Article Figures & Data
Figures
- Figure 1. Stoichiometry of Vps75–Rtt109 complexes.
(A, B) van Holde–Weischet analysis of sedimentation velocity experiments of 7.5 μM Vps75 with 0, 0.5, and 1.0 equivalent (white, light grey, and dark grey, respectively) of Rtt109 at 150 mM NaCl (A, circles) or 300 mM NaCl (B, diamonds). Similar experiments for Rtt109 and H3-H4 are shown in Fig S1. (C) SEC-MALS of 10 μM Vps75 (dotted grey), 10 μM Rtt109 (dotted black), and 10 μM Vps75 with 5, 10, or 20 μM Rtt109 (light grey, dark grey, and black, respectively). Horizontal dashed lines indicate theoretical molecular weights of labeled complexes.
- Figure S1. Rtt109 does not stably interact with H3-H4 or H3DM-H4.
(A, B, C) van Holde–Weischet analysis of sedimentation velocity experiments of Rtt109 alone (A) or with 0 to 2 equivalents of H3-H4 (B) or H3DM-H4 (C). Rtt109 alone is shown in white, and Rtt109 with 0.5, 1, or 2 equivalents of histones is shown in light grey, dark grey, and black, respectively.
- Figure 2. Co-purification of Vps75–Rtt109.
(A, B, C, D) Purification of co-expressed his–Rtt109 and Vps75 by Ni2+-affinity (A, B) and anion exchange (C, D) chromatography. FT is flow-through. (E) SEC-MALS of major peaks at 300 mM NaCl from anion exchange. P1, P2, and P3 are shown in black, dark grey, and light grey, respectively. Horizontal dashed lines indicate theoretical molecular weights of labeled complexes. Optimization of a MgATP wash is shown in Fig S2.
- Figure S2. Removing DnaK from co-expressed Vps75–Rtt109 using MgATP.
(A) SDS–PAGE of anion exchange elution fractions under different conditions. From left to right, no MgATP, 5 mM MgATP wash during Ni2+-affinity chromatography, 5 mM MgATP in lysis and chromatography buffers, and 10 mM MgATP wash during Ni2+-affinity chromatography. The latter two conditions cause a dramatic increase in system pressure. (B) Quantification of the amount of DnaK contamination in each anion exchange fraction. (C) Anion exchange chromatogram of Vps75–Rtt109 without MgATP (black) and with a 5 mM MgATP wash (grey).
- Figure 3. Sedimentation velocity of the Vps75–Rtt109-(H3-H4) complex.
(A, B) van Holde–Weischet analysis of sedimentation velocity experiments of complexes with Vps75, Rtt109, and H3-H4 (A) or H3DM-H4 (B). Vps75 and Rtt109 were mixed 2:2 (top) or 2:1 (bottom). Vps75–Rtt109 is shown in white, and Vps75–Rtt109 and 0.5 or 1 equivalent of histones are shown in light grey and dark grey, respectively. For reference, sedimentation of H3-H4 or H3DM-H4 (faded circles), Rtt109 (faded triangles), and a 2:2 mix of Vps75–(H3-H4) or Vps75–(H3DM-H4) (faded squares) are also shown. Comparison of H3-H4 and H3DM-H4 sedimentation is shown in Fig S3. All experiments were carried out at 150 mM NaCl.
- Figure S3. Sedimentation velocity of H3-H4 and H3DM-H4.
van Holde–Weischet analysis of sedimentation velocity experiments of H3-H4 (white) or H3DM-H4 (black) at 150 mM (circles) or 300 mM (diamonds) NaCl.
- Figure 4. Optimizing solubility of Vps75–Rtt109–(H3-H4) complexes.
Solubility of complexes containing H3-H4 (left) or H3DM-H4 (right) at 6 mg/ml was assayed as a function of NaCl concentration. (A) Image showing visual turbidity. (B) SDS–PAGE of samples before (Input) and after (Soluble) centrifugation. (C) Percent solubility of complexes at 6, 8, and 10 mg/ml based on UV spectroscopy before and after centrifugation (black, grey, and light grey, respectively). Complete data for 8 and 10 mg/ml are shown in Fig S4.
- Figure S4. Optimizing solubility of Vps75–Rtt109–(H3-H4) complexes at 8 and 10 mg/ml.
(A, B) Solubility of complexes containing H3-H4 (left) or H3DM-H4 (right) at 8 mg/ml (A) and 10 mg/ml (B) was assayed as a function of NaCl concentration. The top panel shows visual turbidity. SDS–PAGE shows samples before (input) and after (soluble) centrifugation.
- Figure 5. Stoichiometry and large-scale preparation of Vps75–Rtt109–(H3-H4).
(A, B) SEC-MALS of 20 μM 2:1 Vps75–Rtt109 (black) with 20 or 40 μM (dark grey and light grey, respectively) H3-H4 (A) or H3DM-H4 (B). Horizontal dashed lines indicate theoretical molecular weights of labeled complexes. Molecular weights are listed in Table S2. (C) Gel filtration of large-scale purifications of Vps75–Rtt109–(H3-H4) (black) and Vps75–Rtt109–(H3DM-H4) (grey). SDS–PAGE of peak fractions.
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