Formvar and carbon

PBS containing 50 mM DTT (5 μl) was pipetted directly onto a freshly glow-discharged copper grid that had been coated with formvar and carbon (Electron Microscopy Sciences); the PBS was then blotted off with Whatman #1 filter paper. Next, 5 μl of particle preparation, the plasma membrane preparation, or PBS alone was pipetted onto the grid and allowed to adsorb for 1 min before blotting off with filter paper. Next, 5 μl of 2% uranyl acetate was pipetted onto the grid and blotted off, followed by another 5 μl of 2% uranyl acetate, which was allowed to incubate for 1 min before being blotted off. Grids were imaged using an FEI Tecnai T12 set to 120 kV accelerating voltage equipped with a Gatan 2k × 2 k CCD detector.

TEM images were recorded with FEI Tecnai T12 G2 TWIN transmission electron microscope operating at 120 kV. Samples of SNPs dispersions were deposited on a copper 300 mesh grid, coated with Formvar and carbon (Electron Microscopy Sciences, Fort Washington, PA, USA) and dried for 5 min. Samples were then stained with 1% uranyl acetate solution. Images were taken using TemCam—F214 (Gauting, Germany).

For TEM imaging, samples of protein were diluted to a concentration of 3.8 mg/mL in dialysis buffer and deposited onto a TEM grid (100 mesh copper grid coated with formvar and carbon, Electron Microscopy Sciences), and negatively stained with NanoW (Nanoprobes). They were examined in a Tecnai T-12 transmission electron microscope (FEI). The ultrastructural observations were performed with an aberration corrected FEI Titan 80–300 transmission electron microscope. The observations were made in S/TEM mode using a high angle annular dark field (HAADF) detector. The probe convergence angle was set to 18 mrad and the inner detection angle on the HAADF detector was set to 52 mrad. For high resolution TEM, samples were diluted to 70 μg/mL (Mnx complex) or 35 μg/mL (MnxEF). When protein samples were visualized in the absence of Mn, an aliquot of NanoW (Nanoprobes) was added as a negative stain. Particle sizes were measured using ImageJ software^{64 (link)}. Over 100 particles of MnxEF and over 1000 particles of Mnx complex were measured.

For the oligomer TEM samples, thermal denaturation spin‐down assays were run using citrate synthase. 100 μl of 3.8 μM protein and 7.6 μM ssDNA were thermally denatured together at 60°C for 15 min in 10 mM sodium phosphate, pH 7.5 buffer. The resulting solution was then centrifuged at 16,100 g for 15 min at 4°C to separate the soluble and insoluble fractions. The soluble portion was pipetted off and transferred on ice for TEM analysis.
A positively charged copper mesh grid coated in formvar and carbon (Electron Microscopy Sciences) using the PELCO easiGlow Discharge system was used for each soluble sample. The charged copper grids had 5 μl of sample applied for 20 s and then lightly blotted off using a Whatman filter paper. The grids were then rinsed using 2 drops of MilliQ water, with filter paper blotting for each wash. Finally, the grids were then stained using two drops of a 0.75% uranyl formate solution. The first drop served as a quick wash, followed by 20 s of staining using the second drop. The grids were then blotted and allowed to dry. The TEM images were captured using a FEI Tecnai G2 Biotwin TEM at 80 kV with an AMT side‐mount digital camera. In order to better visualize the intricacies of each oligomer, the images’ contrast and brightness was uniformly enhanced using Adobe Photoshop.