CLC Cl − /H + transporters constrained by covalent cross-linking

Nguitragool and Miller. 10.1073/pnas.0708639104.

Supporting Information

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SI Figure 8
SI Table 2
SI Table 3

Fig. 8. X-ray crystal structure of cross-linked 207C/207C. The cross-linked, purified protein was crystallized with F_AB 10EC3/G4 in 37%(wt/vol) PEG 300, 25 mM NaCl, 50 mM glycine (pH 9.5). Crystals were frozen in liquid N₂, and diffraction data were collected at National Synchrotron Light Source beamline X29A. (A) View of 2F_o - F_c map contoured at 1.3 s (cyan) in the Cl^--binding region of the transporter, along with the corresponding refined model for the cross-linked protein. Orientation is with extracellular side up, as in Fig. 2 Left. Cl^- ions are shown by F_o - F_c Cl^--omit map (yellow) contoured at 1.7 s and are at essentially identical positions in the wild-type protein. (B) 2F_o - F_c map in the region of the loop containing the 207C-207C cross-link and transmembrane I and H helices flanking the loop. The map was contoured at 2.0 s. Ca traces are shown for models of the wild-type (magenta) and cross-linked proteins (yellow). Crystals of wild-type protein analyzed at 3.4 Å in parallel show well defined density, including the I-H loop, in 2F_o - F_c maps with the I-H loop omitted in the model (data not shown).

Table 2. Quantitative thiol measurements

Free thiol was measured by Ellman reaction, as described in Methods. Values represent mean + SE of at least six measurements.

Table 3. Crystallographic statistics

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