Nativemark

Measurements were performed in buffer E (Table S2) using an One^MP mass photometer (Refeyn, Oxford, UK) (Young et al., 2018 (link)). Immediately before analysis, the sample was diluted 1:10 with the aforementioned buffer. Molecular mass was determined in the analysis software provided by the manufacturer using a NativeMark (Invitrogen).

BN-gels were performed using a mini VE cell electrophoresis apparatus (Amersham Pharmacia) according to the method of Schägger [32] (link), [33] (link), as described by Guiral et al. [34] (link) except that the intensity was 10 mA/gel and the voltage 250 V. Running times were 3 h. The gels were run with the blue cathode buffer (50 mM Tricine, 7.5 mM imidazole pH 7.0, 0.02% Coomassie Blue G 250 (Serva Blue G)) until the front line had entered into half of the gels and then the cathode buffer was replaced by the one that contained only 0.002% Serva Blue G. The anode buffer was constituted of 25 mM imidazole pH 7.0. The apparent molecular mass of proteins was estimated using the native molecular mass markers NativeMark (Invitrogen).

Chemicals and reagents were purchased from Sigma‐Aldrich (Steinheim, Germany) unless otherwise stated. Acetonitrile (ACN) was purchased from Biosolve (Valkenswaard, The Netherlands). Sequencing grade trypsin was obtained from Promega (Madison, WI). NativePAGE 3–12% Bis‐Tris protein gels, NativePAGE sample buffer, NativePAGE Running Buffer, NativePAGE Cathode Additive, and NativeMark were purchased from Invitrogen (California, USA). Criterion XT Bis‐Tris Precast Gels (4–12%), XT MOPS Running Buffer, and sample buffer were purchased from Bio‐Rad (California, USA). DSSO was produced in‐house according to a previous protocol (Kao et al, 2011). Oasis HLB 96‐well µElution Plates were purchased from Waters (Massachusetts, USA). GroEL and the major capsid protein gp23 were expressed and purified as previously described. (Quaite‐Randall & Joachimiak, 2000; van Duijn et al, 2005; van Duijn et al, 2006) Complement components C5, C6, and C5b6 were purchased from CompTech (Texas, USA). The fresh bovine heart was obtained from a slaughterhouse.

Mass Photometry was performed in the mass photometry buffer (MP) containing 30 mM HEPES pH 7.8, 150 mM NaCl, 5% glycerol, 1 mM MgCl₂ and 1 mM TCEP. Protein samples (3 μM) were pre-equilibrated for 1 h in the MP buffer. Measurements were performed using Refeyn One (Refyn Ltd, Oxford, UK) mass photometer. Directly before the measurement, the sample was diluted 1:100 with the MP buffer. Molecular mass was determined in Analysis software provided by the manufacturer using a NativeMark (Invitrogen) based standard curve created under the identical buffer composition.

Whole parasite or previously aliquoted enriched mitochondria fraction were mixed with solubilisation buffer (750 mM aminocaproic acid, 50 mM Bis‐Tris–HCl pH 7.0, 0.5 mM EDTA, 1 % (w/v) dodecyl maltoside) and incubated on ice for 5 min. The mixture was centrifuged at 16,000 × g at 4°C for 10 min. The supernatant containing solubilised membrane proteins was transferred into a new microcentrifuge tube and 0.25% (w/v) Coomassie G250 (final concentration) was added. The anode buffer (50 mM Bis‐Tris–HCl pH 7.0) and cathode buffer (50 mM Tricine, 15 mM Bis‐Tris–HCl pH 7.0, 0.02% Coomassie G250 (Serva)) were poured into their respective tank compartment and the appropriate amount of protein (55 μg or 5x10⁵ parasites) was loaded per lane on a NativePAGE™ 4–16% or 3–12% Bis‐Tris Gel (Novex‐ Life technologies). 5 μl NativeMark™ (Invitrogen) was used as a molecular weight marker. Gels were run for ~45 min at 100 V, 4–10 mA at 4°C with cathode buffer containing 0.02% (w/v) Coomassie G250, then for ~2.5 h at 250 V, 15 mA with cathode buffer containing 0.002% (w/v) Coomassie G250, until the dye front reached the bottom of the gel.