Fig. 1.

AFM (a and c) and EM (b) images of ORF3 protein–fibrillarin complexes formed in vitro. (a and b) Mixtures of fibrillarin with wild-type ORF3 protein. (c) Mixture of fibrillarin with the ORF3 L149A mutant, which does not interact with fibrillarin [similar images were obtained when wild-type ORF3 protein was mixed with fibrillarin mutant lacking the GAR domain (Fib2ΔGAR),¹¹ which does not interact with the ORF3 protein]. The recombinant ORF3 protein (ORF3–His; 10 ng) and fibrillarin were mixed in 15 μl of buffer A (10 mM Hepes–KOH, pH 7.6, 100 mM KCl) in a 1:1 molar ratio (15 ng/μl) and incubated at room temperature for 30 min. (a and c) For AFM, mixtures of fibrillarin with ORF3 protein were diluted to ∼ 5 ng/μl in deionized water and 5–10 μl was placed onto freshly cleaved mica strips for 5–15 min. The strips were rinsed with water and dried at room temperature. Imaging of complexes was done in tapping mode by using a Nanowizard® BioAFM (JPK, Berlin, Germany). Silicon beam cantilevers (Veeco Instruments Ltd., Cambridge, UK) with a nominal spring constant of 40 N/m and a resonant frequency of 300 kHz were used. All the AFM experiments were performed in air at room temperature, and the images were captured in constant height mode by using a scan speed of 0.5 Hz. Images, including two-dimensional (2d) and three-dimensional (3d) representations, were processed using JPK software and transferred to Adobe Photoshop for layout. Sample heights and lengths were measured automatically using the JPK software. Cross sections were made around the ring structures along lines connecting centers of granules in the anticlockwise direction starting from granule 1 (as indicated by arrows) to illustrate the heights of the complexes. Periodical height variations represent complexes containing six to eight granules arranged into ringlike structures. (b) For EM, complexes of the ORF3 protein and fibrillarin were negatively stained with 2% sodium phosphotungstate (pH 7.0). The specimens were examined and photographed in a Phillips CM 10 transmission electron microscope. Scale bars represent 20 nm.