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Materials and Methods: DDX5 (3FE2)

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Structure	DDX5
PDB Code	3FE2
Entry clone accession	BC016027
Entry clone source	Mammalian Gene Collection
Tag	N-terminal hexahistidine tag with integrated TEV protease cleavage site: mhhhhhhssgvdlgtenlyfq*sm
Construct sequence	mhhhhhhssgvdlgtenlyfq*smRTAQEVETYRRSKEITVRGHNCPKPVLNFYEANFPANVMDVIARQNFTEPTAIQAQGWPVALSGLDMVGVAQTGSGKTLSYLLPAIVHINHQPFLERGDGPIC LVLAPTRELAQQVQQVAAEYCRACRLKSTCIYGGAPKGPQIRDLERGVEICIATPGRLIDFLECGKTNLRRTTYLVLDEADRMLDMGFEPQIRKIVDQIRPDRQTLMWSATWPKEVRQLAEDFLKDY IHINIGALEL
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 30 ºC overnight. 20 mL of the overnight culture were used to inoculate 1.5 l TB supplemented with 8 g/l glycerol, 50 µg/mL kanamycin and approximately 500 µl 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 (29.6 g wet cell weight) was resuspended in lysis buffer (1.5 mL/g cell pellet), supplemented with 2000 U Benzonase (Merck) and one 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 and diluted to 2 x 68 mL with lysis buffer. 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 (49,000 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, 500 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) Procedure 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 two HiTrap Chelating columns connected in series and washed with IMAC wash1 buffer followed by IMAC wash2 buffer. Bound protein was eluted from the IMAC columns 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 17.6 mg/mL in a volume of 1.2 mL. The purified protein started to precipitate after concentration why precipitate was removed and the protein sample was quickly aliquoted and frozen. Tag removal The N-terminal histidine tag was proteolytically removed by incubating DDX5 with His-tagged TEV protease (van den Berg, S., J. Biotech 121, 291-298 (2006)) at a molar ratio of 30:1 at 4 ºC overnight. The proteolytic reaction went near to completion, as judged by SDS-PAGE. Target protein was purified from tag and protease by passing the reaction mixture over a Ni-charged 1 mL HiTrap Chelating HP column (GE Healthcare) pre-equilibrated with IMAC wash1 buffer. The cleaved protein was concentrated and the buffer was changed to GF buffer containing 2 mM TCEP using a Vivaspin 20 centrifugal filter device with 10,000 MWCO (Sartorius). The final protein concentration was determined to 17.5 mg/mL in a volume of 0.7 mL. The identity of the protein was confirmed by mass spectrometry.
Crystallization	Crystals were obtained by the sitting drop vapour diffusion method using a 96-well plate. 0.2 µl of the protein sample (diluted to 15.8 mg/mL) including 20 mM ADP and 10 mM MgCl₂ was mixed with 0.1 µl of well solution consisting of 0.1 M Bis-Tris pH 6.4, 0.25 M lithium sulfate monohydrate, 17.5% PEG 3350. The plate was incubated at 4 ºC and crystals appeared within 10 hours. Cryo solution containing 0.1 M Bis-Tris pH 5.5, 0.2 M lithium sulfate, 25% PEG 3350, 0.2 M NaCl and 15% glycerol was added directly to the drop. Crystals were mounted and flash-frozen in liquid nitrogen.
Data collection	Data to 2.6 Å resolution was collected from a single crystal at Bessy (BL14-2). Crystal belonged to C2221 space group with cell parameters of a=84.57 Å, b=106.87 Å, c=117.32 Å. The asymmetric unit contains two monomers.
Data processing	The structure was solved by molecular replacement using MOLREP with human DDX3X protein structure (2I4I) residues 3-241 as a search model. ADP was located at the active site. Structure was refined with REFMAC5. Final R-values were R=20.8% and Rfree=27.3%. Coordinates and structure factors are deposited to PDB with accession code 3FE2.