Figure 1.
Photocycle of bacteriorhodopsin.
Different intermediates formed in the photocycle are indicated with timescale of occurrence, sequence and approximate color corresponding to their spectroscopic signatures.
Figure 2.
Ribbon representation of all ground state and cytoplasmically closed state.
All ground state and cytoplasmically closed state (K, L and M1) listed in Table 1 are used. All coordinates are aligned with 1M0L using the LSQMAN program [51]. Models are colored based on the B-factors of that coordinate set using color spectrum provided by Pymol (Color bar is shown in the right corner.). 1CWQ(A) shows a unique shape of the loop around loop EF (“1”).
Table 1.
bR coordinates used in figures.
Figure 3.
Ribbon representation of all cytoplasmically open and later state (M2, N′ and O).
All cytoplasmically open and later state (M2, N′ and O) listed in Table 1 are used. All coordinates are aligned with 1M0L in the same way as in Fig. 2. Eight coordinates (1CWQ(B), 1IW9, 1F4Z, 1C8S, 1P8U(1), 1JV7, 1X0I and 1FBK) are used. 1CWQ(B) displays a unique loop structure around loop EF (“1”). 1JV7 and 1X0I show a unique conformational change around loop DE (“2” and “2′”). 1FBK shows a larger movement in the EF loop region (“3”). Color scheme for B-factor is the same as in Fig. 2.
Figure 4.
Superposition and Cα atom deviation of the helix F region from M1 to N′ intermediates.
(A) Superposition of the helix F region from M1 to N′ intermediates. Residues 171–186 of 1P8H(1), 1P8U(1), 1C8S, and 1FBK are extracted and displayed as “lines” modeled in Pymol. Residue number increases from top (cytoplasmic side) to bottom (periplasmic side) showing larger deviations around the cytoplasmic side. (B) Cα atom deviation of M1 to N′ states from ground state coordinates, 1M0L at each atom position in helix F. 1C8S lacks the cytoplasmic side of helix F.
Figure 5.
Cα atom deviation plots and difference maps between intermediate and ground states calculated at 7 Å.
In the Cα atom deviation plots, red line shows the Cα atom deviation between intermediate and ground state after alignment using LSQMAN. For some plots, there are one or two gaps because there are missing residues either for intermediate or ground states. Blue line shows the deviation between 1FBK and 1FBB as a measure of reference. To calculate difference maps, we used common residues between intermediate and ground state; the retinal was always included. Lipids and water molecules were always excluded.
Figure 6.
Cα atom deviation plots and difference maps between intermediate and ground states calculated at 7 Å (continued).
Figure 7.
Cα atom deviation plots and difference maps between intermediate and ground states calculated at 7 Å (continued).
Figure 8.
Cα atom deviation plots and difference maps between intermediate and ground states calculated at 7 Å (continued).
Figure 9.
Cα atom deviation plots and difference maps between intermediate and ground states calculated at 7 Å (continued).
Figure 10.
Maps calculated using EM data at 7 Å.
Amplitude data for closed and open states from diffraction analysis alone (F(diff, open), F(diff, closed)) were obtained as described in the text. These amplitude data were used to build models for 1FBB and 1FBK [27]. Phase data for closed state (φ(image, closed)) are the same as those of Henderson et al [3]. Phase data for open state (φ(image, open) ) were calculated as described in the text. (A) Projection map of open state, F(diff, open) φ(image, open). Amplitude data is from diffraction patterns and phase data is from images. (B) Projection map of closed state, F(diff, closed) φ(image, closed). Amplitude data is from diffraction patterns and phase data is from images. (C) Difference map, F(model, open) φ(model, closed) - F(model, closed) φ(model, closed). Phase information comes only from the model of the closed (i.e. unilluminated) state. The absence of phase information is a common situation when molecular replacement is used in X-ray or electron crystallography. (D) Difference map, F(diff, open) φ(image, open) - F(diff, closed) φ(image, closed). For both open and closed states, amplitude data is from diffraction patterns and phase data is from images of each state. (E) Difference map, F(model, open) φ(model, open) - F(model, closed) φ(model, closed). All amplitude and phase data are calculated from coordinates 1FBK and 1FBB. The pair of positive (blue) and negative (magenta) peaks correspond to the movements of the cytoplasmic end of helix F, the EF loop and cytoplasmic end of helix G.
Figure 11.
RMSD average of ground, early, and late intermediate state coordinates from 1M0L ground state coordinates.
The height of each vertical bar represents the average value of RMSD in that resolution range (0.25 Å interval). 10 early intermediate and 6 late intermediate coordinates (Table 1) are used for each calculation. 15 ground state coordinates (Table 1) are used for calculation of ground state. All RMSD values used for this figure are listed in Fig. S1. Furthermore, structural distribution of late intermediate coordinates is discussed in Fig. S2.