Molecular replacement then and now

Abstract

The `phase problem' in crystallography results from the inability to directly measure the phases of individual diffracted X-ray waves. While intensities are directly measured during data collection, phases must be obtained by other means. Several phasing methods are available (MIR, SAR, MAD, SAD and MR) and they all rely on the premise that phase information can be obtained if the positions of marker atoms in the unknown crystal structure are known. This paper is dedicated to the most popular phasing method, molecular replacement (MR), and represents a personal overview of the development, use and requirements of the methodology. The first description of noncrystallographic symmetry as a tool for structure determination was explained by Rossmann and Blow [Rossmann & Blow (1962), Acta Cryst. 15, 24-31]. The term `molecular replacement' was introduced as the name of a book in which the early papers were collected and briefly reviewed [Rossmann (1972), The Molecular Replacement Method. New York: Gordon & Breach]. Several programs have evolved from the original concept to allow faster and more sophisticated searches, including six-dimensional searches and brute-force approaches. While careful selection of the resolution range for the search and the quality of the data will greatly influence the outcome, the correct choice of the search model is probably still the main criterion to guarantee success in solving a structure using MR. Two of the main parameters used to define the `best' search model are sequence identity (25% or more) and structural similarity. Another parameter that may often be undervalued is the quality of the probe: there is clearly a relationship between the quality and the correctness of the chosen probe and its usefulness as a search model. Efforts should be made by all structural biologists to ensure that their deposited structures, which are potential search probes for future systems, are of the best possible quality.

Keywords: accuracy; models; molecular replacement; quality.

Figure 1

(a) Number of X-ray structures that have been solved using MR from the inception of the PDB (1970) until 15 February 2013: the total number approaches 78 000. (b) Number of X-ray entries that report ‘molecular replacement’ as the method used to solve the structure as a percentage of the total number of X-ray structures deposited in the PDB to 15 February 2013 : almost 60% of the structures have been solved using MR and in the past two years they account for greater than 70% of all depositions.

Figure 2

Schematic representation of the Patterson refinement procedure as applied to the MR solution of Fab domains. The search model (A) has an Fv and an Fc domain [red and green, connected by a hinge (the elbow angle)]; the relative orientation of the two domains in the search model is different from the orientation in the target (T). The rotation search performed with the intact Fab may provide several solutions, each matching one individual domain (B). These solutions are not easily distinguishable or rankable, and subsequent translation searches usually fail. PC refinement allows sampling of the relative orientation of the Fv and Fc regions, from which the ‘best model’ can be identified (D). This model can then easily generate a solution in the translation search.

Figure 3

Solution of the structure of the ternary complex of Escherichia coli dehydrodipicolinate reductase: following the positioning of the central core, the structure was completed using a single N-terminal domain as a search model and the difference Fourier maps generated from the partial solution as the search space. NCS was then used to place two of the remaining three N-terminal domains. The fourth was built into available density following several rounds of refinement of the partial model. The final model revealed that three of the four subunits are in a closed conformation in the ternary complex, with both cofactor and substrate bound to the enzyme, while the fourth subunit is unliganded and in an open conformation.

Figure 4

(a) Distribution of ‘new’ and total SCOP folds (red and yellow) and ‘new’ and total CATH topologies (purple and green) in the PDB. This graph was generated using the tools available in the ‘PDB Statistics’ page of the RSCB PDB (http://www.rcsb.org; Berman et al., 2000 ▶). There has been no new fold reported since 2008 and no new topology since 2009. (b) Distribution of new ‘all-α’ folds over the years: the large majority were discovered between 1990 and 2000, and between then and now the distribution of folds is basically unchanged. (c) Yearly and total reports for the α–α superhelix fold (as defined in SCOP). Even if the total number of folds has not changed, the number of structures within the fold has increased.

Figure 5

Distribution of R factors (as calculated by REFMAC) versus trial number for the brute-force approach molecular-replacement experiment described in the text. Only one solution clearly differentiated itself from the others (green arrow) and corresponded to a search probe with a sequence identity of less than 13% but a final r.m.s.d. on C^α atoms of 1.18 Å.