Solarus model 950 cleaner

Recombinant TCR B12A and CD155(Sino biological, Inc.) proteins were dialyzed against Hepes buffer (10 mM HEPES, 150 mM NaCl pH 7.5), respectively. To prepare TCR B12A-CD155 complex samples, equal volumes of TCR B12A (4mg/ml) and CD155(4mg/ml) were mixed and chemically cross-linked with 0.04% glutaraldehyde for 30 minutes on ice. The cross-linking reaction was quenched by addition of Tris buffer(pH8) to a final concentration of 10mM. The mixture was subsequently subjected to size-exclusion chromatography (Superdex200 10/300, GE Healthcare) and proteins corresponding to molecular weight of ~100kDa were collected to fractions of 0.5ml. To prepare samples for negative-stained electron microscopy, each fraction was adsorbed to plasma-cleaned (Solarus Model 950 cleaner, Gatan) electron microscope grids coated with continuous carbon film, which were subsequently washed with Hepes buffer and stained with 0.75% uranyl formate. Images were collected using EPU software (FEI) on a Tecnai T12 electron microscope (FEI) fitted with a 4K charge-coupled device camera (Gatan) at an effective pixel size of 0.18 nm in the specimen plane with the magnification of 110K or 67K.

Recombinant NA and Fabs were diluted with buffer (5mM HEPES, 150 mM NaCl pH 7.3) to approximately 0.02 mg/mL and 0.04 mg/mL respectively. To prepare Fab bound samples, equal volumes of NA and Fabs 1F2 or 4F11 were mixed and incubated for 5–10 minutes. The samples were adsorbed to plasma cleaned (Solarus Model 950 cleaner, Gatan Inc., Pleasanton, CA) EM grids coated with continuous carbon film that were subsequently washed with buffer and stained with 0.75% uranyl formate. Images were collected using EPU software (FEI Company, Hillsboro, OR) on a Tecnai T12 electron microscope (FEI Company) fitted with a 4K CCD camera (Gatan Inc.) at an effective pixel size of 0.18 nm in the specimen plane. The software package RELION 1.4 (50 (link)) was used to obtain 3D reconstructions. The maps for unbound NA, and for the complexes with 1F2, and 4F11 were constructed using 47,592, 4,326, and 13,665 particles, respectively and visualized using UCSF Chimera software (51 (link)).