Structural organization of pregenomic RNA and the carboxy-terminal domain of the capsid protein of hepatitis B virus.

Purification of HBV capsids

The pgRNA production, the plasmids coded for HBV Cp183-SSS and Cp183-EEE, and the capsid purification were described previously [15] . Capsids stored at −80°C were disassembled by dialysis at 4°C in the disassembly buffers (1.5 M guanidine HCl, 0.5 M LiCl, 50 mM HEPES at pH 7.5, 10 mM DTT for Cp183-SSS, and 1.5 M guanidine HCl, 1.5 M LiCl, 50 mM Tris at pH 9.5, 10 mM DTT for Cp183-EEE). The encapsidated heterogeneous RNA packaged from E. Coli cells was precipitated by a spin of 20,000× g for 15 min at 4°C. Protein dimers were recovered from the supernatant and purified by size exclusion chromatography using an analytical grade Superose 6 column (GE Lifesciences) equilibrated in disassembly buffer. Fractions containing core protein were identified by SDS-PAGE. Cp183-SSS and Cp183-EEE dimers were either used for the reassembly experiments immediately or stored at 4°C for a short period. Formation of the empty capsids (Cp183 e -SSS and Cp183 e -EEE) was approached by dialyzing the purified dimers in the reassembly buffer (250 mM NaCl, 50 mM HEPES at pH 7.5, 2 mM DTT for Cp183-SSS and 250 mM NaCl, 50 mM Tris pH 7.4, 2 mM DTT for Cp183-EEE). The pgRNA-filled capsids (Cp183 RNA -SSS and Cp183 RNA -EEE) were prepared by reassembling the purified dimers with in vitro transcribed HBV pgRNA at a molar ratio of protein dimer to RNA polymer = 120∶1 in the reassembly buffer (150 mM NaCl, 50 mM HEPES at pH 7.5, 2 mM DTT for Cp183-SSS and 150 mM NaCl, 50 mM Tris at pH 7.4, 2 mM DTT for Cp183-EEE) overnight. Samples for cryo-EM were further concentrated by Amicon Ultra centrifugal filter units (Millipore, MA). The quality and the concentration of the sample were routinely checked by negative stained EM using 2% uranyl acetate.

Cryo-electron microscopy

The sample preparation and cryo-EM operation were followed well established procedures described previously [28] . Briefly, a drop of 3.5 µl sample solution was applied on a glow-discharged Quantifoil holey-carbon grid (R2/2), blotted with filter paper from both sides for 4 s to produce a thin layer of specimen solution across the holes. The grids were quickly plunged into liquid ethane bath cooled by liquid nitrogen in a cryo-container. All processes described above were performed by a FEI Vitrobot. The vitrified specimen on the grid was then transferred to a Gatan 626DH cryo-holder and kept at the low temperature environment (