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- PDB-6xlx: Crystal structure of cancer-associated G301D mutant of U2AF65 bou... -

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Basic information

Entry
Database: PDB / ID: 6xlx
TitleCrystal structure of cancer-associated G301D mutant of U2AF65 bound to AdML splice site
Components
  • DNA/RNA (5'-R(P*UP*UP*(UD)P*UP*U)-D(P*(BRU))-R(P*CP*C)-3')
  • Splicing factor U2AF 65 kDa subunit
KeywordsRNA BINDING PROTEIN/SPLICING / PROTEIN-RNA COMPLEX / RNA SPLICING FACTOR / RNA RECOGNITION MOTIF / POLYPYRIMIDINE TRACT / RNA BINDING PROTEIN-RNA COMPLEX / RNA BINDING PROTEIN / RNA BINDING PROTEIN-SPLICING complex
Function / homology
Function and homology information


U2AF complex / poly-pyrimidine tract binding / pre-mRNA 3'-splice site binding / mRNA 3'-end processing / C2H2 zinc finger domain binding / commitment complex / Transport of Mature mRNA derived from an Intron-Containing Transcript / RNA Polymerase II Transcription Termination / U2-type prespliceosome / molecular function inhibitor activity ...U2AF complex / poly-pyrimidine tract binding / pre-mRNA 3'-splice site binding / mRNA 3'-end processing / C2H2 zinc finger domain binding / commitment complex / Transport of Mature mRNA derived from an Intron-Containing Transcript / RNA Polymerase II Transcription Termination / U2-type prespliceosome / molecular function inhibitor activity / spliceosomal complex assembly / Protein hydroxylation / negative regulation of mRNA splicing, via spliceosome / negative regulation of protein ubiquitination / mRNA Splicing - Major Pathway / positive regulation of RNA splicing / spliceosomal complex / mRNA splicing, via spliceosome / mRNA processing / nuclear speck / enzyme binding / RNA binding / nucleoplasm / 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
RNA / Splicing factor U2AF 65 kDa subunit
Similarity search - Component
Biological speciesHomo sapiens (human)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / FOURIER SYNTHESIS / Resolution: 1.7 Å
AuthorsMaji, D. / Jenkins, J.L. / Kielkopf, C.L.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM070503-15 United States
CitationJournal: J.Biol.Chem. / Year: 2020
Title: Representative cancer-associated U2AF2 mutations alter RNA interactions and splicing.
Authors: Maji, D. / Glasser, E. / Henderson, S. / Galardi, J. / Pulvino, M.J. / Jenkins, J.L. / Kielkopf, C.L.
History
DepositionJun 29, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 7, 2020Provider: repository / Type: Initial release
Revision 1.1Feb 3, 2021Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _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 1.2Oct 18, 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: Splicing factor U2AF 65 kDa subunit
B: DNA/RNA (5'-R(P*UP*UP*(UD)P*UP*U)-D(P*(BRU))-R(P*CP*C)-3')


Theoretical massNumber of molelcules
Total (without water)24,7452
Polymers24,7452
Non-polymers00
Water3,207178
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: isothermal titration calorimetry
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2700 Å2
ΔGint-17 kcal/mol
Surface area10160 Å2
MethodPISA
Unit cell
Length a, b, c (Å)43.492, 61.875, 77.421
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121

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Components

#1: Protein Splicing factor U2AF 65 kDa subunit / U2 auxiliary factor 65 kDa subunit / hU2AF65 / U2 snRNP auxiliary factor large subunit


Mass: 22295.365 Da / Num. of mol.: 1 / Mutation: G301D
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: U2AF2, U2AF65 / Plasmid: PGEX-6P / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: P26368
#2: RNA chain DNA/RNA (5'-R(P*UP*UP*(UD)P*UP*U)-D(P*(BRU))-R(P*CP*C)-3')


Mass: 2449.299 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 178 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN

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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: 2.05 Å3/Da / Density % sol: 40 %
Crystal growTemperature: 277 K / Method: vapor diffusion, hanging drop / pH: 7
Details: 25% PEG 3350, 0.24 M Na malonate pH 7, 5% sucrose, Lauryldimethylamine oxide

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SSRL / Beamline: BL12-2 / Wavelength: 0.9795 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Jul 2, 2019
Details: Rh coated collimating mirrors, K-B focusing mirrors
RadiationMonochromator: Liquid nitrogen-cooled double crystal, non fixed exit slit
Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9795 Å / Relative weight: 1
ReflectionResolution: 1.7→38.71 Å / Num. obs: 44173 / % possible obs: 99.7 % / Redundancy: 5.8 % / CC1/2: 0.998 / Rmerge(I) obs: 0.063 / Rpim(I) all: 0.028 / Net I/σ(I): 13.2
Reflection shellResolution: 1.7→1.73 Å / Redundancy: 5.2 % / Rmerge(I) obs: 0.57 / Mean I/σ(I) obs: 2.4 / Num. unique obs: 1208 / CC1/2: 0.895 / Rpim(I) all: 0.265 / % possible all: 97.1

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Processing

Software
NameVersionClassification
PHENIX1.17.1_3660refinement
XDSMar 15, 2019data reduction
Aimless0.7.4data scaling
PDB_EXTRACT3.25data extraction
PHENIX1.17.1phasing
RefinementMethod to determine structure: FOURIER SYNTHESIS
Starting model: 5EV3

5ev3


Resolution: 1.7→38.71 Å / SU ML: 0.2 / Cross valid method: THROUGHOUT / σ(F): 1.91 / Phase error: 21.5 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.194 2922 6.61 %
Rwork0.1628 41251 -
obs0.1649 44173 99.19 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 131.81 Å2 / Biso mean: 40.9846 Å2 / Biso min: 17.38 Å2
Refinement stepCycle: final / Resolution: 1.7→38.71 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1505 160 0 178 1843
Biso mean---37.95 -
Num. residues----204
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 21

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
1.7-1.730.33381330.30511859199294
1.73-1.760.27491420.26871961210399
1.76-1.790.27091390.25171951209099
1.79-1.820.29231330.22111965209899
1.82-1.860.25561450.21551966211199
1.86-1.90.26871290.203720002129100
1.9-1.940.21191490.19471945209499
1.94-1.990.21321340.181819752109100
1.99-2.050.20651410.179719992140100
2.05-2.110.21031470.177519362083100
2.11-2.180.21461170.17662000211799
2.18-2.250.20831480.170719542102100
2.25-2.340.17711410.165119692110100
2.34-2.450.17661350.165719762111100
2.45-2.580.17871350.15952004213999
2.58-2.740.19691490.162919392088100
2.74-2.950.22161410.15891955209699
2.95-3.250.20381370.1619852122100
3.25-3.720.1461420.144119782120100
3.72-4.680.15351410.125619762117100
4.69-38.710.19891440.15291958210299
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)
14.5699-0.11790.33767.3185-0.96886.20320.094-0.20360.03580.17250.09320.6767-0.20280.0375-0.17080.1492-0.02230.04580.2017-0.08420.2483-18.11240.55414.6016
28.376.1578-2.82948.4242-4.53688.73590.0556-0.14560.47970.5198-0.02410.3206-0.4558-0.127-0.08990.358-0.0291-0.00180.1995-0.07260.2525-9.42854.819520.0019
37.49342.0201-0.50026.63360.14746.73880.0953-0.68310.35820.1084-0.19710.45530.0548-0.08010.11470.39050.0361-0.04090.2122-0.04550.2098-14.75884.827617.1494
41.9545-0.1530.60684.2295-0.2833.2004-0.0592-0.10870.1787-0.00370.0744-0.0551-0.24550.23710.00460.2296-0.04130.00780.2298-0.06820.2068-11.50081.497712.8728
57.5857-0.44990.19846.29140.10785.25720.08891.1030.9362-0.6889-0.2243-0.0627-0.84940.10520.0570.3848-0.0080.01180.33430.05570.3101-15.47852.84572.0065
60.02920.17040.21221.78911.7262.1574-0.5692-0.20981.4003-0.30250.6128-0.6143-0.57870.6088-0.15160.2773-0.0636-0.05330.3726-0.15370.571-2.2304-4.1968.3804
73.5282-0.26590.76861.8969-0.5043.6119-0.0641-0.4561-0.09950.19560.14240.02790.04630.1346-0.11660.20860.03910.02070.262-0.0170.2183-4.3672-19.399212.1517
88.8165-2.7981-0.90186.75442.08770.9958-0.236-0.17980.41230.00890.2962-0.22670.12140.2135-0.05220.20680.0129-0.00070.2686-0.03260.1309-0.5714-15.22319.4215
95.9459-4.8054-5.49876.10523.95845.2115-0.1535-0.2591-0.12160.5169-0.03050.7329-0.20150.13290.04950.3142-0.03980.07360.3580.01970.3073-16.5758-19.44817.6666
103.29450.4628-0.93721.9259-0.51011.49550.0436-0.04920.05220.03270.0073-0.03950.08690.1378-0.01830.22870.0072-0.00840.2495-0.02610.20011.4453-20.72385.2765
117.8114-0.25262.58536.7586-1.82386.61460.0687-0.2634-0.67060.5773-0.11890.41650.3573-0.4375-0.0320.3111-0.00530.09710.1757-0.01980.3114-4.6947-30.3179.7356
124.9435-1.0177-1.90792.01512.90473.8876-0.1378-0.2290.00840.33040.05520.31690.0943-0.13310.05450.1805-0.01590.01830.2472-0.01650.2267-19.406-13.41479.695
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1chain 'A' and (resid 145 through 161 )A145 - 161
2X-RAY DIFFRACTION2chain 'A' and (resid 162 through 175 )A162 - 175
3X-RAY DIFFRACTION3chain 'A' and (resid 176 through 196 )A176 - 196
4X-RAY DIFFRACTION4chain 'A' and (resid 197 through 224 )A197 - 224
5X-RAY DIFFRACTION5chain 'A' and (resid 225 through 243 )A225 - 243
6X-RAY DIFFRACTION6chain 'A' and (resid 244 through 253 )A244 - 253
7X-RAY DIFFRACTION7chain 'A' and (resid 254 through 280 )A254 - 280
8X-RAY DIFFRACTION8chain 'A' and (resid 281 through 290 )A281 - 290
9X-RAY DIFFRACTION9chain 'A' and (resid 291 through 301 )A291 - 301
10X-RAY DIFFRACTION10chain 'A' and (resid 302 through 320 )A302 - 320
11X-RAY DIFFRACTION11chain 'A' and (resid 321 through 340 )A321 - 340
12X-RAY DIFFRACTION12chain 'B' and (resid 2 through 9 )B2 - 9

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