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- PDB-3us5: Crystal structure of a RNA-binding domain of a poly-U binding spl... -

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Basic information

Entry
Database: PDB / ID: 3us5
TitleCrystal structure of a RNA-binding domain of a poly-U binding splicing factor 60KDa (PUF60) from Homo sapiens at 1.38 A resolution
ComponentsPoly(U)-binding-splicing factor PUF60
KeywordsRNA BINDING PROTEIN / Canonical RBD / RRM / Structural Genomics / Joint Center for Structural Genomics / JCSG / Protein Structure Initiative / PSI-BIOLOGY / Partnership for T-Cell Biology / TCELL
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
Poly(U)-binding-splicing factor PUF60
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / SAD / Resolution: 1.38 Å
AuthorsJoint Center for Structural Genomics (JCSG) / Partnership for T-Cell Biology (TCELL)
CitationJournal: To be published
Title: Crystal structure of a RNA-binding domain of a poly-U binding splicing factor 60KDa (PUF60) from Homo sapiens at 1.38 A resolution
Authors: Joint Center for Structural Genomics (JCSG) / Partnership for T-Cell Biology
History
DepositionNov 23, 2011Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 11, 2012Provider: repository / Type: Initial release
Revision 1.1Aug 29, 2012Group: Structure summary
Revision 1.2Nov 8, 2017Group: Refinement description / Category: software / Item: _software.classification / _software.name
Revision 1.3Feb 1, 2023Group: Database references / Derived calculations
Category: database_2 / struct_conn ...database_2 / struct_conn / struct_ref_seq_dif / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_conn.pdbx_leaving_atom_flag / _struct_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

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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
hetero molecules


Theoretical massNumber of molelcules
Total (without water)13,5632
Polymers13,5281
Non-polymers351
Water3,081171
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)43.433, 48.974, 56.298
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121
DetailsCRYSTAL PACKING ANALYSIS SUPPORTS THE ASSIGNMENT OF A MONOMER AS A SIGNIFICANT OLIGOMERIZATION STATE.

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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: 13527.543 Da / Num. of mol.: 1 / Fragment: RRM 3 domain residues 443-559
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: BC011265, FIR, PUF60, ROBPI, SIAHBP1 / Plasmid: SpeedET / Production host: Escherichia Coli (E. coli) / Strain (production host): HK100 / References: UniProt: Q9UHX1
#2: Chemical ChemComp-CL / CHLORIDE ION / Chloride


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


Mass: 18.015 Da / Num. of mol.: 171 / Source method: isolated from a natural source / Formula: H2O
Sequence detailsTHIS CONSTRUCT WAS EXPRESSED WITH AN N-TERMINAL PURIFICATION TAG MGSDKIHHHHHHENLYFQG. THE TAG WAS ...THIS CONSTRUCT WAS EXPRESSED WITH AN N-TERMINAL PURIFICATION TAG MGSDKIHHHHHHENLYFQG. THE TAG WAS REMOVED WITH TEV PROTEASE LEAVING ONLY A GLYCINE (0) FOLLOWED BY RESIDUES 443-559 OF THE TARGET SEQUENCE. THE SEQUENCE NUMBERING MATCHES ISOFORM 1 OF UNIPROT-KB ENTRY Q9UHX1.

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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: 2.21 Å3/Da / Density % sol: 44.42 %
Description: REFLECTIONS IN RESOLUTION BINS 3.71-3.63, 2.28-2.22, AND 1.94-1.90 WERE EXCLUDED DURING DATA INTEGRATION BECAUSE OF ICE RINGS.
Crystal growTemperature: 277 K / Method: vapor diffusion, sitting drop / pH: 8
Details: 2.40M ammonium sulfate, 0.1M TRIS pH 8.0, NANODROP, VAPOR DIFFUSION, SITTING DROP, temperature 277K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: ALS / Beamline: 8.2.2 / Wavelength: 0.9793
DetectorType: ADSC QUANTUM 315 / Detector: CCD / Date: Oct 15, 2011 / Details: KOHZU: Double Crystal Si(111)
RadiationMonochromator: Double Crystal Si(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9793 Å / Relative weight: 1
ReflectionResolution: 1.38→36.95 Å / Num. all: 24223 / Num. obs: 24223 / % possible obs: 95.4 % / Redundancy: 6.9 % / Biso Wilson estimate: 13.374 Å2 / Rsym value: 0.102 / Net I/σ(I): 9.3
Reflection shell

Rmerge(I) obs: 0.011 / Diffraction-ID: 1

Resolution (Å)Redundancy (%)Mean I/σ(I) obsNum. measured allNum. unique allRsym value% possible all
1.38-1.4570.62528636381.064100
1.45-1.5471.12402034420.604100
1.54-1.65722271432520.355100
1.65-1.7873.22125530500.22100
1.78-1.9574.41536622060.15478.7
1.95-2.186.96.51763125490.102100
2.18-2.526.87.11222317900.08979
2.52-3.096.68.61291719510.074100
3.09-4.366.510.9936414400.05693.9
4.36-36.956.512.559029050.04899.8

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Phasing

PhasingMethod: SAD

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Processing

Software
NameVersionClassificationNB
MolProbity3beta29model building
PDB_EXTRACT3.1data extraction
SOLVEphasing
SCALA3.3.20data scaling
REFMAC5.5.0110refinement
MOSFLMdata reduction
RefinementMethod to determine structure: SAD / Resolution: 1.38→36.95 Å / Cor.coef. Fo:Fc: 0.973 / Cor.coef. Fo:Fc free: 0.965 / Occupancy max: 1 / Occupancy min: 0.4 / SU B: 1.87 / SU ML: 0.034 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.06 / ESU R Free: 0.057
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: 1. HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS. 2. A MET-INHIBITION PROTOCOL WAS USED FOR SELENOMETHIONINE INCORPORATION DURING PROTEIN EXPRESSION. THE OCCUPANCY OF THE SE ATOMS IN THE ...Details: 1. HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS. 2. A MET-INHIBITION PROTOCOL WAS USED FOR SELENOMETHIONINE INCORPORATION DURING PROTEIN EXPRESSION. THE OCCUPANCY OF THE SE ATOMS IN THE MSE RESIDUES WAS REDUCED TO 0.75 FOR THE REDUCED SCATTERING POWER DUE TO PARTIAL S-MET INCORPORATION.
RfactorNum. reflection% reflectionSelection details
Rfree0.1813 1213 5 %RANDOM
Rwork0.1425 ---
obs0.1444 24119 95.18 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.4 Å / Solvent model: BABINET MODEL WITH MASK
Displacement parametersBiso max: 51.9 Å2 / Biso mean: 18.1125 Å2 / Biso min: 7.84 Å2
Baniso -1Baniso -2Baniso -3
1-0.06 Å20 Å20 Å2
2--0.44 Å20 Å2
3----0.51 Å2
Refinement stepCycle: LAST / Resolution: 1.38→36.95 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms923 0 1 171 1095
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0150.022956
X-RAY DIFFRACTIONr_bond_other_d0.0020.02644
X-RAY DIFFRACTIONr_angle_refined_deg1.4991.9471293
X-RAY DIFFRACTIONr_angle_other_deg0.93231575
X-RAY DIFFRACTIONr_dihedral_angle_1_deg5.3695125
X-RAY DIFFRACTIONr_dihedral_angle_2_deg36.41925.29451
X-RAY DIFFRACTIONr_dihedral_angle_3_deg10.89915176
X-RAY DIFFRACTIONr_dihedral_angle_4_deg8.791157
X-RAY DIFFRACTIONr_chiral_restr0.1060.2144
X-RAY DIFFRACTIONr_gen_planes_refined0.0060.021096
X-RAY DIFFRACTIONr_gen_planes_other0.0010.02190
X-RAY DIFFRACTIONr_mcbond_it1.6731.5595
X-RAY DIFFRACTIONr_mcbond_other0.621.5246
X-RAY DIFFRACTIONr_mcangle_it2.5612958
X-RAY DIFFRACTIONr_scbond_it3.9153361
X-RAY DIFFRACTIONr_scangle_it5.8784.5331
X-RAY DIFFRACTIONr_rigid_bond_restr1.57631600
X-RAY DIFFRACTIONr_sphericity_free13.8745174
X-RAY DIFFRACTIONr_sphericity_bonded6.94851585
LS refinement shellResolution: 1.38→1.416 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.306 105 -
Rwork0.24 1733 -
all-1838 -
obs--99.78 %

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