Nature Structural & Molecular Biology: doi: /nsmb Supplementary Figure 1

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1 Supplementary Figure 1 Multiple sequence alignments of four Swi2/Snf2 subfamily proteins, ScChd1, SsoRad54 and the RNA helicase Vasa. The sequence alignments of the Swi2/Snf2 subfamily proteins, ScChd1 and SsoRad54 were done with Clustal Omega, which were further aligned with the sequence of Vasa according to structural superposition (PDB code 2DB3). Secondary structural assignments labeled on the top are based on the structure determined in this study, and the helicase motifs are assigned as reported 23. The assignment of HSA domain is based on the reported structure (PDB code 4I6M) 20. The SnAc domain is labeled as an orange bar. The residues numbering is based on the sequence of MtSnf2. The suppressor mutants found in ScSth1 are labeled as magenta dots (in posthsa) and yellow dots (in supph) 18. Blue dots, the DNA-binding residues identified in this study; blue triangles, arginine fingers; red triangle, cancer-associated mutations found in human Brg1 gene 11, Bartlett, C., Orvis, T.J., Rosson, G.S. & Weissman, B.E. BRG1 mutations found in human cancer cell lines inactivate Rb-mediated cell-cycle

2 arrest. J. Cell Physiol. 226, (2011). 51. Medina, P.P. et al. Genetic and epigenetic screening for gene alterations of the chromatin-remodeling factor, SMARCA4/BRG1, in lung tumors. Genes Chromosomes Cancer 41, (2004). 52. Medina, P.P. et al. Frequent BRG1/SMARCA4-inactivating mutations in human lung cancer cell lines. Hum. Mutat. 29, (2008). 53. Wong, A.K. et al. BRG1, a component of the SWI-SNF complex, is mutated in multiple human tumor cell lines. Cancer Res. 60, (2000).

3 Supplementary Figure 2 SAXS measurements of MtSnf2. (a) Three different views of the overlay of the final average ab initio molecular envelope of MtSnf2 ( ) reconstructed from SAXS measurements (grey) with the docked crystal structure. The protein is colored as in Fig.1, and the N-and C-termini are labeled. Additional densities near the N- and C-termini can be attributed to the disordered residues at both ends (the first 13 and the last 48 residues, respectively). (b) Scattering intensity in arbitrary units versus momentum transfer q in Å -1 for MtSnf2. The linear fitting in the Guinier region of scattering curve (inset) indicates that MtSnf2 is monodisperse and homogenuous in solution. (b) The dimensionless Kratkyplot of MtSnf2 has typical feature for folded protein with disordered regions. (d) Pair distance distribution function (PDDF) of MtSnf2 with D max = 117 Å calculated using GNOM (q max =0.30 Å -1 ). (e) Fitting the theoretical scattering curve (red line) of MtSnf2 crystal structure to the experimental scattering curve (black circles), with a chi of 2.1. The theoretical SAXS curve agrees well with the SAXS measurement in solution, further validating the observed conformation in the crystals.

4 Supplementary Figure 3 Comparisons with the SF2 family proteins SsoRad54 and Vasa. (a) Structural alignment of the core1 domains of MtSnf2 (green) and SsoRad54 (grey). The DNA bound by SsoRad54 is shown as ribbon presentation. The DNA-binding sites of the core1 domains of MtSnf2 and SsoRad54 align very well, with K665 and K692 of MtSnf2 located at the same positions as R547 and K573 of SsoRad54. K687 of MtSnf2 is disordered in current structure, and aligned with K568 of SsoRad54 in the primary sequence (Supplementary Figure 1). (b) Structural alignment of the core2 domains of MtSnf2 (cyan) and SsoRad54 (grey). The arginine fingers of SsoRad54 are labeled (R840 and R843). The DNA binds to the surface of the core2 domain of SsoRad54 at a position in conflict with the SnAc domain (orange) of MtSnf2, suggesting MtSnf2 interacts with DNA in a different manner. Instead, biochemical analyses indicated that DNA contacts the core2 domains of both proteins in solution at motif V (around R950 of MtSnf2 and K808 of SsoRad54). R832 of MtSnf2 is also involved in DNA binding, which is absence in SsoRad54. (c) Structural alignment of the core1 domains of MtSnf2 (green) and Vasa helicase (grey). The bound RNA by Vasa is shown as stick presentation. Motif I of MtSnf2 is colored red, with bound sulfate ion as sphere presentation. The DNAbinding sites of MtSnf2 (K665, K687 and K692) identified in this study are in close proximity to the RNA bound by Vasa. (d) Structural alignment of the core2 domains of MtSnf2 (green) and Vasa (grey). Motif VI of MtSnf2 is colored red. The DNA-binding site of R950 in MtSnf2 is close to the RNA bound by Vasa.

5 Supplementary Figure 4 Interactions between post-hsa and supph of MtSnf2. (a) Structure of the posthsa-supph region of MtSnf2. The structure is colored as in Fig.1 with the supph helix in yellow. The suppressor mutants of ScSth1 are mapped to the corresponding positions of MtSnf2 (showed as stick models) 18. Residues are labeled based on the sequence of MtSnf2, and the residues in the parentheses are the corresponding suppressor mutants of ScSth1. Three suppressor mutations are located at posthsa (N384K, D385Yand L392V) and four at supph (E676Q, L680M/V, L681F and K688T). Two mutations at the HSA domain (T373P and K382N) are not present in the current structure. (b) Interactions between supph, posthsa and the core1 domain. The core1 domain is shown as surface presentation, and colored coded by the spectra of blue-to-white (decreasing conservation). The posthsa and supph helices bind to a conserved surface of the core1 domain through hydrophobic interactions, and they also interact with each other through specific H-bonds. (c) Multiple sequence alignments of the Swi2/Snf2 subfamily proteins around the posthsa and supph regions. The suppressor mutations of Sth1 are highlighted in magenta and yellow at the posthsa and supph helices, respectively.

6 Supplementary Figure 5 Chromatin-remodeling activities of various constructs used in this study. (a) Gels of the restriction enzyme-accessibility assays of four core1-core2 interface mutants of MtSnf2 ( ). The cut fractions were quantified and shown in Fig. 3d. Three independent assays were performed and one was showed. (b) Gels of the restriction enzyme-accessibility assays of MtSnf2 ( ) with the WT interface and five DNA-binding mutants. The cut fractions were shown in Fig. 4e. (c) Gels of the restriction enzyme-accessibility assays for ScSnf2 with different N-terminal boundaries. The catalytic core of ScSnf2 ( ), left panel; half HSAcontaining ScSnf2 ( ), middle panel; full HSA-containing ScSnf2 ( ) in complex with Arp7-Arp9-Rtt102 (Snf2 tetramer), right panel. The cut fractions were quantified and shown in Fig. 5c. (d) Gels of the restriction enzyme-accessibility assays of MtSnf2 with different N- terminal boundaries. The catalytic core MtSnf2 ( ), left panel; half HSA-containing MtSnf2 ( ), middle panel. The cut fractions were shown on the right. (e) The ATPase activities of MtSnf2 with different N-terminal boundaries in the absence (-) and presence of DNA/NCP. MtSnf2 ( ), black; MtSnf2 ( ), white. (f) Gels of the restriction enzyme-accessibility assays of MtSnf2 with truncation of the C- terminal flexible tail at different positions. The cut fractions were quantified and shown in Fig. 6e.

7 Supplementary Figure 6 Nucleosome binding activities of various constructs used in this study. (a) EMSA of the nucleosome binding activities of MtSnf2 ( ) with the WT interface and five DNA-binding mutants. 2 nm Cy5-labelled 147 bp NCP was mixed with increasing amounts of proteins (in nm). The samples were resolved by electrophoresis on 4.5% native acrylamide gels, and the fluorescent bands were detected by Typhoon Trio+ imager. With higher concentrations of the proteins, the bound NCPs migrate more slowly, suggesting a heterogeneous population of the protein-bound NCP in the solution, and one NCP might bind more than one molecule of the protein. The star * sign indicates the position of free DNA. The bound fractions were quantified as the disappearance of free NCP relative to the intensity of the whole lane, and shown in Fig. 4c. (b) EMSA of nucleosome binding of three constructs of ScSnf2. The quantification of the bound fractions was showed in Fig. 5a. (c) EMSA of nucleosome binding of MtSnf2 ( ). EMSA of MtSnf2 ( ) was shown in Supplementary Fig. 6a, the left panel (WT interface). Quantification of the bound fractions is shown (right panel). The apparent Kds of the catalytic core MtSnf2 ( ) and the HSA-containing MtSnf2 ( ) are about 150 nm and 190 nm, respectively. (d) EMSA of nucleosome binding of MtSnf2 with truncation of the C-terminal flexible tail at different positions. EMSA of MtSnf2 ( ) was shown in Supplementary Fig. 6a, the left panel (WT interface). The quantification of the bound fractions is showed in Fig. 6c.