Human survival of motor neuron 1, telomeric, Tudor domain, in complex with inhibitor

Target ID:

SMN1

Deposition Date:

Thursday 26th June 2014

Families:

Methyl-lysine readers

Related Diseases:

CancerChromatin and epigenetics

Structure type:

protein-ligand

Download Coordinates:

4QQ6

Authors:

Liu Y, Tempel W, Iqbal A, Walker JR, Bountra C, Arrowsmith CH, Edwards AM, Brown PJ, Min J

Site:

Toronto

4QQ6

tabs

Structure Details

In the complex structure, the tudor domain adopts similar structure as the deposited apo SMN1 structure (1MHN), and the small organic molecule occupies the modified Arginine binding site in deposited complex structure of SMN1 and modified Arginine (4A4E for SMN-sDMA and 4A4G for SMN-aDMA). The amine group of small molecule forms hydrogen bond with the carboxylate oxygen of N132.

Materials and Methods

SMN1

PDB:4QQ6

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:NP_000335.1.
Entry Clone Source:AU98-F11
SGC Clone Accession:SMN1_18 (JMC047:F11): K82-E147
Tag:N-terminal tag: MHHHHHHSSGRENLYFQG
Host:Escherichiacoli BL21 (DE3)

Construct

Prelude:
Sequence:MHHHHHHSSGRENLYFQGKKNTAASLQQWKVGDKCSAIWSEDGCIYPATIASIDFKRETCVVVYTGYGNREEQNLSDLLSPICE
Vector:pET28-MHL

Growth

Medium:
Antibiotics:
Procedure:Lex system, 37 °C 3-4h, 15 °C 18-24h

Purification

Procedure
IMAC: Unclarified lysate was mixed with 3-4 mL of Ni-NTA superflow Resin (Qiagen) per 200 mL lysate. The mixture was incubated with mixing for at least 30 minutes at 4°C. The mixture was then loaded onto an empty comLum (BioRad) and washed with 40 mL wash buffer. Samples were eluted from the resin by exposure to 2-3 column volumes (approx. 10-15 mL) of elution buffer. Concentration of eluted protein was estimated by OD280

Gel filtration chromatography: An XK 16x60 column (GE Healthcare) packed with HighLoad Superdex 75 resin (GE Healthcare) was pre-equilibrated with gel filtration buffer for 1.5 column volumes using an AKTA explorer (GE Healthcare) at a flow rate of 1.0 mL/min. The concentrate sample (approx. 3 mL, concentrated by using 15 mL concentrators with a 3,000 molecular weight cut-off (Amicon Ultra-15, UFC900524, Millipore) at 3750 rpm) from the IMAC step was loaded onto the column at 1 mL/min, and 2mL fractions were collected into 96-well plates (VWR 40002-012) using peak fractionation protocols. Fractions observed by a UV absorption chromatogram to contain the protein were pooled.

Extraction

Procedure
Frozen cell pellet contained in bags (Beckman 369256) obtained from 2L of culture were thawed by soaking in warm water. Each cell pellet was resuspended in 200 mL lysis buffer and homogenized using an Ultra-Turrax T8 homogenizer (IKA Works) at maximal setting for 30-60 seconds per pellet. Cell lysis was accomplished by sonication (Virtis408912, Virsonic) on ice: the sonication protocol was 10 seconds pulse at 80% of maximal frequency (8.0), 10 seconds rest, for 10 minutes total sonication time per pellet.
Concentration:Purified proteins were concentrated using 15 mL concentrators with a 5,000 molecular weight cut-off (Amicon Ultra-15, UFC900524, Millipore) at 3750 rpm, typically resulting in a final concentration around 20 mg/mL.
Ligand
inhibitorMassSpec:
Crystallization:Recombinant human SMN1 tudor domain was mixed with compound at molar ratio of 1:3 and co-crystallized using the sitting drop vapour diffusion method at 18 °C. The crystals were obtained in a buffer containing 2 M NH4SO4, 0.2M K/Na tart, 0.1M Na Citrate, pH 5.6. Crystals were soaked in a cryoprotectant consisting of 100% reservoir solution and 15% glycerol.
NMR Spectroscopy:
Data Collection:
Data Processing: