Cryo-electron microscopy for the study of virus assembly.

HBV sample preparation The Hepatitis B subtype adyw core protein assembly domain and the Cp150 mutant, in which three native cysteine are mutated to alanine and an additional C-terminal cysteine appended, were expressed in E. coli and purified by size exclusion as previously described ( Zlotnick et al., 2007 ). Cp149 and Cp150 capsids were prepared for preliminary Cryo-EM from purified dimer by initiating assembly at 10 μM dimer concentration in 300 mM NaCl, 50 mM HEPES, pH 7.5 and allowing the reaction to proceed overnight. Residual un-assembled dimer was removed by purifying the fresh capsids via size exclusion. Purified capsids were then incubated with HAP-TAMRA at a molar excess overnight. For cryo-EM, capsids were then concentrated to >10 mg/ml.

Synthesis of HAP-TAMRA

Synthesis of HAP13 ( Bourne et al., 2008 ) and HAP-TAMRA are described in the patent literature ( Zlotnick et al., 2014 ). Analysis of the product by HPLC at the 280 nm and 555 nm wavelengths showed a product with ~99% purity; ESI-TOF MS: calculated for C 51 H 50 ClFN 8 O 7 , m/ z 940.35; M + + H, m/ z 941.36; Found, m/ z 941.3; calculated for M + + Na, m/ z 963.4; Found, m/ z 963.3.

Negative stain electron microscopy

Samples from Figure 2a,b represent Cp149 assembly reactions at 50 mM HEPES, 300 mM NaCl, and 10 μM dimer. The reaction in Figure 2b was initiated in the presence of 40 μM HAP-TAMRA. Both samples were adsorbed to the surface of EMS carbon film 300 mesh coper grids, washed with water, and stained with 2% uranyl acetate. Samples were imaged with a JEOL 3200-FS electron microscope operated at 300kV.

Cryo-electron microscopy and image processing

Samples for Cryo-EM were applied to a glow-discharged Quantifoil holey-carbon grids (R2/2). The grids were blotted with filter paper for 4 s before automated plunging into liquid ethane using an FEI vitrobot. The Cp149+HAP TAMRA data from Figure 11 and Figure 4 panel a were imaged using a JEOL 3200-FS electron microscope operated at 300kV with an in-column energy filter. Images were recorded at a nominal magnification of 80,000X, with a pixel size of 1.5 Å, maintaining less than 35e - Å 2 dose, recorded on a Gatan Ultrascan 4000 4k × 4 k CCD detector. The 2D class averages shown in Figure 4 panels b, c, and d were collected on the same JEOL 3200-FS microscope, but with a Direct Electron DE-12 detector, with an image sampling rate of 1.01 Å/pixel. For the high-resolution 3D structure determination, first presented in Figure 5 , samples were imaged on a FEI Titan Krios operated at 300 kV at a nominal magnification of 22,500. Images were recorded on a Gatan K2 Summit detector operating in ‘super-resolution’ mode, resulting in a pixel size of 0.65 Å with a dose of ~33 e - Å 2 . Each exposure was 8 s long, and was collected as 35 individual frames. The data collection process was semi-automated using the Leginon system.( Suloway et al., 2005 ) The final maps are deposited in the EMDB database as 7295 for the T = 3 map and 7294 for the T = 4 map ( Table 1 ). Cryo-EM classification and reconstruction was implemented using standard protocols of the EMAN2, Motioncorr2, and Relion software programs ( Kimanius et al., 2016 ; Zheng et al., 2016 ; Tang et al., 2007 ). Upon convergence of the 3D structures, each map was sharpened by applying a negative B-factor which was obtained using the Guinier fitting procedure implemented in Relion ( Rosenthal and Henderson, 2003 ). To assess the local variability in the quality of the structure a local resolution analysis was employed, also implemented in Relion. The local resolution procedure determined local FSC with a sampling window of 10 Å, using the same negative B-factor obtained at the end of refinement. Model determination The crystal structures of both apo capsid (1qgt)( Wynne et al., 1999b ) and HAP18 bound capsid (5d7y)( Venkatakrishnan et al., 2016 ) were used as starting points for flexible model refinement imposing icosahedral non-crystallographic symmetry constraints, and using the PHENIX and eLBOW software programs ( Afonine et al., 2012 ; Moriarty et al., 2009 ; Emsley et al., 2010 ). The model validation statistics we report were obtained from the final output of the Phenix real space refinement tool. To refine the pixel size of the map, we fit the model of the asymmetric unit into density and measured cross-correlation across pixel sizes. The optimal pixel size was determined to be 1.285 Å, compared to 1.30 Å as determined by the microscope calibration. The final maps and models reflect this change. Structural comparisons and figure creation were carried out in UCSF Chimera. The final structure coordinates are deposited in the protein data bank as 6BVN for the T = 3 structure and 6BVF for the T = 4 structure.

Detection of binding in solution

The final buffer conditions for all binding assays were 300 mM NaCl, 20 mM Tris, 1% DMSO, and pH 7.5 with varying protein and HAP-TAMRA. Size exclusion chromatography assays were performed using a superose 6 30/10 column plumbed into a Shimadzu HPLC equipped with a diode array detector. The HAP-TAMRA absorbance eluting in the capsid fractions was attributed to the capsid bound population. All HAP-TAMRA absorbance eluting later was attributed to free ligand. It is important to note that because the HAP-TAMRA probe is synthesized as a racemic mixture, and because only a single enantiomer is known to bind the HAP pocket, we expected to see at least half of the input HAP-TAMRA eluting as free ligand. When plotting the increase in 520 nm signal in the capsid fraction ( Figure 2d ), the A520 at 7.5 ml was used, corresponding to the pre-established center of the capsid elution volume. Absorbance and fluorescence was measured in a 96-well plate using a 200 µl sample volume by a Biotek Synergy H1 plate reader. Measurements based on fluorescence used an excitation wavelength of 520 nm and monitored emission at 580 nm. For plotting the change in 520/555 nm absorbance ratio, we used the maximum value of the capsid peak at each wavelength. Kinetic assays based on absorbance were sampled in 1 min intervals, where each data point is the value of 520 nm absorbance. To account for the varying presence of excess free probe in the titration time course ( Figure 3b ), the signal is presented as Δ520 nm absorbance compared to a probe-only reference. We defined this as ΔA520 = A520 raw - A520 inert – A520 binding(t=0) where A520 inert accounts for the absorbance of dye that does not participate in binding (because it was either the inactive enantiomer or present in superstoichiometric quantities), and A520 binding(t=0) is the initial absorbance of the fraction of HAP-TAMRA that will participate in binding. This analysis assumes linear binding until saturation, based on the results in Figure 2d , but could underestimate the signal if binding were weaker.

Additional files 10.7554/eLife.31473.017 Transparent reporting form Major datasets The following datasets were generated: Christopher John Schlicksup Joseph Che-Yen Wang Adam Zlotnick 2017 Cryo-EM Structure of Hepatitis B virus T=4 capsid in complex with the fluorescent allosteric modulator HAP-TAMRA http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6BVF Publicly available at the RCSB Protein Data Bank (accession no. 6BVF) Christopher John Schlicksup Joseph Che-Yen Wang Adam Zlotnick 2017 Cryo-EM Structure of Hepatitis B virus T=3 capsid in complex with the fluorescent allosteric modulator HAP-TAMRA http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6BVN Publicly available at the RCSB Protein Data Bank (accession no. 6BVN) Christopher John Schlicksup Joseph Che-Yen Wang Adam Zlotnick 2017 Cryo-EM Structure of Hepatitis B virus T=4 capsid in complex with the fluorescent allosteric modulator HAP-TAMRA http://www.ebi.ac.uk/pdbe/entry/emdb/EMD-7294 Publicly available at the Electron Microscopy Data Bank (accession no. EMD-7294) Christopher John Schlicksup Joseph Che-Yen Wang Adam Zlotnick 2017 Cryo-EM Structure of Hepatitis B virus T=3 capsid in complex with the fluorescent allosteric modulator HAP-TAMRA http://www.ebi.ac.uk/pdbe/entry/emdb/EMD-7295 Publicly available at the Electron Microscopy Data Bank (accession no. EMD-7295)