Egawa et al. 10.1073/pnas.0605911104. |
Fig. 6. The Bchl c assembly models that have all of the head-head (closed diamonds), tail-head (triangles), and tail-tail (open diamonds) contacts were searched in a group of assembly models that were built by translational operations on the two-dimer or four-monomer units colored green. The directions of the translations for generating 2D assemblies are shown by green arrows. If we assume that every Mg atom forms an intermolecular coordination bond, and head and tail parts of every molecule contact with head and/or tail, BChl c assembly structures shown represent all possible models. The assemblies composed of dimers (A-F) and monomers (G-L) are shown. Here, the BChl c ring in the model is allowed to take either orientation of the molecule that flips by 180° along the axis including Mg and C31. Black arrows are parallel to the layers in A, B, D, and E, and parallel to the columns in G and H. The dimers in the same orientation provide only three assembly models A, B, and C. A and B have layers parallel to the chlorin ring planes. As shown by molecular units colored green, in A the lower molecule of the upper dimer fully overlaps with the upper ring in the lower dimers, whereas in B the lower molecule partially overlaps. In C, dimers form uneven-dimer columns. A, B, and C, respectively, give D, E, and F by rotating the lower dimers in the units by 180° along the axes perpendicular to the chlorin rings. G and H form parallel and antiparallel layers. A, C, and E have the three contacts and are equal to Fig. 4 A, B, and C, respectively. Monomers connected by the coordination bonds form layers in I-L. I and J have parallel layers, whereas K and L have antiparallel layers. Although I and K have the three contacts, we have eliminated the models because of the steric hindrance in a layer between the monomer colored green and that surrounded by a dashed line. Therefore, it can be concluded that A, C, E, and H (corresponding to Fig. 4 A, B, C, and D, respectively) represent the models that satisfy the three kinds of contact.
Fig. 7. Ten best-fit structures of 200 calculations for parallel-dimer layers are shown. The coordinates of molecules M2 (dark blue), M3 (light blue), and M4 (magenta) in Fig. 4 E-H can be expressed by the following operations on M1 (colored gray with an Mg atom in green). M2 = Rzyz (83°, -1°, -85°) M1 + (1.5, -5.4, -3.0) Å, M3 = Rzyz(-167°, -1°, 167°) M1 + (-0.4, 1.1, 3.2)Å, and M4 = Rzyz(170°, -5°, -172°) M1 + (-3.2, 8.6, -3.8)Å, where Rzyz is the rotation defined by Euler angles (a, b, g) for the z, y, and z axes, respectively. The x and y axes for molecule M1 are defined by the NA-NC and ND-NB vectors, respectively (Fig. 1A). After these operations, only the side chains at C3 for molecules M2, M3, and M4 were rotated by 180° along the C3-C31 axis. The cylindrical assembly structure in Fig. 5 A and B was generated by bending a planar assembly structure as described in Discussion. The coordinates of the molecules in the planar structure are defined by the above parameters in which b was replaced with 0°.
Table 2. 13C chemical shifts of BChl c in chlorosomes
Carbon no. | Chemical shifts, ppm | Chemical shifts, ppm* | Carbon no. | Chemical shifts, ppm |
1 | 153.7 |
| F1 | 59.4 |
2 | 134.8 |
| F2 | 118.7 |
21 | 14.5 |
| F3 | 139.3 |
3 | 139.7 |
| F31 | 16.3 |
31 | 64.1 | 63.3 | F4 | 41.2 |
32 | 22.5 | 24.9 | F5 | 27.3 |
4 | 144.6 | 143.9 | F6 | 123.9 |
5 | 95.6 | 101.6 | F7 | 135.7 |
6 | 150.5 | 150.2 | F71 | 14.0 |
7 | 132.0 | 132.5 | F8 | 39.4 |
71 | 11.0 | 7.1 | F9 | 24.1 |
8 | 139.7 | 141.4 | F10 | 129.7 |
81 | 29.9 | 28.0 | F11 | 142.2 |
9 | 146.7 |
| F111 | 17.8 |
10 | 105.8 |
| F12 | 17.8 |
11 | 147.4 |
|
|
|
12 | 138.6 |
|
|
|
121 | 18.7 |
|
|
|
13 | 127.8 |
|
|
|
131 | 196.0 |
|
|
|
132 | 48.4 |
|
|
|
14 | 162.4 |
|
|
|
15 | 104.0 |
|
|
|
16 | 154.7 |
|
|
|
17 | 50.2 |
|
|
|
171 | 30.9 |
|
|
|
172 | 29.6 |
|
|
|
173 | 174.5 |
|
|
|
18 | 48.8 | 46.0 |
|
|
181 | 21.1 | 20.4 |
|
|
19 | 167.3 | 165.9 |
|
|
20 | 104.4 | 103.7 |
|
|
201 | 21.5 | 21.9 |
|
|
The carbon numbers are given in Fig. 1A.
*The column shows a second set of correlated peaks.
Table 3. Classification of experimental 13C-13C distances,
Head to head |
| |
C-C correlation | Distance, Å | |
1 | 5 | 2.7 |
1 | 32 | 2.8 |
1 | 6 | 2.6 |
2 | 6 | 1.8 |
21 | 5 | 5.5 |
21 | 6 | 2.1 |
31 | 6 | 3.8 |
31 | 20 | 3.8 |
31 | 71 | 5.5 |
31 | 7 | 4.2 |
4 | 20 | 2.3 |
5 | 201 | 3.3 |
5 | 20 | 5.0 |
7 | 20 | 2.3 |
71 | 18 | 3.5 |
71 | 201 | 4.0 |
Tail to head |
| |
C-C correlation | Distance, Å | |
10 | 6 | 1.7 |
10 | 71 | 4.1 |
11 | 1 | 2.6 |
11 | 31 | 3.8 |
11 | 32 | 2.6 |
11 | 201 | 2.4 |
12 | 1 | 1.7 |
121 | 1 | 2.7 |
121 | 31 | 2.7 |
13 | 4 | 2.1 |
13 | 31 | 3.7 |
13 | 71 | 2.6 |
131 | 1 | 2.4 |
131 | 3 | 1.9 |
131 | 4 | 2.1 |
132 | 5 | 3.0 |
132 | 21 | 4.4 |
132 | 31 | 4.0 |
132 | 31 | 4.0 |
132 | 32 | 2.5 |
132 | 71 | 3.5 |
14 | 3 | 2.3 |
14 | 4 | 2.0 |
15 | 5 | 5.6 |
15 | 6 | 2.1 |
16 | 5 | 3.4 |
16 | 6 | 2.4 |
16 | 31 | 3.6 |
Tail to tail |
| |
C-C correlation | Distance, Å | |
12 | 16 | 2.3 |
121 | 132 | 3.4 |
121 | 16 | 2.8 |
Tail to middle |
| |
C-C correlation | Distance, Å | |
131 | 19 | 2.9 |
14 | 9 | 3.4 |
14 | 18 | 3.7 |
15 | 181 | 2.6 |
Middle to middle |
| |
C-C correlation | Distance, Å | |
81 | 17 | 2.8 |
81 | 18 | 3.1 |
Middle to head |
| |
C-C correlation | Distance, Å | |
1 | 81 | 3.5 |
2 | 18 | 6.0 |
31 | 19 | 4.7 |
31 | 9 | 3.5 |
32 | 9 | 2.6 |
5 | 9 | 2.8 |
71 | 17 | 3.6 |
201 | 81 | 5.3 |
201 | 9 | 2.5 |
Table 4. Z values for the Mann-Whitney test
| Uneven-Dimer Columns | Antiparallel-Dimer Layers | Antiparallel-Monomer Columns |
Parallel-dimer layers | 4.8* | 3.6* | 3.4* |
Uneven-dimer columns | - | -0.32† | −0.12† |
Antiparallel-dimer layers | - | - | -0.14† |
*A nonparametric Mann-Whitney test (1) was conducted to evaluate the significance of the optimized structures in the structure determination. The standard normal random variable, Z, for six structure pairs was obtained by the test. The Z value at a significance level of 0.01 is 2.32 in the one-side test. Thus Z shows that the structure optimized for the parallel-dimer layers agreed with experimental distance constraints better than the other three optimized structures at a confidence level of 99%. This allows discrimination of the parallel-dimer layers from the other models.
†
Because Z for the three structure pairs is between −1.0 and 1.0, none of the three structures is significantly different in the agreement evaluation with the experimental constraints.1. Siegel S, Castellan NJ, Jr (1998) Nonparametric Statistics for the Behavioral Sciences (McGraw-Hill, New York), 2nd Ed.
SI Text
NMR Experiments.
The carbon magnetization was prepared by ramped-amplitude cross-polarization from the proton magnetization with a contact time of 2.2 ms. The 1H and 13C RF amplitudes for p /2 pulses were typically 78 and 50 kHz (71 kHz for SPC-5), respectively. All experiments were carried out at 10°C under MAS. 2D zero-quantum and double-quantum 13C-13C dipolar correlation experiments were performed with RFDR (1) and SPC-5 sequences (2), respectively. The proton CW decoupling amplitude during the mixing periods was 95 kHz (100 kHz for SPC-5). The TPPM decoupling amplitude during the evolution period was 81 kHz. 2D proton-driven 13C spin-diffusion spectra were recorded (3, 4). The experimental data matrices 240(t1) ´ 512(t2) were zero-filled to 1,024 ´ 1,024. Thirty-two free induction decays (FIDs) were accumulated (36 for SPC-5). The experimental time was ~21 h (23 h for SPC-5). FIDs were processed with Felix95 (Accelrys Inc., San Diego, CA). The time-domain data were Fourier-transformed with a sinebell window-function at a 90° phase shift. The 13C chemical shift was referred to the methyl signal of hexamethyl benzene resonating at 16.5 ppm from tetramethylsilane according to International Union of Pure and Applied Chemistry recommendations (5).Structural Calculations.
At the beginning of simulated annealing, 18 BChl c molecules were lined up into an extended arrangement. The monomer molecules in the assembly have the same distance constraints but the two molecules forming dimers have different constraints as specified by n in Eq. 2. The high-temperature annealing was simulated at 10,000 K for 60 ps with 0.003-ps steps. Subsequently, the system was cooled from 10,000 to 0 K for 60 ps. The pseudoenergies caused by the constraints were expressed by square well potential functions with soft asymptotes defined by the experimental distances in SI Table 3 with ± 50% errors (6).1. Bennett AE, Rienstra CM, Griffiths JM, Zhen W, Lansbury PT, Jr, Griffin RG (1998) J Chem Phys 108:9463-9479.
2. Hohwy M, Rienstra CM, Jaroniec CP, Griffin RG (1999) J Chem Phys 110:7983-7992.
3. Szeverenyi NM, Sullivan MJ, Maciel GE (1982) J Magn Reson 47:462-475.
4. Castellani F, van Rossum B, Diehl A, Schubert M, Rehbein K, Oschkinat H (2002)
Nature
420:98-102.5. Harris RK, Becker ED, Cabral de Menezes SM, Goodfellow R, Granger P (2001) Pure Appl Chem 73:1795-1818.
6. Brünger AT, Adams PD, Clore GM, Delano WL, Gros P, Grossekunstleve RW, Jiang J-S, Kuszewski J, Nilges M, Pannu NS, et al. (1998) Acta Crystallogr D 54:905-921.