Journal: eLife
Article Title: MagIC-Cryo-EM, structural determination on magnetic beads for scarce macromolecules in heterogeneous samples
doi: 10.7554/eLife.103486
Figure Lengend Snippet: ( A ) Models of potential cell cycle-dependent H1.8 dynamic binding mechanisms ( B ) Experimental flow of MagIC-cryo-EM analysis for GFP-H1.8 containing complexes isolated from chromosomes assembled in interphase and metaphase Xenopus egg extract. Fluorescence microscopy images indicate localization of GFP-H1.8 to interphase and metaphase chromosomes. DNA and GFP-H1.8 were detected either by staining with Hoechst 33342 or GFP fluorescence, respectively. ( C ) Native PAGE of fragmented interphase and metaphase chromosome sucrose gradient fractions. GFP-H1.8 and DNA were detected with either GFP fluorescence or SYTO-60 staining, respectively. ( D ) Western blot of GFP-H1.8 in interphase and metaphase chromosome sucrose gradient fractions. GFP-H1.8 was detected using anti-GFP antibodies. ( E ) SDS-PAGE of the sucrose gradient fractions 4 and 5 shown in ( C ), demonstrating heterogeneity of the samples. Proteins were stained by gel code blue. Red arrows indicate the H1.8-GFP bands. The full gel image is shown in . ( F ) In silico 3D classification of interphase and metaphase H1.8-bound nucleosomes isolated from chromosomes in Xenopus egg extract. To assess the structural variations and their population of H1.8-bound nucleosomes, ab initio reconstruction and heterogenous reconstruction were employed twice for the nucleosome-like particles isolated by the decoy classification. The initial round of ab initio reconstruction and heterogenous reconstruction classified the particles into three nucleosome-containing 3D models ( A, B, C ). Subsequent ab initio reconstruction and heterogenous reconstruction on the class A, which has weak H1.8 density, yielded three new nucleosome-containing structures, A1, A2, and A3. 3D maps represent the structural variants of GFP-H1.8-bound nucleosomes. Red arrows indicate extra densities that may represent H1.8. Green densities indicate on-dyad H1.8. The bar graphs indicate the population of the particles assigned to each 3D class in both interphase and metaphase particles (gray), interphase particles (blue), and metaphase particles (red). The pipeline for structural analysis is shown in . ( G ) Structures of H1.8-bound nucleosomes isolated from interphase and metaphase chromosomes. Figure 3—source data 1. Full images of gels and membranes shown in . (A) Full gel images used in . (B) Full membrane image used in . (C) Full gel image used in . Figure 3—source data 2. Raw images of gels and membranes shown in .
Article Snippet: For , as primary antibodies, mouse monoclonal antibody against GFP (Santa Cruz Biotechnology, # sc-9996, 1:1000 dilution) and rabbit polyclonal antibody against X. laevis H1.8 ( ; final: 1 μg/mL) were used.
Techniques: Binding Assay, Cryo-EM Sample Prep, Isolation, Fluorescence, Microscopy, Staining, Clear Native PAGE, Western Blot, SDS Page, In Silico, Membrane
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