Glow discharged carbon coated copper grid

Microscopy was conducted at the Emory University Robert P. Apkarian Integrated Electron Microscopy Core. A freshly purified fraction 10 from a Superose 12 purification was diluted 1:100 to approximately 0.01 mg/mL in 10 mM Na2HPO₄/KH₂PO₄ pH 7.2, 200 mM NaCl and 0.02% DDM. These diluted samples (5 μl) were absorbed for 1 min onto glow-discharged, carbon-coated copper grids (Electron Microscopy Sciences, Hatfield, PA, USA). Grids were blotted, washed with three drops of buffer and negatively stained on a drop of freshly prepared and filtered 2% uranyl acetate solution. Samples were imaged at a nominal magnification of 40000x using a JEOL JEM-1400 transmission electron microscope (JEOL, Japan) operating at 80 kV. Electron micrographs were acquired on a 2048 by 2048 charge-couple device camera (UltraScan 1000, Gatan Inc, Pleasanton, CA, USA), yielding a pixel size of 2.6 Å at the specimen level. Reference-free 2D classification was carried out on particles selected using the automated boxing procedure on the EMAN2 images processing software package(Ludtke et al., 1999 (link)). Over 2000 particles were extracted from the micrographs, aligned, and 2D class averaged on EMAN2 to generate the images presented herein. 2D class averages were imported into 3dmod 4.9.12 (part of the IMOD software)(Kremer et al., 1996 (link)) and particle diameter was measured on images of the 2D classes.