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- PDB-5kw1: Crystal Structure of the Two Tandem RRM Domains of PUF60 Bound to... -

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Basic information

Entry
Database: PDB / ID: 5kw1
TitleCrystal Structure of the Two Tandem RRM Domains of PUF60 Bound to a Modified AdML Pre-mRNA 3' Splice Site Analogue
Components
  • DNA/RNA (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


mRNA splice site recognition / alternative mRNA splicing, via spliceosome / regulation of alternative mRNA splicing, via spliceosome / mRNA Splicing - Major Pathway / cell junction / cadherin binding / ribonucleoprotein complex / apoptotic process / DNA binding / RNA binding ...mRNA splice site recognition / alternative mRNA splicing, via spliceosome / 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 ...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/RNA hybrid / DNA/RNA hybrid (> 10) / Poly(U)-binding-splicing factor PUF60
Similarity search - Component
Biological speciesHomo sapiens (human)
unidentified adenovirus
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.1 Å
AuthorsCrichlow, G.V. / Hsiao, H.-H. / Albright, R. / 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 1.1Dec 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 / Experimental preparation / Structure summary
Category: atom_site / exptl_crystal ...atom_site / exptl_crystal / pdbx_entry_details / pdbx_poly_seq_scheme / pdbx_unobs_or_zero_occ_residues / struct_ref_seq
Item: _atom_site.label_seq_id / _exptl_crystal.density_Matthews ..._atom_site.label_seq_id / _exptl_crystal.density_Matthews / _exptl_crystal.density_percent_sol / _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_residues.auth_comp_id / _pdbx_unobs_or_zero_occ_residues.auth_seq_id / _pdbx_unobs_or_zero_occ_residues.label_comp_id / _pdbx_unobs_or_zero_occ_residues.label_seq_id / _struct_ref_seq.db_align_beg / _struct_ref_seq.db_align_end / _struct_ref_seq.pdbx_auth_seq_align_beg / _struct_ref_seq.pdbx_auth_seq_align_end
Revision 2.1Jun 8, 2022Group: Database references / Category: database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Revision 2.2Oct 4, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)55,7154
Polymers55,6803
Non-polymers351
Water1,76598
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2560 Å2
ΔGint-14 kcal/mol
Surface area19760 Å2
MethodPISA
Unit cell
Length a, b, c (Å)62.428, 62.428, 83.313
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number144
Space group name H-MP31

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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: unp residues 118-316 / 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/RNA hybrid DNA/RNA (30-MER)


Mass: 8862.427 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) unidentified adenovirus
#3: Chemical ChemComp-CL / CHLORIDE ION


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


Mass: 18.015 Da / Num. of mol.: 98 / Source method: isolated from a natural source / Formula: H2O
Sequence detailsGuanine 4 in chain C is brominated on carbon C8.

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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: CHESS / Beamline: A1 / Wavelength: 0.9769 Å
DetectorType: ADSC QUANTUM 210 / Detector: CCD / Date: Feb 24, 2010
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9769 Å / Relative weight: 1
ReflectionResolution: 2.1→30 Å / Num. obs: 21264 / % possible obs: 99.3 % / Observed criterion σ(I): -3 / Redundancy: 5.7 % / Rmerge(I) obs: 0.099 / Net I/av σ(I): 14.1 / Net I/σ(I): 13.8
Reflection shellResolution: 2.1→2.15 Å / Redundancy: 5.7 % / Rmerge(I) obs: 0.418 / % possible all: 100

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Processing

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

2qfj


Resolution: 2.1→30 Å / Data cutoff high absF: 10000 / Cross valid method: THROUGHOUT / σ(F): 0
Details: Prior to refinement, the data set was de-twinned, treating the crystal as if it were a perfect twin.
RfactorNum. reflection% reflectionSelection details
Rfree0.237 952 4.7 %random
Rwork0.22 ---
obs-20072 94.5 %-
Solvent computationShrinkage radii: 1 Å / VDW probe radii: 1 Å
Displacement parametersBiso mean: 43.9 Å2
Baniso -1Baniso -2Baniso -3
1--0.01 Å20 Å20 Å2
2---0.01 Å20 Å2
3---0.02 Å2
Refine analyze
FreeObs
Luzzati coordinate error0.33 Å0.28 Å
Luzzati d res low-5 Å
Luzzati sigma a-0.24 Å
Refinement stepCycle: LAST / Resolution: 2.1→30 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3013 79 1 98 3191
Refine LS restraints
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONcns_bond_d0.009
X-RAY DIFFRACTIONcns_angle_deg1.4
LS refinement shellResolution: 2.1→2.23 Å / Rfactor Rfree error: 0.026
RfactorNum. reflection% reflection
Rfree0.349 177 5.1 %
Rwork0.288 3302 -
obs--97.6 %

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