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[…] Chinese hamster (Cricetulus griseus) BiP (residues 28–549) with a T229A mutation () was expressed as a His6-Smt3 fusion protein (UK 1607) in M15 E. coli cells. The bacterial cultures were grown at 37°C to OD600nm 0.8 in LB medium containing 50 µg/ml kanamycin and 100 µg/ml ampicillin and expression was induced with 1 mM IPTG. The cells were further grown at 25°C for 14 hr, harvested and lysed in high-salt buffer (50 mM Tris-HCl pH 7.4, 500 mM NaCl, 1 mM MgCl2, 2 mM PMSF) containing protease inhibitors and DNaseI as described above. The lysate was cleared by centrifugation for 30 min at 25,000 g, passed through a 0.45 µm syringe filter, and supplemented with 25 mM imidazole. The following purification strategies resulted in protein crystals that were suitable for X-ray data collection.Strategy A (PDB 5O4P): The cleared lysate from a 4 l expression culture was passed over a 5 ml Ni-Sepharose HisTrap HP column (GE Healthcare) at 4°C. The column was washed sequentially with 30 ml high-salt buffer and 30 ml low-salt buffer (50 mM Tris, pH7.4, 100 mM NaCl, 1 mM MgCl2) both containing 25 mM imidazole and 1 mM ATP. Bound protein was eluted with low-salt buffer supplemented with 500 mM imidazole (pH 7.4) followed immediately by addition of 10 mM ATP, 10 mM MgCl2 and 1 mM Tris(2-carboxyethyl)phosphine (TCEP). Purified GST-FICDE234G protein and Ulp1 protease were added in a 60:1 and 1000:1 (His6-Smt3-haBiP:X) mass ratio, respectively, and incubated 14 hr at 24°C to simultaneously allow for AMPylation and cleavage of the His6-Smt3 tag. The solution was then supplemented with 100 mM EDTA and the protein was further purified by size-exclusion chromatography using a HiLoad 16/60 Superdex 75 prep grade column (GE Healthcare) equilibrated with GF buffer (5 mM HEPES-KOH pH 7.6, 100 mM NaCl, 0.1 mM EDTA, 0.1 mM TCEP). A Glutathione Sepharose 4B column (GSTrap 4B; GE Healthcare) was connected in series with the gel filtration column to retain GST-FICDE234G protein. The eluted protein was concentrated to >50 mg/ml in presence of 1 mM TCEP and crystallization was performed in 96-well sitting drop plates by combining 150 nl protein solution with 150 nl reservoir solution and equilibration at 20°C against 80 µl reservoir solution. Diffraction quality crystals grew in a solution containing 100 mM HEPES pH 7.5 and 1.5 M Li2SO4. Crystals were soaked in cryosolution [the precipitant solutions containing 20% (v/v) glycerol] and snap frozen in liquid nitrogen. Diffraction data were collected at the Diamond Light Source beamline I02 (or I24, Didcot, United Kingdom; see ) and processed with Mosflm () and Aimless (). The structure was solved by molecular replacement in Phaser () by searching 2 copies of the nucleotide binding domain (PDB 3IUC) and substrate binding domain (PDB 4B9Q). Manual model building was carried out in COOT () and further refinements in refmac5 () and phenix.refine (). The final refinement statistics are summarized in . The structural graphs were generated with PyMOL software (PyMOL version 1.5.0.4; RRID: SCR_000305).Strategy B (PDBs 6EOB and 6EOC): Protein was expressed in a 6 l culture and cell lysis was performed as described in strategy A. The lysate was supplemented with 0.1 mM TCEP and incubated with 6 ml Ni-NTA agarose for 1 hr while slowly rotating at 4°C. The suspended matrix was then transferred to a Glass Econo-column (2.5 × 10 cm; Bio-Rad, Hercules, CA) and washed first with high-salt buffer containing protease inhibitors and then with wash buffer J (50 mM Tris-HCl pH 8, 500 mM NaCl, 30 mM imidazole). Further wash steps were performed with wash buffer K (50 mM Tris-HCl pH 8, 300 mM NaCl, 10 mM imidazole, 5 mM β-mercaptoethanol) sequentially supplemented with (i) 1 M NaCl, (ii) 10 mM MgCl2 and 3 mM ATP, (iii) 0.5 M Tris-HCl pH 8 and (iv) 40 mM imidazole. The retained protein was eluted with elution buffer L (50 mM Tris-HCl pH 7.4, 100 mM NaCl, 250 mM imidazole, 10 mM MgCl2, 10 mM ATP, 1 mM TCEP). After AMPylation with GST-FICDE234G and cleavage of the His6-Smt3 tag with Ulp1 (as described in strategy A) the protein solution was incubated with 1 ml Glutathione Sepharose 4B for 2 hr at room temperature (to bind GST-FICDE234G). The matrix was collected by centrifugation and the supernatant was dialyzed twice for 14 hr against 5 l buffer M (25 mM Tris-HCl pH 7.4, 50 mM NaCl, 5 mM EDTA) and once for 24 hr against 10 l AEX-LS buffer (25 mM Tris-HCl pH 8, 50 mM NaCl). Afterwards, the protein was subjected to anion exchange chromatography using a 5 ml HiTrap Q HP column (GE Healthcare) equilibrated in AEX-LS buffer and bound protein was eluted with AEX-HS buffer (25 mM Tris-HCl pH 8, 1 M NaCl) on a linear gradient (0% to 50% AEX-HS in 20 column volumes). The purest fractions were pooled and incubated with 1.5 ml Ni-NTA agarose beads in presence of 25 mM imidazole for 30 min at 4°C (to bind residual uncleaved protein and free His6-Smt3 tag). To crystallize AMPylated BiP in absence of nucleotide the protein solution was desalted over a HiLoad 16/60 Superdex 75 prep grade column equilibrated with buffer N (10 mM HEPES-KOH pH 7.4, 100 mM NaCl). The protein solution was then supplemented with 1 mM TCEP and concentrated for crystallization. Crystals grown in 20% PEG-1000, 0.1 M NaKHPO4 pH 6.2, 0.1 NaCl (PDB 6EOB) and 5% PEG-1000, 0.2 M Li2SO4, 0.1 M Na2HPO4 pH 4.2 (PDB 6EOC) were used for data collection and the structures were solved as described above.Strategy C (PDB 6EOE): Protein was expressed, purified, and AMPylated as in strategy B. Protein obtained after ion exchange and reverse Ni affinity chromatography was incubated with 14 mM MgCl2 and 10 mM ATP for 2 hr on ice before addition of 100 mM EDTA and immediate gel filtration using a HiLoad 16/60 Superdex 200 prep grade column (GE Healthcare) in buffer TN (25 mM Tris-HCl pH 8, 150 mM NaCl). Fractions of the main elution peak (corresponding to monomeric BiP protein) were pooled, supplemented with 1 mM TCEP, and concentrated for crystallization. The structure of BiP crystallized in 5% PEG-1000, 0.2 M Li2SO4, 0.1 M Na2HPO4 pH 4.2 was solved as described above.Strategy D (PDB 6EOF): Protein was initially purified according to strategy B except that dialysis (after AMPylation and removal of the modifying enzyme) was performed against buffer O (25 mM Tris-HCl pH 7.4, 300 mM NaCl) for 16 hr at 4°C. The dialyzed solution was passed through a column containing 2 ml Q Sepharose High Performance matrix (GE Healthcare) by gravity flow. The flow-trough was concentrated and applied to gel filtration using a Superdex 200 10/300 GL column equilibrated in buffer TN. The purest elution fractions were pooled and dialyzed twice for 24 hr at 4°C against 5 l buffer TN containing 5 mM EDTA (to remove nucleotides) and once against the same buffer without EDTA. The protein solution was then supplemented with 1 mM TCEP and concentrated. The crystallization reactions were set up in presence of 10 mM ADP and 14 mM MgCl2. Crystals grown in 9% PEG-1000, 0.2 M Li2SO4, 0.1 M Na2HPO4 pH 4.4 were used for data collection and the structure was solved as described above. […]

Pipeline specifications

Software tools iMosflm, CCP4, Coot, REFMAC5, PHENIX, PyMOL
Applications Small-angle scattering, Protein structure analysis
Chemicals Adenosine Triphosphate