Temf816 8k 8k cmos camera

Negative Stain Electron Microscopy: uPAR-Fab complexes were prepared by mixing purified uPAR or Fab and uPAR at a 7:1 molar ratio prior to running size-exclusion chromatography on a s200 column using 20 mM HEPES pH 7.5 and 150 mM NaCl as the running buffer. Fractions corresponding to uPAR alone or Fab-uPAR complexes were diluted to a concentration of 0.025 mg/mL. Negative stain EM grids were prepared following established protocols [36 (link)]. Briefly, 2.5 μL of sample was applied to a glow discharged carbon-coated Cu EM grid (Ted Pella Inc., Redding, CA, USA) and stained with 0.75% uranyl formate. The sample was imaged on a T20 microscope (FEI Company, Hillsboro, OR, USA) operated at 200 kV with a nominal magnification of 50,000× using a TemF816 8 K × 8 K CMOS camera (TVIPS GmbH, Gauting, Germany) with a calibrated pixel size of 1.57 Å. All images were further binned by 2 for image processing yielding a pixel size of 3.14 Å. Defocus values were determined using gctf and particles were picked using a Gaussian template with gautomatch [37 (link)]. Particle extraction was done with RELION-2 [38 (link)] and two-dimensional reference-free classification was done using cryoSPARC [39 (link)].

Purified antigens and Fabs were mixed at a 1:1 molar ratio and incubated on ice for one hour. Immediately prior to grid preparation, samples were diluted to either 100 nM for AP2M160 and ESCRT-I complexes or 50 nM for AP2core complexes. Grids of antigen alone and antigen-Fab complexes were prepared for negative-stain EM following established protocols [27 (link)]. Briefly, 2.5 μL of sample was applied to glow-discharged carbon coated Cu EM grids (Ted Pella Inc., Redding, CA) and stained with 0.75% uranyl formate. Negatively stained EM grids were imaged on a T20 microscope (FEI Company) operated at 200 kV with a nominal magnification of 50,000x using a TemF816 8K × 8K CMOS camera (TVIPS GmbH, Gauting, Germany), corresponding to a calibrated pixel size of 1.57 Å on the specimen. All images were binned by 2 for further image processing, resulting in a pixel size of 3.14 Å. Defocus values were determined using gctf and particles were picked using Gautomatch with a Gaussian template [28 (link)]. Two-dimensional class averages were generated with RELION 2 [29 (link)].