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

Nguitragool and Miller. 10.1073/pnas.0708639104.

Supporting Information

Files in this Data Supplement:

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 FAB 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 2Fo - Fc 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 Fo - Fc Cl--omit map (yellow) contoured at 1.7 s and are at essentially identical positions in the wild-type protein. (B) 2Fo - Fc 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 2Fo - Fc maps with the I-H loop omitted in the model (data not shown).





Table 2. Quantitative thiol measurements

Cysteine mutant

Free thiol per subunit

R230C

1.07 ± 0.03

L249C

0.98 ± 0.03

R230C/L249C

0.07 ± 0.02

R230C/L249C/T433C

0.81 ± 0.03

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





Table 3. Crystallographic statistics

Space group

C2

Unit cell parameters

 

 a, Å

233.10

 b, Å

98.36

 c, Å

171.93

 β, °

131.77

Resolution range, Å

50−3.1

No. of reflections

49,855

Completeness, %

99.3 (97.1)

Rmerge, %

8.0 (48.0)

<I>/σI

14.4 (2.3)

Rfree, %

28.0

Rcryst, %

26.2

rmsd from ideal

 

Bond lengths, Å

0.007

Bond angles, °

0.987

Values in parentheses are for the outermost shell.

This Article

  1. Published online before print December 18, 2007, doi: 10.1073/pnas.0708639104 PNAS December 26, 2007 vol. 104 no. 52 20659-20665
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