Structure studio

Purified NbA1 (30 mg/ml) was incubated with the V5 peptide in a 1:3 ratio (protein: peptide). The protein complex was crystallized via the sitting drop vapor diffusion method at 4 °C with a mother liquor composed of Bis-Tris pH 6.5, 19% (w/v) PEG 3350, and 20% (v/v) ethylene glycol. The crystals were flash-frozen in liquid nitrogen, and a full data set was collected using a Rigaku MicroMax-007HF equipped with a copper anode. Images were collected using an R-Axis IV++ detector (Rigaku) and processed using Structure Studio (Rigaku). The structure was solved by molecular replacement using the structure of NbALFA (PDB 6I2G) as search model and Phaser (50 (link)). Following several rounds of NbA1 building and refinement using COOT and Phenix, respectively, V5 was built in the positive Fourier map. The model was completed by adding the molecules and truncating side chains for which no electronic density could be observed. Ramachandran statistics: Nonglycine Ramachandran outliers; 0%, Nonglycine Ramachandran favored; 100%, Molprobity score: 1.65. Statistics of data collection and refinement are summarized in Table1. All structural figures were prepared in PyMOL.

Aggregates of P23T hγD formed under acidic (pH 3) and neutral (pH 7) conditions were pelleted by centrifugation. Aggregates formed under neutral conditions were pelleted at 14,000 g for 20 min in a table-top VWR Galaxy 14D centrifuge, while aggregates formed under acidic conditions were pelleted by ultracentrifugation at 100,000 g for 4 h (Beckman Coulter Optima MAX Ultracentrifuge, with TLA 120.2 rotor). The majority of the supernatant was removed, and the pellet was resuspended in the remaining buffer and packed into a glass capillary (0.7 mm) using a syringe. Capillaries were sealed with wax to retain hydration of the samples. Diffraction data were measured at room temperature with a Rigaku Saturn 944 CCD camera (Tokyo, Japan). A Rigaku FR-E generator (2 kW, spot size 0.07 mm) was used as the X-ray source. Aggregates formed under neutral conditions were exposed for 75 s, while aggregates formed under acidic conditions were exposed for 210 s. Diffraction data were processed and analysed in Structure Studio (Rigaku).