Human Splicing Factor 3a Subunit 1

Target ID:

UBH20

Aliases:

PRP21PRPF21SAP114SF3A120

Deposition Date:

Monday 2nd May 2005

Families:

Ubiquitin Related Biology

Related Diseases:

Signalling

Structure type:

apo

Download Coordinates:

1ZKH

Authors:

J.A.LUKIN,S.DHE-PAGANON,V.GUIDO,A.LEMAK,G.V.AVVAKUMOV,S.XUE,E.M.NEWMAN,F.MACKENZIE,M.SUNDSTROM,A.EDWARDS,C.H.ARROWSMITH,STRUCTURAL GENOMICS CONSORTIUM (SGC)

Site:

Toronto

ICM Datapack:

ICM Datapack

1ZKH

tabs

Structure Details

The spliceosome catalyzes the removal of introns from pre-messenger RNA. It is a large machine composed of more than 50 different proteins and five small nuclear RNA's (snRNA). SF3A1 is a 739 residue, essential protein of the spliceosome, forms a stable heterotrimer with SF3A2 and SF3A3, and contains two swap domains and one ubiquitin like domain. The molecular functions of these domains are unclear. The swap domains could be mediating RNA binding and the ubiquitin like domain could be mediating the interactions with SF3A2, a poorly defined 465 residue protein containing a single zinc-finger element.

We have determined the high resolution structure of SF3A1 and show that the ubiquitin domain is one of the most divergent of the family. Although the ubiquitin fold as well as loop lengths are highly conserved among members, including those of Ubiquitin, NEDD, SUMO, and PARKIN, loop B1-B2 of SF3A1 contains an additional 12 residues. This longer SF3A1 loop wraps back towards the core of the domain, with M714, W720, and L722 forming important interactions. Further experiments are needed to show how the ubiquitin like domain of SF3A1 contributes to the function of the spliceosome.

Materials and Methods

Human SF3A1

PDB:1ZKH

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:GI:53831993
Entry Clone Source:MGC
SGC Clone Accession:ubh20.704.789; plate SDC009:C5
Tag:N-terminal: His-tag with integrated thrombin protease site: MGSSHHHHHHSSGLVPR*GS
Host:E. coli BL21-(DE3) mgk

Construct

Prelude:
Sequence:PVSIKVQVPNMQDKTEWKLNGQVLVFTLPLTDQVSVIKVKIHEATGMPAGKQKLQYEGIFIKDSNSLAYYNMANGAVIHLALKERG
Vector:p28a-LIC

Growth

Medium:M9
Antibiotics:
Procedure:Starter cultures from freshly transformed colonies in 20 ml 2xM9 supplemented with zinc and biotin containing ampicillin and kanamycin. This was diluted into a fresh 500 ml 2xM9 media and was grown at 37 degC to a OD600 of around 1.0 and then induced with IPTG to a final concentration of 1 mM, the temperature is lowered to 15degC.

Purification

Procedure
1.2 mL of qiagen Ni NTA bead slurry (50% beads) were added onto the clarified lysate and mixed at 4C for at least 20 min.

Decant and discard the supernatant. Wash the beads twice with 5 mL washing buffer (20 mM Tris-HCl, pH 8.5, 500 mM NaCl, 10 uM ZnSO4, 30 mM immidazole). Elute the protein with 5 ml of elution buffer (20 mM Tris-HCl, pH 8.5, 500 mM NaCl, 10 uM ZnSO₄, 500 mM immidazole).

Extraction

Procedure
The cell pellets were thawed and resuspended into lysis buffer (20 mM Tris-HCl, pH 8.5, 500 mM NaCl, 10 uM ZnSO4, 15 mM immidazole). The cell pellets were lysed by sonication and the cell debris were centrifuged.
Concentration:
Ligand
MassSpec:
Crystallization:
NMR Spectroscopy:The NMR sample buffer is 10 mM MOPS, 0.01 % NaN₃, 0.01 mM ZnSO₄, 10 mM DTT, 1 mM benzamidine, 1x inhibitor cocktail, 450 mM NaCl, 10% D₂O, pH 6.5.
Data Collection:
Data Processing: