Ah 650 rotor

All preparations were performed in the dark and at 4 °C. B. braunii cells were suspended in ice-cold breaking buffer A and supplemented with 1 mM EDTA and a 1× protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). Cells were disrupted in a 15 mL chamber of a Mini-BeadBeater homogenizer (BioSpec Products, Bartlesville, OK, USA) filled halfway with ice-cold 0.5 mm glass beads for 25 cycles of 15 s beating and 2 min of cooling. Cell debris, intact cells, starch granules, and glass beads settled down as a pellet and were removed by centrifugation at 3000× g at 4 °C for 5 min. The resulting supernatant was collected and centrifuged at 32,600 rpm with a Thermo Scientific AH-650 rotor at 4 °C for 30 min. The pellet of thylakoid membranes was suspended in the supplemented breaking buffer A containing 1% (w/v) n-dodecyl β-d-maltoside (DDM) and solubilized under gentle stirring for 15 min. Subsequently, the solubilized thylakoids were centrifuged at 36,200 rpm with a Thermo Scientific AH-650 rotor at 4 °C for 20 min to obtain stroma membranes as a supernatant; the resulting pellet corresponded to the grana preparation and was discarded. The stromal thylakoid supernatant was stored at −80 °C until use.

VLP budding assays were performed as described in [39 (link)]. Briefly, precleared supernatants from 3X-Flag-M transfected HEK 293T were ultracentrifuged through a 20% (w/v) sucrose at 36,000 rpm for 2 h at 4°C (AH-650 rotor, Thermo Scientific). VLP pellets and cells were resuspended in lysis buffer and subjected to SDS-PAGE and anti-Flag immunoblotting. Relative integrated intensity of VLP/cell lysate bands were quantified and normalized relative to the budding of 3X-Flag-NiV-M.

SPMMV virus-like particles purification was based on previously described protocols^{28 (link)} with modifications. Frozen N. benthamiana infiltrated tissue harvested at 5 days post agroinfiltration was homogenized in 3 volumes of 50 mM Sodium Phosphate buffer (pH 7, containing 0.1% β-mercaptoethanol) and clarified at low-speed (8000 × g, 15 min, 4 °C) before been subjected to ultracentrifugation (100,000 × g, 90 min, 4 °C) in a swinging bucket SureSpin 630 Rotor (Thermo Scientific, Waltham, USA). The pellet was resuspended overnight in phosphate buffer, clarified (2000 × g, 10 min, 4 °C) and loaded on a sucrose gradient 10–40% w/v for ultracentrifugation (100,000 × g, 3 h, 4 °C), in a swinging bucket AH-650 Rotor (Thermo Scientific, Waltham, USA). Fractions of 500 μl were collected and analysed in a 12% SDS-PAGE gel, stained with InstantBlue (Abcam, Cambridge, UK) and subsequent TEM imaging. For cryoEM studies, fractions with the higher VLPs-content were pooled and the excess of salt removed using PD-10 desalting column (GE Healthcare, Chalfont St Giles, UK) equilibrated with sodium phosphate buffer. As above, samples were submitted to cryoEM and other analysis.