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- PDB-5kvy: CRYSTAL STRUCTURE OF THE TWO TANDEM RRM DOMAINS OF PUF60 BOUND TO... -

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Basic information

Entry
Database: PDB / ID: 5kvy
TitleCRYSTAL STRUCTURE OF THE TWO TANDEM RRM DOMAINS OF PUF60 BOUND TO A PORTION OF AN ADML PRE-MRNA 3' SPLICE SITE ANALOG
Components
  • DNA (30-MER)
  • Poly(U)-binding-splicing factor PUF60
KeywordsSPLICING/DNA / TANDEM RRMS / PROTEIN-NUCLEIC ACID COMPLEX / SPLICING FACTOR / SPLICING-DNA complex
Function / homology
Function and homology information


alternative mRNA splicing, via spliceosome / mRNA splice site recognition / regulation of alternative mRNA splicing, via spliceosome / mRNA Splicing - Major Pathway / cell junction / cadherin binding / ribonucleoprotein complex / apoptotic process / DNA binding / RNA binding ...alternative mRNA splicing, via spliceosome / mRNA splice site recognition / regulation of alternative mRNA splicing, via spliceosome / mRNA Splicing - Major Pathway / cell junction / cadherin binding / ribonucleoprotein complex / apoptotic process / DNA binding / RNA binding / nucleoplasm / identical protein binding
Similarity search - Function
Poly-U binding splicing factor, PUF60-like / PUF60, RNA recognition motif 1 / PUF60, RNA recognition motif 2 / PUF60, RNA recognition motif 3 / RNA recognition motif domain, eukaryote / RNA recognition motif / RRM (RNA recognition motif) domain / RNA recognition motif / RNA recognition motif / Eukaryotic RNA Recognition Motif (RRM) profile. ...Poly-U binding splicing factor, PUF60-like / PUF60, RNA recognition motif 1 / PUF60, RNA recognition motif 2 / PUF60, RNA recognition motif 3 / RNA recognition motif domain, eukaryote / RNA recognition motif / 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
DNA / DNA (> 10) / Poly(U)-binding-splicing factor PUF60
Similarity search - Component
Biological speciesHomo sapiens (human)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 1.95 Å
Model detailsprotein / nucleic acid complex
AuthorsHsiao, H.-H. / Crichlow, G.V. / Albright, R.A. / Murphy, J.W. / Lolis, E.J. / Braddock, D.T.
Funding support United States, 1items
OrganizationGrant numberCountry
American Cancer SocietyRSG-0-222-01 United States
Citation
Journal: Plos One / Year: 2020
Title: Unraveling the mechanism of recognition of the 3' splice site of the adenovirus major late promoter intron by the alternative splicing factor PUF60.
Authors: Hsiao, H.T. / Crichlow, G.V. / Murphy, J.W. / Folta-Stogniew, E.J. / Lolis, E.J. / Braddock, D.T.
#1: Journal: EMBO J. / Year: 2008
Title: Dimerization of FIR upon FUSE DNA binding suggests a mechanism of c-myc inhibition
Authors: Crichlow, G.V. / Zhou, H. / Hsiao, H.H. / Frederick, K.B. / Debrosse, M. / Yang, Y. / Folta-Stogniew, E.J. / Chung, H.J. / Fan, C. / De la Cruz, E.M. / Levens, D. / Lolis, E. / Braddock, D.
History
DepositionJul 15, 2016Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 23, 2017Provider: repository / Type: Initial release
Revision 1.1Nov 1, 2017Group: Author supporting evidence / Category: pdbx_struct_assembly_auth_evidence
Revision 1.2Dec 9, 2020Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 2.0Dec 16, 2020Group: Advisory / Atomic model ...Advisory / Atomic model / Database references / Structure summary
Category: atom_site / pdbx_poly_seq_scheme ...atom_site / pdbx_poly_seq_scheme / pdbx_unobs_or_zero_occ_atoms / pdbx_unobs_or_zero_occ_residues / struct_ref_seq
Item: _atom_site.label_seq_id / _pdbx_poly_seq_scheme.auth_mon_id ..._atom_site.label_seq_id / _pdbx_poly_seq_scheme.auth_mon_id / _pdbx_poly_seq_scheme.auth_seq_num / _pdbx_poly_seq_scheme.pdb_mon_id / _pdbx_poly_seq_scheme.pdb_seq_num / _pdbx_unobs_or_zero_occ_atoms.label_seq_id / _pdbx_unobs_or_zero_occ_residues.auth_seq_id / _pdbx_unobs_or_zero_occ_residues.label_seq_id / _struct_ref_seq.db_align_end / _struct_ref_seq.pdbx_auth_seq_align_end
Revision 2.1Oct 4, 2023Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Poly(U)-binding-splicing factor PUF60
B: Poly(U)-binding-splicing factor PUF60
C: DNA (30-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)55,7154
Polymers55,6803
Non-polymers351
Water3,945219
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, light scattering
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)62.438, 62.438, 83.332
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number144
Space group name H-MP31
DetailsAuthors have supported the biological unit analysis by performing size exclusion chromatography and light scattering

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Components

#1: Protein Poly(U)-binding-splicing factor PUF60 / 60 kDa poly(U)-binding-splicing factor / FUSE-binding protein-interacting repressor / FBP- ...60 kDa poly(U)-binding-splicing factor / FUSE-binding protein-interacting repressor / FBP-interacting repressor / Ro-binding protein 1 / RoBP1 / Siah-binding protein 1 / Siah-BP1


Mass: 23408.537 Da / Num. of mol.: 2 / Fragment: tandem RRM domains / Mutation: R123G, C129S, C255A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PUF60, FIR, ROBPI, SIAHBP1 / Plasmid: pET15b / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: Q9UHX1
#2: DNA chain DNA (30-MER)


Mass: 8862.427 Da / Num. of mol.: 1 / Fragment: AdML3' / Source method: obtained synthetically
Details: Sequence from the adenovirus major late pre-mRNA 3'splice site
Source: (synth.) synthetic construct (others)
#3: Chemical ChemComp-CL / CHLORIDE ION / Chloride


Mass: 35.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cl
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 219 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 1.68 Å3/Da / Density % sol: 26.93 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 8.7
Details: 0.1 M TRIS-HCl, 25% PEG 4000, 5-10 mM Barium Chloride Dihydrate, pH 8.7, mixed with 10 mg/ml protein-nucleic acid mixture in 50 mM TRIS-HCl, pH 8.0, 150 mM NaCl, 20 micromolar EDTA.

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 24-ID-C / Wavelength: 0.9795 Å
DetectorType: ADSC QUANTUM 315 / Detector: CCD / Date: Jul 9, 2009
RadiationMonochromator: SI(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9795 Å / Relative weight: 1
ReflectionResolution: 1.95→30 Å / Num. obs: 26419 / % possible obs: 99.7 % / Observed criterion σ(I): -3 / Rmerge(I) obs: 0.059
Reflection shellResolution: 1.95→2 Å / Rmerge(I) obs: 0.452 / Mean I/σ(I) obs: 2.2 / % possible all: 100

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassification
HKL-2000data reduction
HKL-2000data scaling
PHASERphasing
CNSrefinement
PDB_EXTRACT3.2data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 2QFJ

2qfj


Resolution: 1.95→30 Å / Cross valid method: THROUGHOUT / Details: DATA WERE DE-TWINNED DURING REFINEMENT PROCESS
RfactorNum. reflection% reflectionSelection details
Rfree0.232 1139 4.3 %RANDOM, AFTER APPLICATION OF DETWINNING
Rwork0.196 ---
obs-24917 94.2 %-
Displacement parametersBiso mean: 34.49 Å2
Baniso -1Baniso -2Baniso -3
1--0.013 Å20 Å20 Å2
2---0.013 Å20 Å2
3---0.027 Å2
Refine analyze
FreeObs
Luzzati coordinate error0.28 Å0.22 Å
Luzzati d res low-5 Å
Luzzati sigma a0.22 Å0.17 Å
Refinement stepCycle: LAST / Resolution: 1.95→30 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3005 66 24 219 3314
Refine LS restraints
Refine-IDTypeDev idealDev ideal target
X-RAY DIFFRACTIONo_bond_d0.006
X-RAY DIFFRACTIONo_bond_d_na
X-RAY DIFFRACTIONo_bond_d_prot
X-RAY DIFFRACTIONo_angle_d
X-RAY DIFFRACTIONo_angle_d_na
X-RAY DIFFRACTIONo_angle_d_prot
X-RAY DIFFRACTIONo_angle_deg1.34
X-RAY DIFFRACTIONo_angle_deg_na
X-RAY DIFFRACTIONo_angle_deg_prot
X-RAY DIFFRACTIONo_dihedral_angle_d
X-RAY DIFFRACTIONo_dihedral_angle_d_na
X-RAY DIFFRACTIONo_dihedral_angle_d_prot
X-RAY DIFFRACTIONo_improper_angle_d
X-RAY DIFFRACTIONo_improper_angle_d_na
X-RAY DIFFRACTIONo_improper_angle_d_prot
X-RAY DIFFRACTIONo_mcbond_it2.5471.5
X-RAY DIFFRACTIONo_mcangle_it4.3792
X-RAY DIFFRACTIONo_scbond_it
X-RAY DIFFRACTIONo_scangle_it
LS refinement shellResolution: 1.95→2.02 Å
RfactorNum. reflection% reflection
Rfree0.3358 115 -
Rwork0.2727 2280 -
obs--91.5 %

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