International
Tables for
Crystallography
Volume F
Crystallography of biological macromolecules
Edited by M. G. Rossmann and E. Arnold

International Tables for Crystallography (2006). Vol. F, ch. 13.4, pp. 287-288   | 1 | 2 |

Section 13.4.13. Recent salient examples in low-symmetry cases: multidomain averaging and systematic applications of multiple-crystal-form averaging

M. G. Rossmanna* and E. Arnoldb

aDepartment of Biological Sciences, Purdue University, West Lafayette, IN 47907-1392, USA, and  bBiomolecular Crystallography Laboratory, CABM & Rutgers University, 679 Hoes Lane, Piscataway, NJ 08854-5638, USA
Correspondence e-mail:  mgr@indiana.bio.purdue.edu

13.4.13. Recent salient examples in low-symmetry cases: multidomain averaging and systematic applications of multiple-crystal-form averaging

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When averaging molecules that have segmental flexibility, it is essential to be able to define the extents of and noncrystallographic relationships among multiple segments which can flexibly reorient. No general protocol has been described for determining the minimum size or optimal number of segments to use in such cases. If the number of segments used for averaging is too small, then the NCS parameters cannot accurately superpose the entirety of the related segments. If too many segments are used for averaging, the segments may become too small for accurate determination of the NCS parameters. The use of too many segments may also become awkward and somewhat inefficient, since in some program systems the total number of maps that must be stored in a given cycle of averaging is proportional to the number of segments used for averaging. Comparison of atomic models for related segments that have been built or refined independently may provide convenient definitions of envelopes for averaging. In practice, a radius of 2 Å or more (depending upon the stage of structure solution and completeness and expected reliability of the model) may be added around the atoms used to define a molecular mask or envelope used in averaging. As with other averaging procedures, multidomain and multiple-crystal-form averaging approaches generally benefit from updating the molecular masks as structure determination progresses.

Often, a macromolecule can be crystallized in multiple crystal forms. Advances in crystallization technology leading to the frequent occurrence of multiple crystal forms, coupled with the availability of convenient programs, have led to increasing frequency of application of multiple-crystal-form averaging for structure solution.

Proteins, especially those containing more than one folded domain, often contain flexible hinges. As long as the boundaries of and noncrystallographic relationships among the related domains in multiple copies can be determined, then density averaging can be used to improve phasing. Programs such as O can be conveniently used to obtain the initial transformations necessary for correct superposition of related segments. NCS parameters can be refined using routines that either minimize the density differences among related copies or that perform rigid-body refinements of atomic models.

A number of experimental techniques have been described that may permit more widespread application of multiple-domain and multiple-crystal-form averaging. Freezing of macromolecular crystals to liquid-nitrogen temperatures has become a routine approach for enhancing the resolution and quality of macromolecular X-ray diffraction data. With most macromolecular crystals, there is a shrinkage of the `frozen' unit cell relative to the lattice of the `unfrozen' crystals. In many cases, significantly different cell dimensions can also be obtained by using different cryo-protective buffer and salt conditions. These variations can be exploited in a systematic fashion for phasing by electron-density averaging, so long as (1) the shrinkage relationships among the different crystals are not merely isotropic and (2) the boundaries and NCS parameters among related segments can be determined. Perutz (Perutz, 1946[link]; Bragg & Perutz, 1952[link]) recognized the potential utility of such shrinkage stages for crystallographic phasing in studies of haemoglobin crystals with varying degrees of hydration.

Recent examples of structure solutions involving multidomain and multiple-crystal-form averaging include studies of HIV reverse transcriptase (RT) (Ren et al., 1995[link]; Ding et al., 1995[link]). Studies of HIV RT by Stuart and coworkers involved multidomain and multiple-crystal-form averaging using different soaking solutions (Esnouf et al., 1995[link]; Ren et al., 1995[link]), in some cases with dramatically improved diffraction resolution. Arnold and coworkers have applied multidomain and multiple-crystal-form averaging to studies of HIV RT, including a systematic application of averaging electron density between `frozen' and `unfrozen' crystal forms (Ding et al., 1995[link]; Das et al., 1996[link]). Tong et al. (1997[link]) recently described electron-density averaging among multiple closely related crystal forms of the human cytomegalovirus protease that were obtained by treatment of the crystals with different soaking buffers containing differing levels of precipitants, such as salt and polyethylene glycol.

References

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