SASDD25: 1:1 Mixture between Protein sex-lethal mutant (Sxl10GS) and RNA d...

Basic information

• Entry: Database: SASBDB / ID: SASDD25

Sample

1:1 Mixture between Protein sex-lethal mutant (Sxl10GS) and RNA decaneucleotide UGU8

• Protein sex-lethal mutant (protein), Sxl10GS, Drosophila melanogaster
• RNA decaneucleotide UGU8 (RNA), UGU8, synthetic construct

Function / homology

Function:

sex determination, primary response to X:A ratio / germarium-derived cystoblast division / epithelium regeneration / female sex determination / somatic sex determination / female germ-line sex determination / oocyte differentiation / imaginal disc growth / regulation of stem cell division / sex determination / negative regulation of RNA export from nucleus / poly-pyrimidine tract binding / sex-chromosome dosage compensation / sex differentiation / alternative mRNA splicing, via spliceosome / negative regulation of receptor signaling pathway via JAK-STAT / poly(A) binding / positive regulation of smoothened signaling pathway / pre-mRNA binding / regulation of mRNA splicing, via spliceosome / reciprocal meiotic recombination / poly(U) RNA binding / regulation of alternative mRNA splicing, via spliceosome / oogenesis / negative regulation of mRNA splicing, via spliceosome / negative regulation of translational initiation / mRNA regulatory element binding translation repressor activity / positive regulation of RNA splicing / mRNA 3'-UTR binding / mRNA 5'-UTR binding / negative regulation of translation / protein stabilization / ribonucleoprotein complex / mRNA binding / protein-containing complex / RNA binding / nucleus / cytosol / cytoplasm

Domain/homology:

Sex-lethal splicing factor / Paraneoplastic encephalomyelitis antigen / 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

Component:

Protein sex-lethal

• Biological species: Drosophila melanogaster (fruit fly); synthetic construct (others)
• Citation: Journal: ACS Comb Sci / Year: 2018; Title: A General Small-Angle X-ray Scattering-Based Screening Protocol Validated for Protein-RNA Interactions.; Authors: Po-Chia Chen / Pawel Masiewicz / Vladimir Rybin / Dmitri Svergun / Janosch Hennig / ; Abstract: We present a screening protocol utilizing small-angle X-ray scattering (SAXS) to obtain structural information on biomolecular interactions independent of prior knowledge, so as to complement affinity-based screening and provide leads for further exploration. This protocol categorizes ligand titrations by computing pairwise agreement between curves, and separately estimates affinities by quantifying complex formation as a departure from the linear sum properties of solution SAXS. The protocol is validated by sparse sequence search around the native poly uridine RNA motifs of the two-RRM domain Sex-lethal protein (Sxl). The screening of 35 RNA motifs between 4 to 10 nucleotides reveals a strong variation of resulting complexes, revealed to be preference-switching between 1:1 and 2:2 binding stoichiometries upon addition of structural modeling. Validation of select sequences in isothermal calorimetry and NMR titration retrieves domain-specific roles and function of a guanine anchor. These findings reinforce the suitability of SAXS as a complement in lead identification.

Contact author

• Po-chia Chen (EMBL, European Molecular Biology Laboratory (EMBL) - Heidelberg, Heidelberg, Germany)

Models

• Model #1877: ; Type: mix / Software: (2.1) / Radius of dummy atoms: 1.90 A / Chi-square value: 0.777 / P-value: 0.240677; Search similar-shape structures of this assembly by Omokage search (details)
• Model #1878: ; Type: mix / Software: (2.1) / Radius of dummy atoms: 1.90 A / Chi-square value: 0.777 / P-value: 0.240677; Search similar-shape structures of this assembly by Omokage search (details)
• Model #1879: ; Type: mix / Software: (2.1) / Radius of dummy atoms: 1.90 A / Chi-square value: 0.777 / P-value: 0.240677; Search similar-shape structures of this assembly by Omokage search (details)

Sample

• Sample: Name: 1:1 Mixture between Protein sex-lethal mutant (Sxl10GS) and RNA decaneucleotide UGU8; Specimen concentration: 1 mg/ml / Entity id: 1008 / 1009
• Buffer: Name: 50% dilution of protein buffer {10 mM KP, 50 mM NaCl, 10 mM DTT pH 6} with {milliQ-water pH 7} suspended RNA; pH: 6

Entity #1008

Name: Sxl10GS / Type: protein / Description: Protein sex-lethal mutant / Formula weight: 20.432 / Num. of mol.: 2 / Source: Drosophila melanogaster / References: UniProt: P19339
Sequence:

GAMASNTNLI VNYLPQDMTD RELYALFRAI GPINTCRIMR DYKTGYSFGY AFVDFTSEMD SQRAIKVLNG ITVRNKRLKV SYARPGGGSG SGGGGSGESI KDTNLYVTNL PRTITDDQLD TIFGKYGSIV QKNILRDKLT GRPRGVAFVR YNKREEAQEA ISALNNVIPE GGSQPLSVRL AEEHGK

Entity #1009

Name: UGU8 / Type: RNA / Description: RNA decaneucleotide UGU8 / Formula weight: 3.119 / Num. of mol.: 2 / Source: synthetic construct
Sequence:

UGUUUUUUUU

Experimental information

• Beam: Instrument name: ESRF BM29 / City: Grenoble / 国: France / Type of source: X-ray synchrotron / Wavelength: 0.09919 Å / Dist. spec. to detc.: 2.867 mm
• Detector: Name: Pilatus 1M

Scan

Title: 1:1 Mixture between Protein sex-lethal mutant (Sxl10GS) and RNA decaneucleotide UGU8
Measurement date: Apr 12, 2017 / Storage temperature: 20 °C / Cell temperature: 20 °C / Exposure time: 0.5 sec. / Number of frames: 10 / Unit: 1/nm /

	Min	Max
Q	0.0308	4.9444

Distance distribution function P(R)

Sofotware P(R): GNOM 4.5a / Number of points: 295 /

	Min	Max
Q	0.2087	2.997
P(R) point	1	295
R	0	8.878

Result

Type of curve: single_conc
Comments: This synchrotron session suffered from misalignment of the beamstop resulting in parasitic scattering at low angles. Note that the uploaded EOM ensemble is chosen as the 2:2-complex with one RNA monomer as fully bound by two Sxl10GS molecules in the canonical position from Protein data bankk (PDB) entry 1B7F.pdb. The second RNA monomer is placed in the canonical position bound to the free RRM2, resulting in three rigid bodies: RRM2+RNA, RRM1+RRM2+RNA, and RRM1.

	Experimental	Porod
MW	24 kDa	-
Volume	-	40.96 nm3

	P(R)	P(R) error	Guinier	Guinier error
Forward scattering, I0	77.67	0.08	76.65	0.13
Radius of gyration, Rg	2.63 nm	0.004	2.54 nm	0.07

	Min	Max
D	-	8.88
Guinier point	51	92