Tables for
Volume B
Reciprocal space
Edited by U. Shmueli

International Tables for Crystallography (2006). Vol. B. ch. 4.5, pp. 479-480   | 1 | 2 |

Section Other techniques

R. P. Millanea* Other techniques

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Aside from the techniques for structure determination described in the previous sections, a variety of other techniques have been applied to specific problems where the methods described above are not suitable. This situation usually arises where the diffraction data available are far too few, by themselves, to determine the individual atomic coordinates of a structure, even with the usual stereochemical constraints. Often only relatively low-resolution data are available, but they can be supplemented by either a low-resolution or high-resolution model of either a whole molecule or relatively large subunits. Structure determination often amounts to positioning the molecules or subunits within a larger assembly. The results can be quite precise, depending on the information available. The problem is almost always one of refinement or optimization, since it invariably involves optimizing some kind of model directly against the fibre diffraction data. The problem is usually twofold: (1) parameterizing the model with few enough parameters to obtain a usable data-to-parameter ratio, but retaining enough degrees of freedom to represent the important structural features; and (2) devising an optimization procedure that will locate the global minimum of the resulting complicated cost function. There have been numerous such applications in fibre diffraction, and rather than attempt to be exhaustive or detailed, I will briefly mention a few of the more prominent applications and techniques.

The structure of the bacteriophage Pf1 was determined at 7 Å resolution using a model in which the α-helical segments of the structure were represented by rods of electron density of appropriate dimensions and spacings (Makowski et al., 1980[link]). The positions and orientations of the rods were refined in an iterative procedure that alternated between real space and reciprocal space and also incorporated solvent levelling. Neutron fibre diffraction data have been collected from specifically deuterated phages and, starting with a model of the kind described above, iterative application of difference maps (between the deuterated and native data) was used to locate 15 (of the 46) residues, allowing construction of a model of the coat protein (Stark et al., 1988[link]; Nambudripad et al., 1991[link]).

Pf1 undergoes a temperature-induced structural transition that involves a small change in the helix symmetry. The low-temperature form has 7113 helix symmetry with a c repeat of 216.5 Å, and the high-temperature form (that discussed in the previous paragraph) has 275 helix symmetry and a c repeat of 78.3 Å. These two symmetries are very similar since [71/3 \simeq 27/5] and [216.5/71 \simeq 78.3/27], i.e. the rotations and translations from one subunit to the next are very similar in both structures.

The structure of the low-temperature form of Pf1 has been determined at 3.3 Å resolution by starting with an α-helical polyalanine model (Marvin et al., 1987[link]) and alternating rounds of molecular-dynamics refinement and model rebuilding based on [(2F_o - F_c)] maps and omit maps (Gonzalez et al., 1995[link]). The structure of the high-temperature form of Pf1 was determined using data to 3 Å resolution, starting with a model based on the low-temperature form, making small adjustments to satisfy the slightly different helix symmetry, and refining the model using molecular dynamics (Welsh et al., 2000[link]).

The bacteriophage Pf3 is related to Pf1 but does not undergo a structural transition, and fibre diffraction patterns are similar to those from the high-temperature form of Pf1. An α-helical polyalanine model of Pf3 based on the Pf1 structure was used to separate and phase the Bessel terms, which were then used to calculate [(5F_o - 4F_c)] maps. These maps were used to align and position the polypeptide chain, and the resulting model was refined by molecular dynamics (Welsh et al., 1998[link]).

The R-type bacterial flagellar filament structure (that has a very high molecular weight subunit) has been determined at 9 Å resolution by X-ray fibre diffraction (Yamashita et al., 1998[link]). Accurate intensities were taken from high-quality X-ray diffraction patterns and combined with phases obtained from electron cryomicroscopy, and solvent levelling was used to refine the phases.

Some studies of muscle provide a good example of the use of low-resolution fibre diffraction data, coupled with high-resolution crystal structures of some of the component molecules, to determine the structure of a complex. Holmes et al. (1990)[link] constructed a model of F-actin based on the crystal structure of the monomer, G-actin, and 8 Å fibre diffraction data, by either treating the monomer as a rigid body or dividing it into four separate rigid domains, and using a search procedure followed by least-squares refinement. The results gave the orientation of the actin monomer in the actin helix. This structure has since been refined using a genetic algorithm (Lorenz et al., 1993[link]) and normal-mode analysis (Tirion et al., 1995[link]). The genetic algorithm involved a Monte Carlo method of selecting subdomains to be refined and nonlinear least squares to obtain the best fit for the selected domains. In the normal-mode analysis, the model was parameterized in terms of its low-frequency vibrational modes to allow low-energy conformational changes and reduce the number of parameters which were optimized against the fibre diffraction data using nonlinear least squares.

Squire et al. (1993)[link] have refined a low-resolution model of the muscle thin-filament structure that consists of four spheres representing each of the F-actin monomer subdomains and five spheres (fixed relative to each other) representing tropomyosin. Steric restraints were placed on the actin subdomain and thin-filament structures. The positions of the actin subdomains and the orientation of the tropomyosin were refined using a search procedure against fibre diffraction data from both `resting' and `activated' muscle at 25 Å resolution. More recent work has used a low-resolution model of the myosin head (based on the single-crystal atomic structure), a search procedure and simulated-annealing refinements to study myosin head configuration (Hudson et al., 1997[link]) and myosin rod packing (Squire et al., 1998[link]).


First citationGonzalez, A., Nave, C. & Marvin, D. A. (1995). Pf1 filamentous bacteriophage: refinement of a molecular model by simulated annealing using 3.3 Å resolution X-ray fiber diffraction data. Acta Cryst. D51, 792–804.Google Scholar
First citationHolmes, K. C., Popp, D., Gebhard, W. & Kabsch, W. (1990). Atomic model of the actin filament. Nature (London), 347, 44–49.Google Scholar
First citationHudson, L., Harford, J. J., Denny, R. C. & Squire, J. M. (1997). Myosin head configuration in relaxed fish muscle: resting state myosin heads must swing axially by up to 150 Å or turn upside down to reach rigor. J. Mol. Biol. 273, 440–455.Google Scholar
First citationLorenz, M., Popp, D. & Holmes, K. C. (1993). Refinement of the F-actin model against X-ray fibre diffraction data by the use of a directed mutation algorithm. J. Mol. Biol. 234, 826–836.Google Scholar
First citationMakowski, L., Caspar, D. L. D. & Marvin, D. A. (1980). Filamentous bacteriophage Pf1 structure determined at 7 Å resolution by refinement of models for the α-helical subunit. J. Mol. Biol. 140, 149–181.Google Scholar
First citationMarvin, D. A., Bryan, R. K. & Nave, C. (1987). Pf1 inovirus. Electron density distribution calculated by a maximum entropy algorithm from native fiber diffraction data to 3 Å resolution and single isomorphous replacement data to 5 Å resolution. J. Mol. Biol. 193, 315–343.Google Scholar
First citationNambudripad, R., Stark, W. & Makowski, L. (1991). Neutron diffraction studies of the structure of filamentous bacteriophage Pf1. J. Mol. Biol. 220, 359–379.Google Scholar
First citationSquire, J., Cantino, M., Chew, M., Denny, R., Harford, J., Hudson, L. & Luther, P. (1998). Myosin rod-packing schemes in vertebrate muscle thick filaments. J. Struct. Biol. 122, 128–138.Google Scholar
First citationSquire, J. M., Al-Khayat, H. A. & Yagi, N. (1993). Muscle thin-filament structure and regulation. Actin sub-domain movements and the tropomyosin shift modelled from low-angle X-ray diffraction. J. Chem. Soc. Faraday Trans. 89, 2717–2726.Google Scholar
First citationStark, W., Glucksman, M. J. & Makowski, L. (1988). Conformation of the coat protein of filamentous bacteriophage Pf1 determined by neutron diffraction from magnetically oriented gels of specifically deuterated virions. J. Mol. Biol. 199, 171–182.Google Scholar
First citationTirion, M., ben Avraham, D., Lorenz, M. & Holmes, K. C. (1995). Normal modes as refinement parameters for the F-actin model. Biophys. J. 68, 5–12.Google Scholar
First citationWelsh, L. C., Symmons, M. F. & Marvin, D. A. (2000). The molecular structure and structural transition of the α-helical capsid in filamentous bacteriophage Pf1. Acta Cryst. D56, 137–150.Google Scholar
First citationWelsh, L. C., Symmons, M. F., Sturtevant, J. M., Marvin, D. A. & Perham, R. N. (1998). Structure of the capsid of Pf3 filamentous phage determined from X-ray fiber diffraction data at 3.1 Å resolution. J. Mol. Biol. 283, 155–177.Google Scholar
First citationYamashita, I., Hasegawa, K., Suzuki, H., Vonderviszt, F., Mimori-Kiyosue, Y. & Namba, K. (1998). Structure and switching of bacterial flagellar filaments studied by X-ray fiber diffraction. Nature Struct. Biol. 5, 125–132.Google Scholar

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