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- PDB-2mkc: Cooperative Structure of the Heterotrimeric pre-mRNA Retention an... -

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Basic information

Entry
Database: PDB / ID: 2mkc
TitleCooperative Structure of the Heterotrimeric pre-mRNA Retention and Splicing Complex
Components
  • Pre-mRNA leakage protein 1
  • Pre-mRNA-splicing factor CWC26
  • U2 snRNP component IST3
KeywordsSPLICING / spliceosome / Snu17p / Bud13p / Pml1p / heterotrimer / cooperativity / RES / RRM / PROTEIN BINDING / Ist3p
Function / homology
Function and homology information


maintenance of RNA location / RES complex / generation of catalytic spliceosome for first transesterification step / U2-type prespliceosome assembly / U2 snRNP / precatalytic spliceosome / spliceosomal complex assembly / Prp19 complex / mRNA export from nucleus / spliceosomal complex ...maintenance of RNA location / RES complex / generation of catalytic spliceosome for first transesterification step / U2-type prespliceosome assembly / U2 snRNP / precatalytic spliceosome / spliceosomal complex assembly / Prp19 complex / mRNA export from nucleus / spliceosomal complex / mRNA splicing, via spliceosome / mRNA binding / RNA binding / nucleus / cytoplasm
Similarity search - Function
Ist3-like, RNA recognition motif / : / Bud13 / : / Pre-mRNA-splicing factor of RES complex / : / Forkhead associated domain / Forkhead-associated (FHA) domain profile. / FHA domain / Forkhead-associated (FHA) domain ...Ist3-like, RNA recognition motif / : / Bud13 / : / Pre-mRNA-splicing factor of RES complex / : / Forkhead associated domain / Forkhead-associated (FHA) domain profile. / FHA domain / Forkhead-associated (FHA) domain / SMAD/FHA domain superfamily / 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
U2 snRNP component IST3 / Pre-mRNA-splicing factor CWC26 / Pre-mRNA leakage protein 1
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodSOLUTION NMR / simulated annealing
AuthorsWysoczanski, P. / Schneider, C. / Xiang, S. / Munari, F. / Trowitzsch, S. / Wahl, M.C. / Luhrmann, R. / Becker, S. / Zweckstetter, M.
CitationJournal: Nat.Struct.Mol.Biol. / Year: 2014
Title: Cooperative structure of the heterotrimeric pre-mRNA retention and splicing complex.
Authors: Wysoczanski, P. / Schneider, C. / Xiang, S. / Munari, F. / Trowitzsch, S. / Wahl, M.C. / Luhrmann, R. / Becker, S. / Zweckstetter, M.
History
DepositionFeb 4, 2014Deposition site: BMRB / Processing site: RCSB
Revision 1.0Sep 3, 2014Provider: repository / Type: Initial release
Revision 1.1Oct 1, 2014Group: Database references
Revision 1.2Oct 22, 2014Group: Database references
Revision 1.3Jun 14, 2023Group: Data collection / Database references / Other
Category: database_2 / pdbx_database_status ...database_2 / pdbx_database_status / pdbx_nmr_software / pdbx_nmr_spectrometer / struct_ref_seq_dif
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_database_status.status_code_nmr_data / _pdbx_nmr_software.name / _pdbx_nmr_spectrometer.model / _struct_ref_seq_dif.details
Revision 1.4May 15, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2 / Item: _database_2.pdbx_DOI

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: U2 snRNP component IST3
B: Pre-mRNA leakage protein 1
C: Pre-mRNA-splicing factor CWC26


Theoretical massNumber of molelcules
Total (without water)19,8393
Polymers19,8393
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)20 / 500target function
RepresentativeModel #1closest to the average

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Components

#1: Protein U2 snRNP component IST3 / Increased sodium tolerance protein 3 / U2 snRNP protein SNU17


Mass: 13495.979 Da / Num. of mol.: 1 / Fragment: UNP residues 25-138
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Strain: ATCC 204508 / S288c / Gene: IST3, SNU17, YIB5W, YIR005W / Plasmid: pETM-11 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: P40565
#2: Protein/peptide Pre-mRNA leakage protein 1


Mass: 2514.782 Da / Num. of mol.: 1 / Fragment: UNP residues 22-42
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Strain: ATCC 204508 / S288c / Gene: PML1, YLR016C, L1591 / Production host: Escherichia coli (E. coli) / References: UniProt: Q07930
#3: Protein/peptide Pre-mRNA-splicing factor CWC26 / Bud site selection protein 13 / Complexed with CEF1 protein 26 / Synthetic lethal with CLF1 protein 7


Mass: 3828.191 Da / Num. of mol.: 1 / Fragment: UNP residues 215-245
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Strain: ATCC 204508 / S288c / Gene: BUD13, CWC26, SLC7, YGL174W, G1642 / Production host: Escherichia coli (E. coli) / References: UniProt: P46947
Compound detailsAUTHORS STATE THAT THE ISOTOPICALLY LABELLED BIOSYNTHETIC PEPTIDES HAVE GS CLONING ARTIFACT AT THE ...AUTHORS STATE THAT THE ISOTOPICALLY LABELLED BIOSYNTHETIC PEPTIDES HAVE GS CLONING ARTIFACT AT THE N-TERMINI, WHICH WERE USED PRIMARILY TO DERIVE THE DATA THAT LEAD TO THE RELEVANT PARTS OF THE STRUCTURE. HOWEVER, THE NATURAL ISOTOPIC ABUNDANCE SYNTHETIC PEPTIDES WERE ALSO USED FOR SOME NMR EXPERIMENTS. THEY DO NOT HAVE GS CLONING ARTIFACT. INSTEAD, THEY HAVE ACETYL GROUP AT THE N-TERMINUS AND AMIDE AT THE C-TERMINUS.

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Experimental details

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Experiment

ExperimentMethod: SOLUTION NMR
NMR experiment
Conditions-IDExperiment-IDSolution-IDType
1112D 1H-15N HSQC
1212D 1H-15N HSQC
1312D 1H-15N HSQC
1412D 1H-15N HSQC
1522D 1H-15N HSQC
1622D 1H-15N HSQC
1722D 1H-15N HSQC
1822D 1H-15N HSQC
1932D 1H-15N HSQC
11032D 1H-15N HSQC
11132D 1H-15N HSQC
11232D 1H-15N HSQC
11313D HNCO
11413D HNCA
11523D HNCA
11633D HNCA
11743D HN(CA)CB
11813D HN(CO)CA
11923D HN(CO)CA
12033D HN(CO)CA
12113D (H)CCH-TOCSY
12223D (H)CCH-TOCSY
12333D (H)CCH-TOCSY
12413D 1H-15N NOESY
12523D 1H-15N NOESY
12633D 1H-15N NOESY
12753D 1H-15N NOESY
12813D 1H-13C NOESY aliphatic
12923D 1H-13C NOESY aliphatic
13033D 1H-13C NOESY aliphatic
13153D 1H-13C NOESY aliphatic
13213D 1H-13C NOESY aromatic
13323D 1H-13C NOESY aromatic
13433D 1H-13C NOESY aromatic
13553D (H)CCH-TOCSY
13613D 1H-13C NOESY filtered/edited
13723D 1H-13C NOESY filtered/edited
1381(HB)CB(CGCD)HD
13913D CCH-TOCSY
14033D CBCA(CO)NH
14133D 1H-15N NOESY ARG-centered

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Sample preparation

Details
Solution-IDContentsSolvent system
125 mM sodium phosphate, 250 mM NaCl, 1 mM sodium azide, 0.9-1.3 mM [U-13C; U-15N] Snu17p, 1.1-1.5 mM Bud13p, 1.4-1.9 mM Pml1p, 90% H2O/10% D2O90% H2O/10% D2O
225 mM sodium phosphate, 250 mM NaCl, 1 mM sodium azide, 1.0-1.3 mM Snu17p, 1.2-1.5 mM Bud13p, 0.8-1.0 mM [U-13C; U-15N] Pml1p, 90% H2O/10% D2O90% H2O/10% D2O
325 mM sodium phosphate, 250 mM NaCl, 1 mM sodium azide, 1.0-1.3 mM Snu17p, 0.8-1.0 mM [U-10% 13C; U-99% 15N] Bud13p, 1.5-1.9 mM Pml1p, 90% H2O/10% D2O90% H2O/10% D2O
425 mM sodium phosphate, 250 mM NaCl, 1 mM sodium azide, 0.9-1.3 mM [U-13C; U-15N; U-2H] Snu17p, 1.1-1.5 mM Bud13p, 1.4-1.9 mM Pml1p, 90% H2O/10% D2O90% H2O/10% D2O
525 mM sodium phosphate, 250 mM NaCl, 1 mM sodium azide, 1.0-1.3 mM [U-13C; U-15N; U-2H] Snu17p, 0.8-1.0 mM [U-10% 13C; U-99% 15N] Bud13p, 1.5-1.9 mM Pml1p, 90% H2O/10% D2O90% H2O/10% D2O
Sample
Conc. (mg/ml)UnitsComponentIsotopic labelingConc. range (mg/ml)Solution-ID
25 mMsodium phosphate-11
250 mMNaCl-21
1 mMsodium azide-31
mMSnu17p-4[U-13C; U-15N]0.9-1.31
mMBud13p-51.1-1.51
mMPml1p-61.4-1.91
25 mMsodium phosphate-72
250 mMNaCl-82
1 mMsodium azide-92
mMSnu17p-101.0-1.32
mMBud13p-111.2-1.52
mMPml1p-12[U-13C; U-15N]0.8-1.02
25 mMsodium phosphate-133
250 mMNaCl-143
1 mMsodium azide-153
mMSnu17p-161.0-1.33
mMBud13p-17[U-10% 13C; U-99% 15N]0.8-1.03
mMPml1p-181.5-1.93
25 mMsodium phosphate-194
250 mMNaCl-204
1 mMsodium azide-214
mMSnu17p-22[U-13C; U-15N; U-2H]0.9-1.34
mMBud13p-231.1-1.54
mMPml1p-241.4-1.94
25 mMsodium phosphate-255
250 mMNaCl-265
1 mMsodium azide-275
mMSnu17p-28[U-13C; U-15N; U-2H]1.0-1.35
mMBud13p-29[U-10% 13C; U-99% 15N]0.8-1.05
mMPml1p-301.5-1.95
Sample conditionspH: 6.8 / Pressure: ambient / Temperature: 308 K

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NMR measurement

NMR spectrometer
TypeManufacturerModelField strength (MHz)Spectrometer-ID
Bruker AvanceBrukerAVANCE9001
Bruker AvanceBrukerAVANCE8002
Bruker AvanceBrukerAVANCE7003
Bruker AvanceBrukerAVANCE6004
Bruker AvanceBrukerAVANCE8005

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Processing

NMR software
NameVersionDeveloperClassification
CYANA3Guntert, Mumenthaler and Wuthrichstructure solution
CNS1.2Brunger, Adams, Clore, Gros, Nilges and Readrefinement
TopSpin3.1Bruker Biospinprocessing
NMRPipeDelaglio, Grzesiek, Vuister, Zhu, Pfeifer and Baxprocessing
Analysis_CCPNCCPNdata analysis
RefinementMethod: simulated annealing / Software ordinal: 1 / Details: WATER REFINEMENT (RECOORD)
NMR constraintsNOE constraints total: 4105 / NOE intraresidue total count: 769 / NOE long range total count: 1597 / NOE medium range total count: 775 / NOE sequential total count: 964 / Hydrogen bond constraints total count: 50 / Protein chi angle constraints total count: 0 / Protein other angle constraints total count: 0 / Protein phi angle constraints total count: 115 / Protein psi angle constraints total count: 115
NMR representativeSelection criteria: closest to the average
NMR ensembleAverage torsion angle constraint violation: 0.62 ° / Conformer selection criteria: target function / Conformers calculated total number: 500 / Conformers submitted total number: 20 / Maximum lower distance constraint violation: 0 Å / Maximum torsion angle constraint violation: 3.2 ° / Maximum upper distance constraint violation: 0.307 Å / Torsion angle constraint violation method: CNS
NMR ensemble rmsDistance rms dev: 0.0101 Å / Distance rms dev error: 0.0008 Å

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