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Figure 4.
Figure 4. Two crystal structures of the E71A mutant. (a)
Electron density map of residues 60–84 from two diagonally
symmetric subunits for the crystal form of E71A with residue
Asp80 flipped upward (flipped E71A). Sticks, polypeptide chain;
blue mesh, 1-  contour
of the 2F[o] – F[c] electron density map for the protein;
magenta mesh, 2- contour
of the ions; black dotted ovals, cavities created by the absence
of the Glu71 side chain. (b) Single-subunit line representation
of the P-loop of the flipped (red) and nonflipped (black) E71A
structures overlaid onto the wild-type structure^32 (PDB entry
1K4C; gray) highlights the conformational rearrangements in the
backbone of the selectivity filter (residues 75–79). Insets,
the side chain conformational changes in Asp80 and Trp67 as
fitted to the 2- contour
of the simulated annealing omit map. The omit maps were
calculated for residues 79–84 and 67, respectively; atoms
within 3.5 Å of selected residues were also omitted in the
calculation. The density attributed to the alternate rotamer of
the Trp67 side chain in the flipped X-ray structure is clearly
distinct from the density of the lipid observed near this
position in the WT X-ray structure^33. (c) One-dimensional
electron density profiles for the two crystal conformations of
the E71A mutant (flipped and nonflipped). Top, F[o] – F[c]
omit maps of K^+ ions in the selectivity filter shown relative
to the protein model. The electron density maps are shown as a
6- contour
for the flipped E71A conformer and as 7- (blue)
and 4- (cyan)
contours for the nonflipped E71A conformer. Different contour
levels were chosen for the purpose of visual clarity. Bottom,
one-dimensional electron density profile along the central
symmetry (z) axis is shown using the ion in the cavity as z = 0.
Gray-shaded peaks represent the profile for the wild-type
channel (PDB entry 1K4C at 2.0 Å). Numbers and E at top
denote the K^+-binding sites (S0–S4 and S[ext],
respectively).(d) Comparison of crystallographic B-factors for
P-loop residues (63–83) from the WT KcsA structure (bottom
chart) and the nonflipped E71A mutant (E71A-NF, top chart).
Black dotted line represents the mean value for all atoms in the
P-loop. Vertical capped lines represent the  3
values
for each data set.
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