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- PDB-3v4m: Crystal structure of a RNA binding domain of a U2 small nuclear r... -

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Basic information

Entry
Database: PDB / ID: 3v4m
TitleCrystal structure of a RNA binding domain of a U2 small nuclear ribonucleoprotein auxiliary factor 2 (U2AF) from Mus musculus at 1.80 A resolution
ComponentsSplicing factor U2AF 65 kDa subunit
KeywordsRNA BINDING PROTEIN / Canonical RNA binding protein / RNA 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


mRNA 3'-end processing / RNA Polymerase II Transcription Termination / Transport of Mature mRNA derived from an Intron-Containing Transcript / Protein hydroxylation / mRNA Splicing - Major Pathway / U2AF complex / poly-pyrimidine tract binding / pre-mRNA 3'-splice site binding / Prp19 complex / C2H2 zinc finger domain binding ...mRNA 3'-end processing / RNA Polymerase II Transcription Termination / Transport of Mature mRNA derived from an Intron-Containing Transcript / Protein hydroxylation / mRNA Splicing - Major Pathway / U2AF complex / poly-pyrimidine tract binding / pre-mRNA 3'-splice site binding / Prp19 complex / C2H2 zinc finger domain binding / commitment complex / U2-type prespliceosome / spliceosomal complex assembly / negative regulation of mRNA splicing, via spliceosome / negative regulation of protein ubiquitination / RNA splicing / positive regulation of RNA splicing / spliceosomal complex / mRNA splicing, via spliceosome / nuclear speck / enzyme binding / nucleus
Similarity search - Function
U2 snRNP auxilliary factor, large subunit, splicing factor / 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
Splicing factor U2AF 65 kDa subunit
Similarity search - Component
Biological speciesMus musculus (house mouse)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MAD / Resolution: 1.8 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 U2 small nuclear ribonucleoprotein auxiliary factor 2 (U2AF) from Mus musculus at 1.80 A resolution
Authors: Joint Center for Structural Genomics (JCSG) / Partnership for T-Cell Biology (TCELL)
History
DepositionDec 15, 2011Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 13, 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 / pdbx_struct_conn_angle ...database_2 / pdbx_struct_conn_angle / 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 / _pdbx_struct_conn_angle.ptnr1_auth_comp_id / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_asym_id / _pdbx_struct_conn_angle.ptnr1_label_comp_id / _pdbx_struct_conn_angle.ptnr1_label_seq_id / _pdbx_struct_conn_angle.ptnr3_auth_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_asym_id / _pdbx_struct_conn_angle.ptnr3_label_comp_id / _pdbx_struct_conn_angle.ptnr3_label_seq_id / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _struct_conn.pdbx_leaving_atom_flag / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id
Revision 1.4Nov 20, 2024Group: Data collection / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / pdbx_entry_details / pdbx_modification_feature
Item: _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Splicing factor U2AF 65 kDa subunit
B: Splicing factor U2AF 65 kDa subunit
hetero molecules


Theoretical massNumber of molelcules
Total (without water)24,4435
Polymers24,3492
Non-polymers943
Water2,666148
1
A: Splicing factor U2AF 65 kDa subunit
B: Splicing factor U2AF 65 kDa subunit
hetero molecules

A: Splicing factor U2AF 65 kDa subunit
B: Splicing factor U2AF 65 kDa subunit
hetero molecules


Theoretical massNumber of molelcules
Total (without water)48,88510
Polymers48,6974
Non-polymers1886
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation10_665-y+1,-x+1,-z+1/61
Buried area4870 Å2
ΔGint-79 kcal/mol
Surface area20150 Å2
MethodPISA
2


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1260 Å2
ΔGint-37 kcal/mol
Surface area11250 Å2
MethodPISA
Unit cell
Length a, b, c (Å)60.192, 60.192, 239.686
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number179
Space group name H-MP6522
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21B

NCS domain segments:
Dom-IDComponent-IDEns-IDRefine codeAuth asym-IDAuth seq-ID
1116A368 - 475
2116B368 - 475
DetailsCRYSTAL PACKING ANALYSIS SUGGESTS THE ASSIGNMENT OF A DIMER AND TETRAMER AS THE SIGNIFICANT OLIGOMERIZATION STATES.

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Components

#1: Protein Splicing factor U2AF 65 kDa subunit / U2 auxiliary factor 65 kDa subunit / U2 snRNP auxiliary factor large subunit


Mass: 12174.353 Da / Num. of mol.: 2 / Fragment: RNA binding domain
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: BC043071, U2af2, U2af65 / Plasmid: SpeedET / Production host: Escherichia Coli (E. coli) / Strain (production host): HK100 / References: UniProt: P26369
#2: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Cl
#3: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Na
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 148 / Source method: isolated from a natural source / Formula: H2O
Has protein modificationY
Sequence detailsTHIS CONSTRUCT WAS EXPRESSED WITH A PURIFICATION TAG MGSDKIHHHHHHENLYFQG. THE TAG WAS REMOVED WITH ...THIS CONSTRUCT WAS EXPRESSED WITH A PURIFICATION TAG MGSDKIHHHHHHENLYFQG. THE TAG WAS REMOVED WITH TEV PROTEASE LEAVING ONLY A GLYCINE (0) FOLLOWED BY RESIDUES 372-475 OF THE TARGET SEQUENCE. THE SEQUENCE NUMBERING IS BASED ON THE UNIPROTKB ID P26369 ISOFORM THAT MATCHES THE CANONICAL HUMAN ISOFORM FROM UNIPROT ID P26368.

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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: 2.57 Å3/Da / Density % sol: 52.21 %
Crystal growTemperature: 277 K / Method: vapor diffusion, sitting drop / pH: 7.3
Details: 4.0M NaFormate, No Buffer pH 7.3, 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: 0.9537,0.9793
DetectorType: ADSC QUANTUM 315 / Detector: CCD / Date: Sep 8, 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
20.97931
ReflectionResolution: 1.8→29.19 Å / Num. obs: 25066 / % possible obs: 100 % / Observed criterion σ(I): -3 / Biso Wilson estimate: 21.384 Å2 / Rmerge(I) obs: 0.078 / Net I/σ(I): 17.88
Reflection shell

Diffraction-ID: 1

Resolution (Å)Highest resolution (Å)Rmerge(I) obsMean I/σ(I) obsNum. measured obsNum. unique obs% possible all
1.8-1.860.8542.2238804213100
1.86-1.940.63.3277234888100
1.94-2.030.3865.2257704528100
2.03-2.130.2497.9240984238100
2.13-2.270.18510.4269024714100
2.27-2.440.14412.9251534397100
2.44-2.690.10416.8262564587100
2.69-3.070.06225.9253904434100
3.07-3.870.03541.3258174562100
3.870.02652.225711457099.6

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Phasing

PhasingMethod: MAD

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Processing

Software
NameVersionClassificationNB
MolProbity3beta29model building
PDB_EXTRACT3.1data extraction
SHELXphasing
SHARPphasing
XSCALEDecember 6, 2010data scaling
REFMAC5.6.0116refinement
XDSdata reduction
SHELXDphasing
RefinementMethod to determine structure: MAD / Resolution: 1.8→29.19 Å / Cor.coef. Fo:Fc: 0.957 / Cor.coef. Fo:Fc free: 0.946 / Occupancy max: 1 / Occupancy min: 0.37 / SU B: 4.296 / SU ML: 0.069 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.113 / ESU R Free: 0.108
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: 1. HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS. 2. ATOM RECORD CONTAINS SUM OF TLS AND RESIDUAL B FACTORS. ANISOU RECORD CONTAINS SUM OF TLS AND RESIDUAL U FACTORS. 3. WATERS WERE ...Details: 1. HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS. 2. ATOM RECORD CONTAINS SUM OF TLS AND RESIDUAL B FACTORS. ANISOU RECORD CONTAINS SUM OF TLS AND RESIDUAL U FACTORS. 3. WATERS WERE EXCLUDED FROM AUTOMATIC TLS ASSIGNMENT. 4. 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 I INCORPORATION. 5.A SODIUM (NA) AND CHLORIDE (CL) FROM THE CRYSTALLIZATION ARE MODELED INTO THE STRUCTURE.
RfactorNum. reflection% reflectionSelection details
Rfree0.2069 1269 5.1 %RANDOM
Rwork0.1774 ---
obs0.1788 24964 99.88 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso max: 104.42 Å2 / Biso mean: 26.0161 Å2 / Biso min: 9.69 Å2
Baniso -1Baniso -2Baniso -3
1--0.07 Å2-0.03 Å20 Å2
2---0.07 Å20 Å2
3---0.1 Å2
Refinement stepCycle: LAST / Resolution: 1.8→29.19 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1658 0 3 148 1809
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0120.0221843
X-RAY DIFFRACTIONr_bond_other_d0.0010.021319
X-RAY DIFFRACTIONr_angle_refined_deg1.2891.9852519
X-RAY DIFFRACTIONr_angle_other_deg0.81733227
X-RAY DIFFRACTIONr_dihedral_angle_1_deg5.0095247
X-RAY DIFFRACTIONr_dihedral_angle_2_deg32.66823.54893
X-RAY DIFFRACTIONr_dihedral_angle_3_deg13.9315346
X-RAY DIFFRACTIONr_dihedral_angle_4_deg15.6751518
X-RAY DIFFRACTIONr_chiral_restr0.0780.2274
X-RAY DIFFRACTIONr_gen_planes_refined0.0060.0212086
X-RAY DIFFRACTIONr_gen_planes_other0.0010.02387
Refine LS restraints NCS

Dom-ID: 1 / Ens-ID: 1 / Number: 1339 / Refine-ID: X-RAY DIFFRACTION

Auth asym-IDTypeRms dev position (Å)Weight position
ALOOSE POSITIONAL1.065
BLOOSE THERMAL4.4510
LS refinement shellResolution: 1.8→1.847 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.298 73 -
Rwork0.268 1470 -
all-1543 -
obs--100 %
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
11.17660.32880.1990.98180.17451.79180.01170.0139-0.08190.15760.06740.13310.3526-0.4022-0.07910.0902-0.06440.00940.11220.03050.039914.014613.013911.2729
22.4498-0.08860.81370.4952-0.14791.0711-0.05620.07480.2590.0124-0.0395-0.0097-0.00710.10660.09570.0019-0.0015-0.00470.08460.03280.046613.938135.2573-0.9255
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A0 - 475
2X-RAY DIFFRACTION2B372 - 475

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