Functionalized graphene-oxide grids enable high-resolution cryo-EM structures of the SNF2h-nucleosome complex without crosslinking

Abstract

Single-particle cryo-EM is widely used to determine enzyme-nucleosome complex structures. However, cryo-EM sample preparation remains challenging and inconsistent due to complex denaturation at the air-water interface (AWI). Here, to address this issue, we develop graphene-oxide-coated EM grids functionalized with either single-stranded DNA (ssDNA) or thiol-poly(acrylic acid-co-styrene) (TAASTY) co-polymer. These grids protect complexes between the chromatin remodeler SNF2h and nucleosomes from the AWI and facilitate collection of high-quality micrographs of intact SNF2h-nucleosome complexes in the absence of crosslinking. The data yields maps ranging from 2.3 to 3 Å in resolution. 3D variability analysis reveals nucleotide-state linked conformational changes in SNF2h bound to a nucleosome. In addition, the analysis provides structural evidence for asymmetric coordination between two SNF2h protomers acting on the same nucleosome. We envision these grids will enable similar detailed structural analyses for other enzyme-nucleosome complexes and possibly other protein-nucleic acid complexes in general.

Fig. 1. Functionalized GO grids protect SNF2h-nucleosome complexes from the air-water interface.

a Schematic illustrating functionalized graphene-oxide (GO) grids. GO is layered on top of a commercial Quantifoil holey carbon grid. The GO surface is then functionalized with either single-strand DNA (ssDNA) or thiol-poly(acrylic acid-co-styrene) (TAASTY) co-polymer to attract SNF2h-nucleosome complexes away from the air-water interface. b Representative micrograph out of 7423 micrographs of the SNF2h-nucleosome complex on a ssDNA GO grid. Representative 2D classes of particles picked from a ssDNA GO grid are also shown. c Chemical structure of the TAASTY co-polymer. d Representative micrograph out of 2,020 micrographs of the SNF2h-nucleosome complex on a TAASTY GO grid. Representative 2D classes of particles picked from a TAASTY GO grid are also shown.

Fig. 2. High-resolution SNF2h-nucleosome structures determined using functionalized GO grids.

a Coulomb potential map of a consensus SNF2h-nucleosome complex at ~2.3 Å global resolution determined with datasets collected on ssDNA GO and TAASTY GO grids. In all figures, histones H3, H4, H2A, and H2B are colored as light blue, green, yellow, and orange, respectively. SNF2h is colored in darker blue. The two strands of DNA are colored as light and dark gray. b Coulomb potential map of SNF2h bound to nucleosome at the inactive position at ~2.7 Å resolution. A filtered map at lower contour is shown as a shadow with the position of flanking DNA marked. c Coulomb potential map of the double-bound SNF2h-nucleosome complex at ~2.8 Å resolution. A filtered map at lower contour is shown as a shadow with the position of flanking DNA marked.

Fig. 3. SNF2h rocks on the nucleosome and intermittently contacts SHL6.

a Two coulomb potential maps representing the endpoints of eigenvector 2 from 3D variability analysis of the single-bound SNF2h-nucleosome particles. On one end (structure 2A), SNF2h is stably bound to nucleosome and contacts DNA at the SHL6 position. On the other end (structure 2B), SNF2h becomes more dynamic and dissociates from DNA at SHL6 while rocking slightly upward toward the histone octamer core. b Density for nucleotide in structures 2A (threshold level = 0.37) and 2B (threshold level = 0.23). Clear extra density is observed for Mg²⁺ and BeF_x ions in structure 2A (denoted as gray and green circles, respectively), but in structure 2B only possible density for Mg²⁺ is observed (gray circle). c (left) ATP hydrolysis assays visualizing hydrolysis using thin-layer chromatography. In the experiments shown, [nucleosome] = 320 nM, [SNF2h] = 15 nM, and [γ-³²P-ATP] was in trace. SNF2h^SHL6_ALA hydrolyzes ATP at a much slower timescale compared to wild-type SNF2h. Source data are provided as a Source Data file. (right) ATPase rate constants determined for wild-type SNF2h and SNF2h^SHL6_ALA at [nucleosome] = 80 nM or 320 nM. Data are presented as mean values +/- SD. n = 3 independent ATPase experiments. Source data are provided as a Source Data file. d Model for SNF2h conformational changes during ATP-dependent translocation of DNA across a nucleosome. SNF2h is initially in a ground state stably bound to both ATP and DNA at SHL6. SNF2h then rocks upwards and detaches from DNA at SHL6, entering a state primed for ATP hydrolysis (step 1). SNF2h hydrolyzes ATP, which causes the top ATPase lobe of SNF2h to shift upward and promote partial translocation of DNA (step 2). Exchange of ADP for ATP finishes DNA translocation and resets SNF2h for subsequent rounds of ATP-dependent remodeling (step 3).

Fig. 4. Coordinated asymmetric actions of SNF2h protomers on a nucleosome.

a Two coulomb potential maps representing the endpoints of one principal component from 3D variability analysis of the double-bound SNF2h-nucleosome particles. On one end (structure db-2A), one SNF2h is stably bound to the nucleosome, while the other SNF2h is more dynamic. On the other end (structure db-2B), the SNF2h that was stably bound to the nucleosome becomes more dynamic, while the SNF2h that was more dynamic becomes more static. b (left) Nucleosome remodeling assays visualizing SNF2h-mediated centering of end-positioned nucleosomes using native PAGE. Top and bottom symbols to the right of the gels denote centered and end-positioned nucleosomes, respectively. In the experiments shown, [nucleosome] = 15 nM, [SNF2h] = 500 nM, and [ATP] = 4 mM. The bottom two time courses are both with the mutant SNF2h^SHL6_ALA, with one containing longer time points. Source data are provided as a Source Data file. (right) Quantification of the percent of nucleosomes that remain end-positioned at equilibrium for wild-type SNF2h and SNF2h^SHL6_ALA. Data are presented as mean values +/- SD. n = 3 independent nucleosome remodeling experiments. Source data are provided as a Source Data file. c Model for SNF2h conformations while coordinating actions on a nucleosome based on all available data. In the DNA-length sensing state, each SNF2h protomer can either be in a static ground state or dynamic pre-activated state while sensing for flanking DNA using its HAND-SANT-SLIDE (HSS) domain. The protomer that is able to sense flanking DNA will undergo further conformational change to reach an activated state that promotes ATP hydrolysis and DNA translocation. Exchange of ADP for ATP resets SNF2h to a ground state, and the other SNF2h protomer then has first priority to again search for flanking DNA in a dynamic, pre-activated state.