Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
[Preprint]. 2025 Jan 7:2024.01.21.576499.
doi: 10.1101/2024.01.21.576499.

MagIC-Cryo-EM: Structural determination on magnetic beads for scarce macromolecules in heterogeneous samples

Yasuhiro Arimura  1   2 , Hide A Konishi  1 , Hironori Funabiki  1
Affiliations

MagIC-Cryo-EM: Structural determination on magnetic beads for scarce macromolecules in heterogeneous samples

Yasuhiro Arimura et al. bioRxiv. .

Abstract

Cryo-EM single-particle analyses typically require target macromolecule concentration at 0.05~5.0 mg/ml, which is often difficult to achieve. Here, we devise Magnetic Isolation and Concentration (MagIC)-cryo-EM, a technique enabling direct structural analysis of targets captured on magnetic beads, thereby reducing the targets' concentration requirement to < 0.0005 mg/ml. Adapting MagIC-cryo-EM to a Chromatin Immunoprecipitation protocol, we characterized structural variations of the linker histone H1.8-associated nucleosomes that were isolated from interphase and metaphase chromosomes in Xenopus egg extract. Combining Duplicated Selection To Exclude Rubbish particles (DuSTER), a particle curation method that excludes low signal-to-noise ratio particles, we also resolved the 3D cryo-EM structures of nucleoplasmin NPM2 co-isolated with the linker histone H1.8 and revealed distinct open and closed structural variants. Our study demonstrates the utility of MagIC-cryo-EM for structural analysis of scarce macromolecules in heterogeneous samples and provides structural insights into the cell cycle-regulation of H1.8 association to nucleosomes.

PubMed Disclaimer

Conflict of interest statement

Declaration of interests YA, HAK, and HF have filed a patent application encompassing aspects of MagIC-cryo-EM (PCT/US2023/03315). HF is affiliated with the Graduate School of Medical Sciences, Weill Cornell Medicine, and Cell Biology Program at the Sloan Kettering Institute.

Figures

Figure 1.
Figure 1.. Single particle cryo-EM analysis of poly-nucleosomes attached to magnetic beads
(A) Schematic of a pilot cryo-EM experiment on magnetic beads. Biotin-labeled 19-mer nucleosome arrays attached to 50 nm streptavidin-coated magnetic nanobeads were loaded onto the cryo-EM grid. (B) Representative medium magnification micrographs. The magnetic beads are seen as black dots (red arrows). (C) Left; a representative high magnification micrograph. The micrograph was motion-corrected and low-pass filtered to 5 Å resolution. Right; green circles indicate the nucleosome-like particles selected by Topaz, and the blue areas indicate the halo-like scattering. (D) The 3D structure of the nucleosome bound on magnetic beads.
Figure 2.
Figure 2.. MagIC-Cryo-EM structural determination of low-quantity and low-purity targets
(A) Schematic depicting the principle steps of MagIC-cryo-EM. (B) Graphical representation of the MagIC-cryo-EM beads with 3HB and SAH spacers and GFP nanobody target capture module. (C) Schematic of MagIC-cryo-EM for in vitro reconstituted H1.8-GFP bound nucleosomes isolated from an excess of H1.8-free nucleosomes. (D) Native PAGE analysis of H1.8-GFP bound nucleosomes and unbound nucleosomes in the input. DNA staining by SYTO-60 is shown. (E) A handmade humidity chamber used for the 5 min incubation of the cryo-EM grids on the magnet. The humidity chamber was assembled using a plastic drawer. Wet tissues are attached to the side walls of the chamber, which is sealed with a plastic cover to maintain high humidity. Two pieces of neodymium magnets are stacked. A graphene grid is held by a non-magnetic vitrobot tweezer and placed on the magnets. 4 μL of sample is applied on the grid and incubated for 5 min. (F) Micrograph montage of the grids without using magnetic concentration. The GFP-nanobody-MagIC-cryo-EM beads (4 μL of 12.5 pM beads) were applied on the graphene-coated Quantifoil R 1.2/1.3 grid and vitrified without incubation on a magnet. (G) Micrograph montage of the grids without using magnetic concentration. The GFP-nanobody-MagIC-cryo-EM beads (4 μL of 12.5 pM beads) were applied on the graphene-coated Quantifoil R 1.2/1.3 grid and vitrified with 5 min incubation on two pieces of 40 x 20 mm N52 neodymium disc magnets. (H) Quantitative analysis of the percentage of holes containing MagIC-cryo-EM beads. Each data point represents the percentage of holes containing MagIC-cryo-EM beads on each square mesh. (I) Quantitative analysis of the average number of MagIC-cryo-EM beads per hole. Each data point represents the average number of MagIC-cryo-EM beads per hole on each square mesh. The edges of the boxes and the midline indicates the 25th, 50th, and 75th percentiles. Whiskers indicate the maximum and lowest values in the dataset, excluding outliers. For the quantification, 11 square meshes with 470 holes without magnetic concentration and 11 square meshes with 508 holes with 5 min incubation on magnets were used. (J) Representative motion corrected micrographs of in vitro reconstituted H1.8-GFP nucleosomes captured by MagIC-cryo-EM beads. The micrographs were low-pass filtered to 10 Å resolution. Green circles indicate the nucleosome-like particles picked by Topaz. (K) 3D structure of the in vitro reconstituted H1.8-GFP-bound nucleosome determined through MagIC-cryo-EM. The pipeline for structural analysis is shown in Figure S2.
Figure 3.
Figure 3.. MagIC-Cryo-EM structural determination of H1.8-bound nucleosomes from interphase and metaphase chromosomes in Xenopus egg extract.
(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 Figure S4A. (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 Figure S5A. (G) Structures of H1.8-bound nucleosomes isolated from interphase and metaphase chromosomes.
Figure 4.
Figure 4.. MagIC-cryo-EM and DuSTER reconstructed cryo-EM structures of interphase-specific H1.8-bound NPM2.
(A) Schematic of DuSTER workflow. (B) 2D classes before and after particle curation with DuSTER. More 2D classes are shown in Figure S10B–S10E. (C) 3D cryo-EM structure of interphase-specific H1.8-containing complex. C5 symmetry was applied during structural reconstruction. The complete pipeline is shown in Figures S8, S10, and S11. (D) MS identification of proteins that cofractionated with H1.8 in sucrose gradient fraction 4 from interphase chromosomes shown in Figure 3C. Portions of MagIC-cryo-EM beads prepared for cryo-EM were subjected to MS. Proteins shown in red are the proteins that comprise the GPF nanobody-MagIC-cryo-EM beads. Proteins shown in blue represent signals from H1.8-GFP. (E) Western blot of NPM2 in the sucrose gradient fractions of interphase and metaphase chromosome fragments. (F) The structural comparison of the crystal structure of the pentameric NPM2 core (PDB ID: 1K5J), and AF2 predicted structure of the pentameric NPM2 core, and MagIC-cryo-EM structures of NPM2-H1.8. The MagIC-cryo-EM structures indicate NPM2 in the NPM2-H1.8 complex forms pentamer.
Figure 5.
Figure 5.. Structural variations of NPM2 bound to H1.8.
(A) Structural differences between the opened and closed forms of NPM2. Left panels show cryo-EM maps of the opened and closed forms of NPM2 with H1.8. Middle panels show the atomic models. The right panel shows the zoomed-in view of the open form (green) and closed form (gray) of the NPM2 protomer. In the closed form, β8 runs straight from the sepal side to the petal side. In the open form, the C-terminal portion of β8 is bent outward to the rim. (B) Putative H1.8 density (red arrow) in the averaged NPM2-H1.8 structure. (C) The NPM2 surface that contacts the putative H1.8 density (corresponding to aa 42-44) is shown in orange. The H1.8-binding sites are accessible in the open form while they are internalized in the closed form. Note that C-terminal acidic tracts A2 and A3 (Figure S13A) are not visible in the cryo-EM structure but are likely to contribute to H1.8 binding as well in both open and closed forms. (D) Model of the mechanism that regulates the amount of the H1.8 in interphase and metaphase nucleosome.
Figure 6.
Figure 6.. Advantages of MagIC-cryo-EM over conventional cryo-EM methods.
(A) The on-bead-cryo-EM approach reduces preparation steps (for example, target isolation, enrichment, and buffer exchange), which can lead to sample loss. (B) Sample loss during the grid-freezing process is reduced by magnet-based enrichment of the targets on cryo-EM grids. (C) The magnetic beads are easily identified in medium - magnification montage maps, enabling the selection of areas where targets exist prior to high-magnification data collection. (D) Targets are highly concentrated around the beads, ensuring that each micrograph contains more than 100 usable particles for 3D structure determination.
See this image and copyright information in PMC

Similar articles

See all similar articles

References

    1. Azinas S., and Carroni M. (2023). Cryo-EM uniqueness in structure determination of macromolecular complexes: A selected structural anthology. Curr. Opin. Struct. Biol. 81, 102621. 10.1016/j.sbi.2023.102621. - DOI - PubMed
    1. Natchiar S.K., Myasnikov A.G., Kratzat H., Hazemann I., and Klaholz B.P. (2017). Visualization of chemical modifications in the human 80S ribosome structure. Nature 551, 472–477. 10.1038/nature24482. - DOI - PubMed
    1. Arimura Y., Shih R.M., Froom R., and Funabiki H. (2021). Structural features of nucleosomes in interphase and metaphase chromosomes. Mol. Cell 81, 4377–4397. 10.1016/j.molcel.2021.08.010. - DOI - PMC - PubMed
    1. Arimura Y., and Funabiki H. (2022). Structural Mechanics of the Alpha-2-Macroglobulin Transformation. J. Mol. Biol. 434, 167413. 10.1016/j.jmb.2021.167413. - DOI - PMC - PubMed
    1. Leesch F., Lorenzo-Orts L., Pribitzer C., Grishkovskaya I., Roehsner J., Chugunova A., Matzinger M., Roitinger E., Belačić K., Kandolf S., et al. (2023). A molecular network of conserved factors keeps ribosomes dormant in the egg. Nature 613, 712–720. 10.1038/s41586-022-05623-y. - DOI - PMC - PubMed

Publication types