Nanosprint5 camera

Wandering 3^rd instar larvae were dissected and fixed in 1% glutaraldehyde and 4% paraformaldehyde in 1% (0.1M) sodium cacodylate buffer overnight at 4°C. Samples were postfixed in 1% osmium tetroxide and 1.5% potassium ferrocyanide for 1 h, then 1% aqueous uranyl acetate for 0.5 h. Stepwise dehydration was conducted for 10 min each in 30%, 50%, 70%, 85%, and 95% ethanol, followed by 2× 10 min in 100% ethanol. Samples were transferred to 100% propylene oxide for 1 h, then 3:1 propylene oxide and 812 TAAB Epon Resin (epon, TAAB Laboratories Equipment Ltd.) for 1 h, then 1:1 propylene oxide:epon for 1 h and then left overnight in a 1:3 mixture of propylene oxide:epon. Samples were then transferred to fresh epon for 2 h. Samples were then flat-embedded and polymerized at 60°C for 48 h, and remounted for sectioning. 70-μm-thin sections were cut on a Leica UC6 Ultramicrotome (Leica Microsystems), collected onto 2×1 mm slot grids coated with formvar and carbon, and then poststained with lead citrate (Venable and Coggeshall, 1965). Grids were imaged using a FEI Morgagni transmission electron microscope (FEI) operating at 80 kV and equipped with an AMT Nanosprint5 camera.

wt IcmF, Q341A IcmF, or K344A IcmF was thawed on ice and diluted to 20 ng/μL in IcmF SEC buffer (20 mM Hepes pH 8, 50 mM NaCl). Each of the samples except samples with GTP were incubated for 30 min on ice with the corresponding nucleotide (GDP or GMPPCP) before grid preparation. The final concentration of any additives (nucleotides: GDP, GTP, or GMPPCP; cofactor: AdoCbl or OHCbl; MgCl₂) in the samples were 500 μM. For the samples containing GTP, the GTP was added, and the protein solution immediately applied to the grid. For the samples containing AdoCbl and exposed to light, after incubation for 30 min in the dark, the protein solution was then exposed to a white light for 15 min before application on the grid.
Carbon-coated 300 mesh copper EM grids (Electron Microscopy Services) were glow discharged for 1 min at −15 mA. An aliquot (5 μl) of the protein solution was applied to the grid; after approximately 1 min, the solution was blotted and immediately replaced with solution of 2% uranyl acetate (VWR). The stain solution was blotted and replaced twice, then allowed to stand for 1 min before the final blot, and then was dried. All blotting was done manually using filter paper (Whatman, grade 40). The specimens were imaged with an AMT Nanosprint5 camera on a FEI Morgagni electron microscope operated at 80 kV. Images were collected at 18,000× magnification.