_status.status_code_mr ..._pdbx_database_status.status_code_cs / _pdbx_database_status.status_code_mr / _pdbx_nmr_software.name / _pdbx_nmr_spectrometer.model
Revision 1.3May 15, 2024Group: Data collection / Database references / Derived calculations
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / struct_sheet
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_sheet.number_strands

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: SPLICING FACTOR U2AF 65 KDA SUBUNIT
B: 5'-R(*UP*UP*UP*UP*UP*UP*UP*UP*UP)-3'


Theoretical massNumber of molelcules
Total (without water)24,2212
Polymers24,2212
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)10 / 125LOWEST ENERGY
RepresentativeModel #1

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Components

#1: Protein SPLICING FACTOR U2AF 65 KDA SUBUNIT / U2 AUXILIARY FACTOR 65 KDA SUBUNIT / HUMAN U2AF65 / U2 SNRNP AUXILIARY FACTOR LARGE SUBUNIT / HU2AF(65)


Mass: 21510.525 Da / Num. of mol.: 1
Fragment: TANDEM RRM1 AND RRM2 DOMAINS JOINED BY NATIVE LINKER, RESIDUES 148-342
Source method: isolated from a genetically manipulated source
Details: STRUCTURE BASED ON DATA FROM TEN CYSTEINE MUTANTS (N155C, A164C, A171C, L187C, A188C, T209C, D273C, S281C, A287C AND A318C) COVALENTLY MODIFIED BY 3-(2-IODOACETAMIDO)-2,2,5,5, TETRAMETHYL-1- ...Details: STRUCTURE BASED ON DATA FROM TEN CYSTEINE MUTANTS (N155C, A164C, A171C, L187C, A188C, T209C, D273C, S281C, A287C AND A318C) COVALENTLY MODIFIED BY 3-(2-IODOACETAMIDO)-2,2,5,5, TETRAMETHYL-1-PYRROLIDINYLOXY RADICAL (IODOACETAMIDO-PROXYL)
Source: (gene. exp.) HOMO SAPIENS (human) / Plasmid: PETM-11 / Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): BL21 / Variant (production host): PLYSS / References: UniProt: P26368
#2: RNA chain 5'-R(*UP*UP*UP*UP*UP*UP*UP*UP*UP)-3'


Mass: 2710.535 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) HOMO SAPIENS (human)

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Experimental details

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Experiment

ExperimentMethod: SOLUTION NMR
NMR experiment
Conditions-IDExperiment-IDSolution-IDType
1111H
12115N HSQC (PRE)
2311H
24115N TROSY (HN-N RDC)
3513D HNCO (N-CO RDC)
NMR detailsText: THE STRUCTURE WAS DETERMINED USING A MULTI-STEP PROTOCOL AS DESCRIBED IN REMARK 3.

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Sample preparation

DetailsContents: 90% WATER/10% D2O
Sample conditions
Conditions-IDIonic strengthpHPressure (kPa)Temperature (K)
170 mM6.51.0 atm295.0 K
270 mM6.51.0 atm295.0 K
370 mM6.51.0 atm295.0 K

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NMR measurement

NMR spectrometer
TypeManufacturerModelField strength (MHz)Spectrometer-ID
Bruker AVANCEBrukerAVANCE6001
Bruker AVANCEBrukerAVANCE6002
Bruker AVANCEBrukerAVANCE6003

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Processing

NMR software
NameVersionDeveloperClassification
CNS1.2BRUNGER,ADAMS,CLORE,DELANO,GROS,GROSSE- KUNSTLEVE,JIANG,KUSZEWSKI,NILGES,PANNU, READ,RICE,SIMONSON,WARRENrefinement
Sparkystructure solution
RefinementMethod: MODIFIED ARIA / Software ordinal: 1
Details: STRUCTURE CALCULATION INCLUDED DISTANCE RESTRAINTS BASED ON PARAMAGNETIC RELAXATION ENHANCEMENT DATA FROM TEN INDEPENDENT CYSTEINE MUTANTS (N155C, A164C, A171C, L187C, A188C, T209C, D273C, ...Details: STRUCTURE CALCULATION INCLUDED DISTANCE RESTRAINTS BASED ON PARAMAGNETIC RELAXATION ENHANCEMENT DATA FROM TEN INDEPENDENT CYSTEINE MUTANTS (N155C, A164C, A171C, L187C, A188C, T209C, D273C, S281C, A287C AND A318C) COVALENTLY MODIFIED BY 3-(2-IODOACETAMIDO)-2,2,5,5, TETRAMETHYL-1-PYRROLIDINYLOXY RADICAL (IODOACETAMIDO- PROXYL). THE STRUCTURE WAS DETERMINED USING A MULTI-STEP PROTOCOL. FIRST WITH REFINEMENT OF INDIVIDUAL STRUCTURED DOMAINS FOR RRM1 AND RRM2 OF U2AF65. THE INITIAL COORDINATES OF THE ISOLATED DOMAINS WERE BASED ON PDB ENTRY 2G4B BY SICKMIER ET AL. 2006 (SEE REMARK 1 REFERENCE). SECOND STEP WAS ADDITION AND RANDOMIZATION OF FLEXIBLE AND LINKER RESIDUES. THE THIRD STEP WAS MODEL CALCULATION USING RDC ORIENTATION, PRE-BASED DISTANCE, TALOS DIHEDRAL AND HYDROGEN BOND RESTRAINTS WITH THE RRM1 AND RRM2 DOMAINS RESTRAINED TO THEIR INITIAL STARTING STRUCTURES. INTER- MOLECULAR PROTEIN-RNA DISTANCE RESTRAINTS WERE BASED ON ATOMIC DETAILS FROM PDB ENTRY 2G4B AND ASSUMED AN EIGHT- URIDINE BINDING MODE FOR U2AF65 RRM1-RRM2.
NMR ensembleConformer selection criteria: LOWEST ENERGY / Conformers calculated total number: 125 / Conformers submitted total number: 10

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