Tecnai f30 transmission electron microscope

The emission scanning electron microscope (SEM) images were taken using a 20 kV MIRA4 LMH field emission SEM (TESCAN, Brno, Czech). Transmission electron microscopy (TEM) images were observed with a FEI Tecnai F30 transmission electron microscope (FEI, Hillsboro, OR, USA) operating at 100 kV. Crystal structures were determined with the aid of a SmartLab3 kW X-ray diffractometer (XRD) with Cu Kα radiation (λ = 1.5406 Å) at 40 kV and 30 mA (Rigaku, Tokyo, Japan). The Renishaw inVia Raman microscope (Renishaw, UK) and UV-3600iPLUS UV-vis-NIR spectrophotometer (Shimadzu Scientific Instruments, Kyoto, Japan) were used to acquire the Raman spectra and the extinction spectra, respectively. Nicolet iS50 (Thermo Fisher Scientific, Waltham, MA, USA) FTIR spectra were obtained for this study. XPS test was performed on Thermo Scientific K-Alpha using an Al Kα excitation source at 6 mA and 12 kV (Thermo Fisher Scientific, USA). A TCP-5100-VDV inductively coupled plasma–mass spectrometer (ICP-MS) was used to analyze the composition quantitatively (NYSE: A, Malaysia). Malvern Zetasizer Nano ZSE analyzer was used to determine the hydrodynamic size and zeta potential (Malvern, UK).

Immune complex were prepared by mixing CVA6 A-particle (1.4 mg ml⁻¹) with 1.2-fold excess of Fab-1D5 (MW = ~50 kDa, 108 a.a. and 119 a.a. corresponding to light and heavy chains of the variable domain) fragment (equivalent to a molar ratio of 1:72), then incubated at 37 °C for 2 h. Aliquots (3 μl) of purified samples of procapsid, A-particle or immune complex were deposited onto glow discharged holey carbon Quantifoil Cu grid (R2/2, 200 mesh, Quantifoil Micro Tools) inside an FEI Mark IV Vitrobot at a humidity level of 100%. After 6s blotting, the grid was plunge-frozen into liquid ethane cooled by liquid nitrogen, and then examined under low-dose conditions at 300 kV with an FEI Tecnai F30 transmission electron microscope. All images were recorded on a Falcon II direct electron detector (seven-frame movie mode) with the defocus settings ranging between 1.5 and 3.0 μm underfocus and at a nominal magnification of 93,000 (corresponding to a pixel size of 1.128 Å). The total electron dose was set to 25 e⁻ Å⁻² with an exposure time of 1 s. The FEI EPU automated data collection software was used for all data acquisition, and micrographs with excessive drift or astigmatism were discarded. A total of 312 micrographs for CVA6 procapsid, 203 micrographs for the A-particle and 1085 micrographs for the immune complex were selected for further image processing.

Colloidal gold particles (15 nm; Sigma-Aldrich) were attached to both sides of semi-thick sections collected on copper slot grids to serve as fiducial markers for subsequent image alignment. For dual-axis electron tomography58 (link), series of tilted views were recorded using a TECNAI F30 transmission electron microscope (FEI Company, Eindhoven, The Netherlands) operated at 300 kV. Images were captured every 1° over a ±60° range and a pixel size of 2.3 nm using a Gatan US1000 CCD camera (2k × 2k). For each serial section two montages of 2 × 3 frames were collected and combined to a supermontage using the IMOD software package to cover the pole-to-pole distance of the spindles59 (link). For image processing, the tilted views were aligned using the positions of the Colloidal gold particles as fiducial markers. Tomograms were computed for each tilt axis using the R-weighted back-projection algorithm60 (link). For double-tilt data sets two montages, each consisting of six tomograms, were aligned to each other and combined to a supermontage58 (link). To cover a large volume of the pole-to-pole region of each mitotic spindle, we recorded on average 24 consecutive serial sections per spindle.