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Leschziner et al. 10.1073/pnas.0700706104.

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Fig. 5. Microscopy data. (A) Areas of micrographs from negatively stained RSC prepared by the sandwich method, collected at -45° and + 45°. (B) Examples of plots of distribution of in-plane rotation angles for images in a "good" (Left) and a "bad" (Right) class. The plots are equivalent to viewing the "tilted" images (i.e., those used for reconstruction) along their common axis of rotation (which is normal to the plane of the class average). Each line seen in the plots corresponds to the edge of an image. The plot on the right shows a large wedge of missing images, indicating a non-random distribution of in-plane rotation angles. (C) Maps of Euler angle distribution of the 0° data after projection-matching refinement of the open (Left) and closed (Right) conformations of RSC. The plots show a hemisphere of Euler space. The location of each point represents the set of Euler angles of a given reference and its size is proportional to the number of images assigned to that reference.

Fig. 6. Electrophoretic Mobility Shift Assays of RSC and mono- or dinucleosomes. Radiolabeled mono- (lanes 1 and 2) or dinucleosomes (lanes 3 and 4) were incubated either in the presence of RSC (lanes 2 and 4) or mock treated (lanes 1 and 3) at 30°C for 20 min. All samples were incubated in the absence of ATP. The samples were then resolved on a native polyacrylamide gel and autoradiographed.

Fig. 7. ATP-dependent translocation by RSC on a nucleosomal substrate. Translocation by RSC (10 nM) was measured under constant tension (4 pN) in the presence of 1 mM ATP. The trace shows a single translocation event leading to the formation of a large loop followed by cooperative loop dissipation. The translocation velocity obtained by fitting the curve with a straight line is shown.

Movie 1. Movie of the open conformation of RSC with a high resolution nucleosome modeled within its cavity. This is an animation of the structure shown in Fig. 4.

SI Text

Preparation of negatively stained samples, by the sandwich method, was as follows. Typically, 5 ml of ~0.1 mg/ml RSC in 20 mM Tris·HCl pH 7.5; 150 mM NaCl; 2 mM EGTA; 0.01% Nonidet P-40; 1 mM DTT; 2% glycerol and 6% trehalose were applied to a freshly glow-discharged (5 sec) copper mesh grid with a holey carbon support and a continuous layer of thin carbon on top and adsorbed for 2 min. The grid was rinsed, successively, in four 75 ml drops of freshly-made 2% uranyl formate without blotting. After the fourth rinse, the drop was allowed to sit on the grid for 1 min before a second layer of carbon, which had been floated on a well of 2% uranyl formate, was picked up. The grid was blotted very gently at this point leaving behind a clearly visible layer of liquid and air-dried.

Data were collected and analyzed as previously reported (1). Briefly, a DNA molecule was bound to two polysterene beads which were held by a micropipette and a force-measuring optical trap. A feedback mechanism controls the position of the micropipette relative to the trap to maintain a constant force (as was the case in the experiments described here). The positions of the beads are tracked during the experiment and their distance is used as a measure of the end-to-end distance in the DNA. The presence of a single nucleosome in the DNA template was confirmed by stretching it at constant speed until the high forces required to mechanically disrupt nucleosomes are reached. At this point, a single ripping signal should appear in the force-extension curves indicating the disruption of a single nucleosome.

Mononucleosomes were assembled on a radiolabeled 173 bp 5S DNA. A portion was ligated to a separate mononucleosome pool bearing cohesive DNA ends, to form dinucleosomes. Mononucleosomes and dinucleosomes were purified separately by sucrose gradient sedimentation. Mononucleosomes or dinucleosomes (1 mM) were incubated with pure RSC (10 mM, or mock treated) and incubated in buffer (10 mM Tris·acetate pH 7.9, 10 magnesium acetate, 50 mM potassium acetate, 1 mM DTT, 0.5 mg/ml BSA) at 30°C for 20 min. Reactions were then electrophoresed in a 3.2% native gel (38.9:1.1 acrylamide:bis), 15 mM TrisCl pH 8, 1 mM EDTA for sereral hours at 4°C in buffer containing 10 mM Tris·Cl pH 8, 1 mM EDTA. The gel was then dried and autoradiographed. The particular nucleosomes shown here bore tags on their amino terminal tails (Myc-H3 and Flag-H2A), but show identical affinities for RSC as untagged nucleosomes (data not shown).

1. Schnitzler GR, Cheung CL, Hafner JH, Saurin AJ, Kingston RE, Lieber CM (2001) Mol Cell Biol 21:8504-8511.

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