Figure 1.
Flowchart of PoreWalker stepwise algorithm.
Figure 2.
Changes of the pore axis during PoreWalker calculation for the ASIC1 acid-sensing ion channel (PDBcode 2qts [46]).
Step 1: the 3D structure as submitted to the program. Step 2. Preliminary definition of the pore axis (shown in red). Step 3. Definition of pore centre and translation of the pore axis so that it passes through it (axis shown in red). Step 4–8. Iterative identification of local pore centres (red spheres) and variations in the position of the pore axis (green) during the refinement of the pore axis. Step 9. PoreWalker output.
Figure 3.
PoreWalker output for the bovine aquaporin-1 water channel (1j4n).
(A) visualization of a pore section showing pore-lining residues and pore centres at 3Å steps: the section of the structure was obtained by cutting the protein structure along the xy-plane, where the x-axis corresponds to the pore-axis, and y-coordinate>0 only are displayed. Pore-lining atoms and residues are coloured in orange and blue, respectively. The remaining part of the protein is shown in green. Red spheres represent pore centres at given pore heights and their diameters correspond to 1/10 of the pore diameter calculated at that point; (B) representation of the shape of the pore and shape characterization string: the pore is simplified as a stack of building blocks going from the most negative to the most positive coordinate along the pore axis (x-axis). D, U and S indicate conical frustums of decreasing diameter, conical frustums of increasing diameter and cylinders, respectively; (C) pore diameter profile at 3Å steps corresponding the pore shape in (B). Pore axis (X-Coord): the position along the pore axis is shown as x-coordinate in Å. Dia (Ang): pore diameter value in Å; (D) visualization of a pore section showing the position of the biggest spheres (pore centres) that can be built along the channel at 1Å steps: the section of the structure was obtained by cutting the protein structure along the xy-plane. The protein structure is coloured in green; (E) diagram of the regularity of the pore cavity as number of lines that can approximate the positions of the pore centres at 1Å steps (PRINCIP). The pore is represented as a series of consecutive straight and wiggly lines representing channel areas where pore centres can (straight) or cannot (wiggly) be fitted to a line, going form the most negative to the most positive coordinate along the pore axis (x-axis). Vertical lines describe either the only low-RMSD areas throughout a pore or low-RMSD areas that are co-linear. Diagonal lines represent low-RMSD areas, which are different from the low-RMSD areas other identified along the channel. Curve lines indicate areas where pore centres are highly spread; (F) pore diameter profile at 1Å steps. Pore axis (X-Coord): the position along the pore axis is shown as x-coordinate in Å. Dia (Ang): pore diameter value in Å; (G)–(I) horizontal sections of the pore at the pore height highlighted in purple in (H) viewed from the bottom, i.e. the most negative coordinate along the pore axis (G) and from the top, i.e. the most positive coordinate along the pore axis (I).
Figure 4.
PoreWalker visual representations.
Images show xz-plane sections, z-coordinate>0 only, with the x-axis corresponding to the pore axis. (A) KirBac1.1 potassium channel (1p7b); (B) bovine aquaporin-1 (1j4n); (C) KcsA potassium channel (1k4c); (D) MthK calcium-gated potassium channel (1lnq); (E) CorA Mg2+ transporter (2iub); (F) MscS voltage-modulated mechanosensitive channel (2oau); (G) ASIC1 acid-sensing ion channel (2qts); (H) SecYE-beta protein-conducting channel (2yxr); (I) Kv1.2 voltage-gated potassium channel (2a79); (J) sodium-potassium channel (2ahy); (K) Amt-1 ammonium channel (2b2f); (L) nicotinic acetylcholine receptor (2bg9). Pore-lining atoms and residues are coloured in orange and blue, respectively. The rest of the protein is shown in green. Red spheres indicate pore centres at 3Å steps and their size is proportional to the pore diameter in that point. Black arrows indicate the main pore axis as identified by visual analysis of the structure.
Figure 5.
PoreWalker results and the molecules of solute found in the 3D structures.
(A)–(B) PoreWalker and HOLE results obtained for the Amt-1 ammonium channel (2b2f). (A)-Vertical view. PoreWalker cavity is shown as y-coordinate>0 only of the protein section along the xy-plane, where the x-axis corresponds to the pore axis. Pore-lining atoms and residues are coloured in orange and blue, respectively, and the rest of the protein is shown in light grey. Red spheres represent pore centres at 3Å steps and their size is proportional to the pore diameter at that point. HOLE cavity is shown as purple surface. Xenon atoms, indicating the presence of cavities within the protein structure, are shown in light blue. (B)-Top view. The colour scheme is as in (A). White dots indicate the external loop, which divides the channel top gate into multiple exits. (C)–(F)-Comparison of PoreWalker results with the actual position of solute molecules for the SoPiP2;1 water channel (C–D, 1z98) and the sodium potassium channel (E–F, 2ahy). Pore visualizations follow the colour scheme in (A). Water molecules are shown in yellow, Na+ ions in purple and Ca2+ ions in green. (C) and (E) display PoreWalker sections as in (A). (D) and (F) show the pore only, represented as surface of the pore-lining atoms and residues only.
Table 1.
List of protein structures in the test set.
Figure 6.
Comparisons of channel cavities identified by PoreWalker and HOLE and corresponding 1Å step profile diameters.
(A)&(D) MthK potassium channel (1lnq), (B)&(E) MscS voltage-modulated mechanosensitive channel (2oau); (C)&(F) bovine aquaporin-0 (1ymg), (G)&(J) ASIC1 acid-sensing ion channel (2qts); (H)&(K) Amt-1 ammonium channel (2b2f); (I)&(L) SoPiP2;1 water channel (1z98). PoreWalker cavities are shown as y-coordinate>0 only of the protein section along the xy-plane, where the x-axis corresponds to the pore axis. Pore-lining atoms and residues are coloured in orange and blue, respectively, and the rest of the protein is shown in light grey. Red spheres represent pore centres at 3Å steps and their size is proportional to the pore diameter at that point. HOLE cavities are shown as purple surface. PoreWalker and HOLE profile diameters are shown as solid and dotted lines, respectively. Corresponding R2 values are 0.958, 0.951, 0.814, 0.450, 0.017 and 0.000 for 1lnq, 2oau, 1ymg, 2qts, 2b2f and 1z98, respectively.
Figure 7.
PoreWalker results for the 3D-structures of the KcsA K+ channel at low (1k4c) and high (1bl8) concentration of K+.
(A),(C)-PoreWalker visual representation (xz-plane section, z-coordinate>0 only, x-axis corresponding to the pore axis of KcsA K+ channel at low (1k4c, A) and high (1bl8, C) concentration of K+. Pore-lining atoms and residues are coloured in orange and blue, respectively, and the rest of the protein is shown in green. Red spheres represent pore centres at 3Å steps and their size is proportional to the pore diameter at that point. IN and OUT indicate the cytoplasmic and periplasmic side of the pore, respectively. (B)-Diameter profiles calculated by PoreWalker standard protocol (3Å steps, solid line), HOLE (1Å steps, dotted line) and PoreWalker-1Å steps (dashed line). The internal pore, the internal cavity and the selectivity filter are highlighted in orange, blue and red, respectively. (D)-Diameter profiles calculated by PoreWalker standard protocol for the low-K+ (solid line) and high-K+ (dotted line) KcsaA channel structures. The entrance of the selectivity filter is shown in green and is found at the pore height highlighted by a green box in the channel structures (A for the low-K+ channel and C for the high-K+ channel). (E)–(F) Different conformation of the Thr75s lining the entrance of the selectivity filter in the low-K+ (E) and high-K+ (F) channels. Backbone and sidechain oxygens are coloured in dark pink and red, respectively, and their atomic volume is shown by dots. Red spheres represent the pore centres at the entrance of the selectivity filter and their size is proportional to the pore diameter predicted for that point (0.92Å and 3.63Å for the low-K+ (E) and high-K+ (F) channels, respectively).
Figure 8.
Superpositions of the selectivity filter 3D-structures of the low-K+ (green/red/blue colour scheme) and high-K+ (white/orange/blue colour scheme) KcsA channels.
(A) the whole filter (C-alpha RMSD = 0.38Å, all atom RMSD = 0.58Å), (B) G77s only, top view (all atom RMSD = 0.33Å), (C) V76s only, top view (all atom RMSD = 0.58Å), (D) T75s only, top view (all atom RMSD = 0.99Å).
Figure 9.
Superposition of the 3D-structures of Thr75s from the low-K+ (green/red/blue colour scheme) and high-K+ (white/orange/blue colour scheme) KcsA channels, top view.
Water molecules from the low-K+ structure are shown in light blue. Violet dots represent a K+ ion. Thr side chain atoms and the four water molecules interacting with them are shown as opaque. Thr backbone atoms and the remaining four water molecules of the K+ hydration shell are shown as transparent.