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- PDB-3uwt: Crystal structure of a RNA binding domain of poly-U binding splic... -

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Basic information

Entry
Database: PDB / ID: 3uwt
TitleCrystal structure of a RNA binding domain of poly-U binding splicing factor 60KDa (PUF60) from Homo sapiens at 2.50 A resolution
ComponentsPoly(U)-binding-splicing factor PUF60
KeywordsRNA BINDING PROTEIN / RNA recognition motive / RRM / RNA binding domain / splicing / 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 / MAD / Resolution: 2.5 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 poly-U binding splicing factor 60KDa (PUF60) from Homo sapiens at 2.50 A resolution
Authors: Joint Center for Structural Genomics (JCSG) / Partnership for T-Cell Biology (TCELL)
History
DepositionDec 2, 2011Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 11, 2012Provider: repository / Type: Initial release
Revision 1.1Oct 21, 2015Group: 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)22,3142
Polymers22,2781
Non-polymers351
Water48627
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)64.173, 64.173, 80.337
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number152
Space group name H-MP3121

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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: 22278.254 Da / Num. of mol.: 1 / Fragment: RNA binding domain
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: BC008875, 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.: 27 / Source method: isolated from a natural source / Formula: H2O
Sequence detailsTHIS CONSTRUCT (RESIDUES 118-316) WAS EXPRESSED WITH A PURIFICATION TAG MGSDKIHHHHHHENLYFQG. THE ...THIS CONSTRUCT (RESIDUES 118-316) WAS EXPRESSED WITH A PURIFICATION TAG MGSDKIHHHHHHENLYFQG. THE TAG WAS REMOVED WITH TEV PROTEASE LEAVING ONLY A GLYCINE (0) FOLLOWED BY THE TARGET SEQUENCE. RESIDUE NUMBERING IS BASED ON ISOFORM 1 OF UNIPROTKB 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: 2.14 Å3/Da / Density % sol: 42.62 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 9.5
Details: 0.2M sodium chloride, 1.26M ammonium sulfate, 0.1M CHES pH 9.5, NANODROP, VAPOR DIFFUSION, SITTING DROP, temperature 293K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: ALS / Beamline: 8.2.2 / Wavelength: 0.9537,0.9796,0.9793
DetectorType: ADSC QUANTUM 315 / Detector: CCD / Date: Oct 16, 2011 / Details: KOHZU: Double Crystal Si(111)
RadiationMonochromator: Double Crystal Si(111) / Protocol: MAD / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelength
IDWavelength (Å)Relative weight
10.95371
20.97961
30.97931
ReflectionResolution: 2.5→29.798 Å / Num. all: 6937 / Num. obs: 6937 / % possible obs: 99.8 % / Redundancy: 6.5 % / Biso Wilson estimate: 68.061 Å2 / Rsym value: 0.094 / Net I/σ(I): 11.4
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Mean I/σ(I) obsNum. measured allNum. unique allRsym value% possible allRmerge(I) obs
2.5-2.576.91.635325141.37799.9
2.57-2.646.80.732544781.00399.60.014
2.64-2.716.7132054750.72499.70.014
2.71-2.86.81.332004730.59599.80.014
2.8-2.896.81.929824390.40699.80.014
2.89-2.996.72.528714300.30499.80.014
2.99-3.16.83.228874260.2371000.014
3.1-3.236.74.626914020.16399.80.014
3.23-3.376.65.826133950.1399.80.014
3.37-3.546.67.425023770.09899.80.014
3.54-3.736.35.822213530.11899.90.014
3.73-3.956.49.622223480.07199.90.014
3.95-4.236.41120153140.0611000.014
4.23-4.566.412.919193010.0599.90.014
4.56-56.11217282810.05199.80.014
5-5.595.910.614922510.05599.90.014
5.59-6.465.69.113102340.07199.70.014
6.46-7.916.18.812091970.0799.90.014
7.91-11.186.212.19701570.05299.70.014
11.18-29.7985.513.9508920.04594.70.014

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Phasing

PhasingMethod: MAD

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Processing

Software
NameVersionClassificationNB
MolProbity3beta29model building
PDB_EXTRACT3.1data extraction
SHELXphasing
SHARPphasing
SCALA3.3.20data scaling
BUSTER-TNT2.10.0refinement
MOSFLMdata reduction
SHELXDphasing
BUSTER2.10.0refinement
RefinementMethod to determine structure: MAD / Resolution: 2.5→29.798 Å / Cor.coef. Fo:Fc: 0.9446 / Cor.coef. Fo:Fc free: 0.9143 / Occupancy max: 1 / Occupancy min: 0.5 / Cross valid method: THROUGHOUT / σ(F): 0
Details: 1. 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 ...Details: 1. 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. 2. ATOM RECORD CONTAINS SUM OF TLS AND RESIDUAL B FACTORS. ANISOU RECORD CONTAINS SUM OF TLS AND RESIDUAL U FACTORS. 3. CHLORIDE (CL) FROM THE CRYSTALLIZATION SOLUTION HAS BEEN MODELED IN THE SOLVENT STRUCTURE. 4. THE REFINEMENT WAS RESTRAINED WITH THE MAD PHASES. 5. RAMACHANDRAN OUTLIERS AT RESIDUES 150 AND 290 ARE SUPPORTED BY ELECTRON DENSITY.
RfactorNum. reflection% reflectionSelection details
Rfree0.2399 327 4.74 %RANDOM
Rwork0.2025 ---
obs0.2042 6896 99.18 %-
Displacement parametersBiso max: 148.14 Å2 / Biso mean: 71.9912 Å2 / Biso min: 32.52 Å2
Baniso -1Baniso -2Baniso -3
1--4.1785 Å20 Å20 Å2
2---4.1785 Å20 Å2
3---8.3569 Å2
Refine analyzeLuzzati coordinate error obs: 0.427 Å
Refinement stepCycle: LAST / Resolution: 2.5→29.798 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1496 0 1 27 1524
Refine LS restraints
Refine-IDTypeNumberRestraint functionWeightDev ideal
X-RAY DIFFRACTIONt_dihedral_angle_d720SINUSOIDAL6
X-RAY DIFFRACTIONt_trig_c_planes37HARMONIC2
X-RAY DIFFRACTIONt_gen_planes232HARMONIC5
X-RAY DIFFRACTIONt_it1544HARMONIC20
X-RAY DIFFRACTIONt_nbd
X-RAY DIFFRACTIONt_improper_torsion
X-RAY DIFFRACTIONt_pseud_angle
X-RAY DIFFRACTIONt_chiral_improper_torsion203SEMIHARMONIC5
X-RAY DIFFRACTIONt_sum_occupancies
X-RAY DIFFRACTIONt_utility_distance
X-RAY DIFFRACTIONt_utility_angle
X-RAY DIFFRACTIONt_utility_torsion
X-RAY DIFFRACTIONt_ideal_dist_contact1771SEMIHARMONIC4
X-RAY DIFFRACTIONt_bond_d1544HARMONIC20.01
X-RAY DIFFRACTIONt_angle_deg2091HARMONIC21.14
X-RAY DIFFRACTIONt_omega_torsion2.89
X-RAY DIFFRACTIONt_other_torsion2.38
LS refinement shellResolution: 2.5→2.79 Å / Total num. of bins used: 5
RfactorNum. reflection% reflection
Rfree0.2922 103 5.31 %
Rwork0.2308 1835 -
all0.2341 1938 -
obs--99.18 %
Refinement TLS params.Method: refined / Origin x: 10.5861 Å / Origin y: 36.9312 Å / Origin z: 9.2179 Å
111213212223313233
T-0.2767 Å20.0924 Å20.0136 Å2--0.0076 Å2-0.0808 Å2---0.193 Å2
L2.8485 °2-0.306 °2-0.8064 °2-4.5125 °2-0.2793 °2--3.2206 °2
S0.0157 Å °0.341 Å °0.2655 Å °-0.1349 Å °-0.0479 Å °-0.0563 Å °-0.1806 Å °-0.1963 Å °0.0322 Å °
Refinement TLS groupSelection details: { A|0 - 313 }

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