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)].
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