Supplemental Data

Files in this Data Supplement:

• Supplemental Figure 1 - Supplemental Figure 1. N-terminal residues of ToxA. The 2F_o – F_c electron density map contoured at 1.0 ρ_rms shows unambiguous density for residues 61 through 64 situated at the trimer interface in the form-II crystals. The residues, including the pyroglutamate 61 (Pca61), are labeled and the orientation and the coloring scheme are identical to that of Figure 5A. The figure was generated using Pymol (www.pymol.org).
• Supplemental Figure 2 - Supplemental Figure 2. Gel filtration analysis. Size-exclusion chromatography elution profiles of ToxA (curve A) and His-tagged proToxA (curve B) shows the existence of predominantly monomeric species. Arrows above the profiles indicate the elution time of the standard proteins: Peak 1-Bovine serum albumin (67 kDa), Peak 2-Ovalbumin (43 kDa), Peak 3-Rnase A (13.7 kDa), and Peak 4-Aprotinin (6.5 kDa).
• Supplemental Figure 3 - Supplemental Figure 3. Sedimentation equilibrium analysis of ToxA. (A) The 30 data sets resulting from three independent scans of four different ToxA concentrations at three different speeds are shown as dots and the curves are the best global fit of all of the data to a monomer-trimer model. (B) The residuals of the fits for each of the 30 data sets.
• Supplemental Figure 4 - Supplemental Figure 4. Sedimentation equilibrium analysis of proToxA. (A) The 9 data sets from three independent scans of proToxA at three different speeds with the data fit to a single-component ideal model are shown. (B) The residuals of the fits for each of the 9 data sets.
• Supplemental Figure 5 - Supplemental Figure 5. The trimerization interface of ToxA. A stereo representation showing the interactions at one of the trimer interfaces. The orientation and the coloring scheme are identical to that of Figure 5A and the 3-fold axis is highlighted by a gray triangle. The residues involved in the trimer interactions are represented as ball-and-stick models. To maintain clarity the side chain atoms of Phe147, Tyr150, Leu152 and Gln154 are not shown and residues Thr126, Ala151, Leu152, Gln154, Gly155, Gly157, waters 31 and 41 are not labeled. Arg128, Asp149, and Glu177 from all three chains form a symmetrical hydrogen-bonding network with water molecules and a Ni⁺² at the 3-fold. The burial of many hydrophobic residues also contributes to the stability of the trimer interface. Hydrogen bonds bridging the different monomers are represented by dotted gray lines. Residues having ≥ 40 å² surface area buried at the trimer interface are (in parentheses are buried surface areas in å²): Pca61 (62), Ser63 (47), Thr 126 (55), Arg128 (65), Arg140 (91), Tyr144 (123), Glu145 (74), Leu146 (97), Asp149 (48), Ala151 (55), Ile153 (117), Arg156 (115), Ser158 (55) and Glu177 (74). The figure was prepared using Molscript (Kraulis, 1991) and Raster3D (Merritt and Bacon, 1997).
• Supplemental Figure 6 - Supplemental Figure 6. Residues on a crystallographic 2-fold axis. A stereo diagram showing the 2_Fo – F_c electron density contoured at 0.7 ρ_rms at the crystallographic 2-fold axis near chain B of the form-II crystals. The residues that have been modeled with half-occupancies are indicated as stick models and labeled. The identical residues from the crystallographically related molecule that share the space in the crystal are shown as a semi-transparent model and also labeled. One chain makes a parallel interaction with strand β2 and the other is anti-parallel. The way this disorder works is that half of the molecules in the crystal have one of the chains ordered and half have the other chain ordered. This means that the crystallographic symmetry is broken locally, but is maintained for the crystal as a whole. The 2-fold axis is shown for reference. The figure was prepared using BobScript (Esnouf, 1999) and Raster3D (Merritt and Bacon, 1997).