Morgagni electron microscope

Thin-sections were post-stained with 2% lead citrate in water and examined using a Morgagni electron microscope (FEI). Thick sections (300 nm) of HPF-FS samples were placed on a slot grid covered with a formvar film and decorated with 10 nm protein-A gold particles on both sides for image alignment. Grids were placed in a high-tilt holder (Fischione Model 2020) and dual-axis ET were carried out using a Tecnai F30 (FEI) electron microscope (operated at 300 kV) equipped with a field emission gun and a 4084×4084 pixels CCD camera (Eagle, FEI). Tomographic tilt ranges were typically from +60° to −60° with an angular increment of 1° pixel size ranging from 0.5 to 1 nm. Alignments, 3D reconstructions, and merging of serial tomograms were done with IMOD software suite [53] (link). The volume segmentations were performed with the Amira 4.1 visualization package (Visage Imaging, Berlin, Germany).

Virus samples were diluted to 0.5 mg/ml in PBS and heated at 1°C/min to 52 or 55°C, cooled to 4°C, and 8 μl were applied to glow discharged carbon-coated grids and left for 10 min. The grids were washed with 2% sodium phosphotungstic acid (PTA), pH 7.6, followed by 10 min incubation with PTA and blotting with filter paper. The stained samples were imaged in a FEI Morgagni electron microscope (80 kV, 110 k magnification). Identification of (sub)viral particles was as in Kumar and Blaas (2013) (link); native virions appear as impermeable bright spheres. A particles are permeable for the stain, making them appear as bright rings with a grainy interior. Empty B particles present as bright circles encasing a uniformly dark core stemming from the negative stain accumulating in the void (Figure 2D - white arrows). In total, ten distinct fields from two independent treatments were evaluated (∼1000 particles per treatment), and the mean and standard deviation calculated. Two-way ANOVA with multiple correlation analysis reveals that the difference from 52 to 55°C is a statistically significant change in the population of all the subviral particles (p < 0.0001).

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.

The purified Holliday junction complex was checked for homogeneity by screening negatively-stained samples on a Morgagni electron microscope (FEI, Hillsboro, Oregon). For cryo preparation, we applied 3.0 μL of complex solution to a C-flat 1.2/1.3 Cu grid (400 mesh) (Protochips, Raleigh, NC), which had been glow discharged at 20 mA for 30 s. Grids were plunge-frozen with a Vitrobot Mark II (FEI) at 85% humidity, offset -2, blot time 7 s. Images were recorded on a Tecnai F30 electron microscope (FEI) operated at 300 kV and using a liquid-nitrogen cooled 626 cryo-specimen holder (Gatan Inc., Pleasanton, CA). We used the semi-automated acquisition program SerialEM (Mastronarde, 2005 (link)) to record 1359 movies with a Falcon II direct detector (FEI) at an underfocus set between 2.7 – 4.2 μm. Each movie consisted of 25 frames, collected in a 2 s exposure of 35.5 electrons/Ų. The nominal magnification was 78,000x, corresponding to a magnification of 100,000x on the detector and 1.4 Å pixel size on the specimen. Movie frames were aligned and summed using motioncorr (Li et al., 2013 (link)).

Late stage embryos of the desired genotype were prepared for TEM analysis essentially as described [65 (link)]. Following washing with 0.1 M cacodylate buffer, tissue was stained with 1% osmium tetroxide for 1 hour, dehydrated in a graded ethanol series and propylene oxide and embedded in Poly/BedR 812 (Polysciences, Inc., Eppelheim, Germany). Semi-thin cross sections of each 1 μm were made on a Leica Ultracut S ultramicrotome. Sections were stained with toluidine blue for 3 minutes at 70°C. Image acquisition was on a Zeiss Axiophot microscope system. Ultrathin sections were contrasted with uranyl acetate and lead citrate and examined with a FEI Morgagni electron microscope. Digital images were taken with a Morada CCD camera and the iTEM software (Olympus Soft Imaging Solutions GmbH, Münster, Germany).