Initial crystallization conditions for 3D5/His_683 were identified by sparse matrix screening (Hampton Research HR2-139 and HR2-138). For 3D5/EE_48.A and 3D5/EE_48.K, we initially used conditions that resulted in diffraction quality crystals of the parent 3D5/EE_48 scFv, but included several optimization steps to increase the diffraction quality of the second generation scFvs. Variables optimized include protein concentration, protein:drop ratio, temperature, buffer concentration, salt, and polyethylene glycol (PEG) molecular mass in the mother liquor. Ultimately, the best crystals of 3D5/His_683 (8mg/ml in 10 mM Hepes pH 7.5, 150 mM NaCl, HBS) were grown at room temperature with 0.2M KI, 0.001M Guanidinium HCl, 18% PEG 8000 (Hampton Research). These crystals appeared in 2-3 days and grew to a maximal size of 120-150 μm within 1 week. Crystals of 3D5/EE_48.K (7.5mg/ml in HBS) were grown at 4°C with 0.1M Tris pH 8.5, 0.2M Li2(SO4), 3% 6-aminohexanoic acid, 24% PEG 8000; crystals appeared in 4-5 days and grew to a maximal size of 30-40 μm within 2 weeks. Crystals of 3D5/EE_48.A (7.5mg/ml in HBS) were grown at 4°C with 0.1M BisTris pH 6.5, 0.2M Mg(OAc)2, 21% PEG 8000, crystals appeared in 4-5 days and grew to a maximal size of 20-30 μm within 3-4 weeks. All crystals were grown utilizing a 1:1 reservoir:protein ratio.
Peg 8000
Manufactured by Hampton Research
Sourced in United States
PEG 8000 is a polyethylene glycol with an average molecular weight of 8,000 Daltons. It is a water-soluble, non-ionic polymer commonly used as a precipitating agent in protein crystallization experiments.
Automatically generated - may contain errors
Lab products found in correlation
Hyperflask, by Corning (1 mentions)
Crystal screen 1, by Hampton Research (1 mentions)
Hek293t cell, by American Type Culture Collection (1 mentions)
Phhs lot 4405c, by Lonza (1 mentions)
Power primary hep medium, by Takara Bio (1 mentions)
Rat tail collagen type 1, by Corning (1 mentions)
Fetal bovine serum (fbs), by American Type Culture Collection (1 mentions)
Fetal bovine serum (fbs), by Atlanta Biologicals (1 mentions)
7 protocols using peg 8000
1
Optimized Crystallization Conditions for scFvs
2
Quantifying HBV DNA Production
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HepAD38 cells were grown in DME/F12 medium containing 4% FBS and 1% dimethyl sulfoxide (DMSO) in HyperFlasks (Corning). Cell culture supernatants were collected every 6 days and were used for HBV concentration by precipitation with 10% polyethylene glycol (PEG) 8,000 (Hampton Research). The genome copy numbers of HBV DNA were determined by a real-time PCR method.
3
HBV Infection Assay in NTCP-expressing Cells
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HC9AT cells expressing NTCP receptors were infected with HBV-containing medium obtained from HepG2.2.15 cells. Briefly, HC9AT cells (in DMEM-F12 containing 5% FBS, 3% DMSO, and 1× Pen-Strep) were seeded in a 24-well plate containing collagen-coated coverslips. At 18 to 20 h postseeding, the medium was removed and cells were overlaid with viral supernatant diluted to a final concentration of 62.5 or 500 viral genome equivalents per cell. This infection medium additionally contained 5% PEG8000 (Hampton Research, CA, USA) and 2% DMSO. Plates were shaken for 4 h at 350 rpm at room temperature and then incubated at 37°C and 5% CO2. For treatment with myrcludex B (MyrB, a kind gift from Stephan Urban, University Hospital Heidelberg, Germany), cells were incubated with 500 nM MyrB for 1 h before adding the viral inoculum and then for 24 h with the viral inoculum. At 24 h postinfection, the viral inoculum was removed and the cells were washed three times with 1× PBS and maintained in DMEM-F12 supplemented with 5% FBS, 1× Pen-Strep, and 1% DMSO. Cells were fixed at day 3, 4, 6, 8, or 10 postinfection and immunostained as described below.
4
Crystallization of K5–K14-Cys367A 2B Heterocomplexes
5
Crystallization of S39E HDAC8-Droxinostat Complex
6
Crystallization and structural determination of PCPBAe1
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Crystallization screens were conducted using the sitting drop vapor diffusion method at room temperature (22°C) by mixing protein with the crystallization reservoir solutions at a 1:1 volume ratio. Crystals appeared after about 2 wk in the condition Crystal Screen 1(0.2 M ammonium sulphate, 0.1 M sodium cacodylate trihydrate, pH 6.5, 30% wt/vol PEG 8000) (Hampton Research). The diffraction quality crystals were immersed in mother liquor solution supplemented with 25% ethylene glycol as a cryoprotectant for data collection. A complete native 2.0 Å resolution diffraction data set was collected at 100K using Rigaku MicroMax-007 HF equipped with Saturn 944+ CCD detector, National University of Singapore. Data were processed with the HKL-2000 program (38 (link)). The structure was determined using molecular replacement (39 (link)) using the coordinates of CPAHa (PDB: 1JQG ) (10 (link)) and the final model was refined at 2.08 Å resolution. The structure has good stereochemical parameters evaluated with PROCHECK (Table S2). PyMOL and CHIMERA (40 (link)) were used to prepare all structure-related figures.
Table S2
7
Establishing HBV-Producing Cell Lines
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A puromycin-resistant HepG2NTCP cell line (HepG2NTCP-P3) was described in our previous work [25 (link)]. The HBV-producing cell line HepAD38 was described previously [77 (link)]. Both HepG2NTCP and HepAD38 were grown in DME/F12 medium containing 10% of fetal bovine serum (FBS, Atlanta Biologicals). Primary human hepatocytes (PHHs, lot#4405C) were purchased from Lonza and were cultured in Power Primary HEP medium (Takara, San Francisco, CA). HEK293T cells were from ATCC and grown in DMEM containing 10% of FBS as previously reported [78 (link)]. Cell culture flasks and plates were coated with 50 μg/mL of rat tail collagen type I (Corning). HBV obtained from HepAD38 cells were concentrated by precipitation with 10% polyethylene glycol (PEG) 8,000 (Hampton Research). The genome copy numbers of HBV DNA were quantified by a real-time PCR method.
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