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Materials and Methods: NUMBL (3F0W)

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Structure	NUMBL
PDB Code	3F0W
Entry clone source	Mammalian Gene Collection
SGC clone accession	NUMBLA-k019
Tag	N-terminal hexahistidine tag with integrated TEV protease cleavage site: mhhhhhhssgvdlgtenlyfq*sm
Construct sequence	mhhhhhhssgvdlgtenlyfq*smASRPHQWQADEDAVRKGTCSFPVRYLGHVEVEESRGMHVCEDAVKKLKAMGRKSVKSVLWVSADGLRVVDDKTKDLLVDQTIEKVSFCAPDRNLDKAFSYICR DGTTRRWICHCFLALKDSGERLSHAVGCAFAACLERKQRREK
Vector	pNIC-BSA4
Expression host	E.coli BL21(DE3) R3 pRARE, where R3 denotes a derivative of BL21(DE3) resistant to a strain of T1 bacteriophage (SGC Oxford) and the pRARE plasmid originating from the Rosetta strain (Novagen) supplies tRNAs for rare codons.
Growth method	Cells from a glycerol stock were grown in 20 mL TB supplemented with 8 g/l glycerol, 100 µg/mL kanamycin and 34 µg/mL chloramphenicol at 37 ºC overnight. The overnight culture (20 mL) was used to inoculate 1.5 l TB supplemented with 8 g/l glycerol, 50 µg/mL kanamycin and approximately 0.2 mL Dow Corning anti-foam RD emulsion 63213 4D (BDH Silicone Products). The culture was grown in a LEX bioreactor system (Harbinger Biotechnology) at 37 ºC until OD₆₀₀ reached ~2. The bottle was down-tempered to 18 ºC over a period of 1 hour before target expression was induced by addition of 0.5 mM IPTG. Expression was allowed to continue overnight and cells were harvested the following morning by centrifugation (4,400 x g, 10 min, 4 ºC). The resulting cell pellet (15.2 g wet cell weight) was resuspended in lysis buffer (2 mL/g cell pellet), supplemented with 2000 U Benzonase (Merck) and 1 tablet of Complete EDTA-free protease inhibitor (Roche Applied Science). The cell suspension was stored at -80 ºC.
Extraction buffers	Lysis buffer: 100 mM HEPES, 500 mM NaCl, 10% glycerol, 10 mM imidazole, 0.5 mM TCEP, pH 8.0
Extraction procedure	The cell suspension was quickly thawed in water. Cells were disrupted by sonication (Vibra-Cell, Sonics) at 80% amplitude for 3 min effective time (pulsed 4s on, 4s off) and cell debris was removed by centrifugation (49,00 x g, 20 min, 4 ºC). The supernatant was decanted and filtered through a 0.45 µm flask filter.
Purification buffers	IMAC wash1 buffer: 20 mM HEPES, 500 mM NaCl, 10% glycerol, 10 mM imidazole, 0.5 mM TCEP, pH 7.5 IMAC wash2 buffer: 20 mM HEPES, 500 mM NaCl, 10% glycerol, 25 mM imidazole, 0.5 mM TCEP, pH 7.5 IMAC elution buffer: 20 mM HEPES, 500 mM NaCl, 10% glycerol, 500 mM imidazole, 0.5 mM TCEP, pH 7.5 Gel filtration (GF) buffer: 20 mM HEPES, 300 mM NaCl, 10% glycerol, 0.5 mM TCEP, pH 7.5
Purification procedure	Columns IMAC: Ni-charged 1 mL HiTrap Chelating HP (GE Healthcare) Gel filtration column: HiLoad 16/60 Superdex 75 Prep Grade (GE Healthcare) Purification: Purification of the protein was performed as a two step process on an ÄKTAxpress system (GE Healthcare). Prior to purification, columns were equilibrated with IMAC wash1 buffer and gel filtration buffer, respectively. The filtered lysate was loaded onto the Ni-charged HiTrap Chelating column and washed with IMAC wash1 buffer followed by IMAC wash2 buffer. Bound protein was eluted from the IMAC column with IMAC elution buffer and automatically loaded onto the gel filtration column. Fractions containing the target protein were pooled and fresh TCEP was added to a final concentration of 2 mM. The protein was subsequently concentrated using an Amicon Ultra-15 centrifugal filter device with 10,000 NMWL (Millipore) to 13.4 mg/mL in a volume of 1.8 mL.
Crystallization	The crystal was obtained by the hanging drop vapour diffusion method using a 24-well plate containing 500 µl well solution. 1 µl of the protein solution (13.4 mg/mL) was mixed with 1 µl of well solution consisting of 0.1 M sodium acetate trihydrate, pH 4.7, 0.1 M lithium sulfate monohydrate and 24% PEG 8000. The plate was incubated at 4 ºC and crystals appeared within 2 days. The crystal was quickly transferred to cryo solution containing 0.1 M sodium acetate trihydrate, pH 4.5, 0.1 M lithium sulfate monohydrate, 30% PEG-8000 and 17 % glycerol and flash frozen in liquid nitrogen.
Data collection	Data to 2.7 Å resolution was collected from a single crystal at ESRF (ID23-1). The crystal belonged to space group P3221 with cell parameters of a=b=55.1 Å and c=99.4 Å. Data was 99.1% complete and has Rmerge of 6.6%.
Data processing	The structure was solved by molecular replacement using MOLREP with NMR model of mouse Numb protein (1WJ1) as a search model. The asymmetric unit consisted of one polypeptide chain. Structure was refined with Phenix and finally with REFMAC5. Three TLS groups were used for the protein chain according to suggestion of TLSMD server. Final model has a good geometry as analyzed with Molprobity and final R-values were R=24.2% and Rfree=28.1%. The coordinates and structure factors were deposited in PDB with accession code 3F0W.