research papers
Using electron-microscopy images as a model for molecular replacement
aYork Structural Biology Laboratory, Chemistry Department, University of York, York YO10 5DD, England
*Correspondence e-mail: e.dodson@ysbl.york.ac.uk
This review addresses the technical problems encountered while using models based on T = 4 icosohedral symmetry.
to generate initial phases for crystallographic studies. The test cases used were the gp6 portal protein with 13-fold rotational symmetry and the human hepatitis virus HepB, a viral assembly withKeywords: molecular replacement; electron microscopy.
1. Introduction
The electron microscope provides a way of visualizing the structures of large macromolecular assembles. To complete a three-dimensional reconstruction of a single particle, images of the specimen viewed from different angles are aligned and the model built up. If the same assembly can be crystallized, the electron-microscopy (EM) model and phases can be used as a starting point for the X-ray
Before this can be performed, the EM model must be correctly positioned in the in other words, the molecular-replacement solution must be obtained.Molecular replacement is a technique for matching observed crystal diffraction intensities to those predicted by a suitable model correctly orientated and positioned within the AMoRe, MOLREP) or by using standard software for calculating structure factors, which then provide the input to an MR package such as ALMN. It is convenient to generate these assuming that the model lies at the origin of a P1 cell with `crystal' axes at least twice the model diameter. Many packages such as AMoRe use model structure factors which are more finely sampled in i.e the `crystal' into which the model is placed has axes three or four times the model diameter (Lattman, 1985; Navaza, 2001). This means that the structure factors corresponding to different orientations of the model can be obtained by interpolation from the initial set. This saves a great deal of computer time without a serious loss of accuracy and allows many orientations to be explored rapidly. The structure factors generated by the model at a given position in the are the sum of symmetry-equivalent reflections with phase corrections appropriate for the space group.
Usually, the model is described using coordinates and the predicted intensity pattern can be generated either within the molecular-replacement program (The usual procedure for generating structure factors for large molecules first generates an `atom map' where the contribution from all atoms is summed onto a regular grid. Once such a map has been constructed, the structure factors can be generated very quickly using the inverse fast Fourier transform. Since electron microscopists conventionally construct `maps' from their phase estimations, these too can generate a structure-factor list in a suitable `cell' for input to the existing MR packages.
In addition to the technical issues to be resolved, in any interdisciplinary research we face the serious problem of properly understanding the difficulties and the conventions of our partners. The CCP4 Study Weekend on Low Resolution Phasing in 2000 had brought together electron microscopists and crystallographers and the Proceedings [Acta Cryst. (2000), D56, 1205–1357] give an excellent starting point with good descriptions of the techniques. The comprehensive review by Baker et al. (1999) is also very helpful.
Another difficulty is the paucity of test data sets. There is not yet a mechanism for the deposition of the experimental results of et al., 1999), R. A. Crowther (Bottcher et al., 1997) and A. Leslie (Wynne et al., 1999) for providing images and X-ray data for these studies.
I am very indebted to E. Orlova (Orlova2. Problems
Technical problems to consider are as follows.
3. Test cases
3.1. Bacteriophage SPP1 portal protein (gp6)
This portal protein, located at one single vertex of the icosahedral viral capsid, is a central component acting at different steps of tailed bacteriophage's assembly. It is a circular multi-subunit assembly composed of 13 identical subunits through which DNA movements occur and where the phage tail is attached. This protein has been a subject of electron-microscopy single-particle reconstruction studies which have led to the determination of the structure of native gp6 and of the mutant gp6 SizA (Fig. 1) (Tavares et al., 1992; Orlova et al., 1999 and references therein).
gp6 has also been crystallized in C2221, with unit-cell parameters 173.42, 222.61, 420.12 Å (Jekow et al., 1998), and X-ray data has now been collected to 3.4 Å. With the current technology at the ESRF it was possible to record all the low-resolution data to 80 Å. The unit-cell volume requires that there be only one assembly of gp6 in the sited at a general position in the unit cell.
3.2. Human hepatitis B virus capsid (HepB)
This viral assembly consists of 240 subunits arranged with T = 4 icosahedral symmetry. Its structure was determined first by single-particle electron cryomicroscopy at 7.4 Å resolution (Bottcher et al., 1997) and then crystallized and solved using X-ray techniques (Wynne et al., 1999). The is C2, with unit-cell parameters 538.4, 354.8, 370.1 Å, β = 132.3°. The unit-cell volume required that the crystal contained only one half of the icosahedra and that the complete unit was generated by the crystallographic twofold axis. This meant that the particle be positioned on the twofold rotation axis and limited the MR problem to orientating the particle relative to the crystal axes.
4. Methodology
4.1. Self-rotation searches
Since the expected results for the self rotations were known (a 13-fold rotation axis for gp6 and T = 4 viral symmetry for HepB), we were able to use self-rotation functions to test various parameters, such as the sphere radii, and the best resolution limits. The outer range was restricted to the limit available from the EM model (about 12 Å for gp6 and 7.4 Å for HepB). The inner resolution limit was varied to give the strongest signal from the protein symmetry, i.e where the protein contribution to the X-ray amplitudes dominated that from the solvent (Fig. 2). For gp6, the maps generated using data from 80–50 Å showed no 13-fold symmetry at all, indicating that this shell of data is dominated by the solvent diffraction. This low-resolution information helped to determine the translation parameters and was valuable for phase-extension procedure, but was not useful for the rotation search.
4.2. Cross-rotation searches
As described above, structure factors were generated from both the EM maps. The 〈F〉 distribution for these are shown in Fig. 3 for comparison with the 〈F〉 distribution of the X-ray amplitudes. The cross rotation requires that these distributions are similar for the measured and calculated amplitudes; for more conventional studies, this is usually controlled by modifying the relative temperature-factor corrections. It is often advantageous to sharpen both data sets to increase the signal from the outer resolution shells, but matching the EM and X-ray distributions is tricky. In many cases, the strongest X-ray observations at low resolution have been lost; the increases sharply, increasing the risk that the reflections will overload the detector and be rejected during data processing. However, we found the best results were obtained using data in the range 30–15 Å, after applying a relative temperature factor of 400 Å to sharpen the calculated EM amplitudes. Although EM amplitudes had been generated to 10 Å, the 15–10 Å data did not improve the signal, presumably because the reliability of the EM results falls off. The best sphere radius was 50 Å, which covered a reasonable volume of the assembly without including too much of the central cavity. We knew the direction of the 13-fold axis in the crystal from the self-rotation results; it lay in the bc* plane at 25° to c*. This meant that the 13 cross-rotation peaks should satisfy ω = 25, Φ = 90° and occur at regular intervals of 27.7° round κ. AMoRe, MOLREP and ALMN all gave clear answers, with the best results obtained after scaling the EM images by a factor of 1.01.
The HepB case was simpler. The X-ray self rotation showed the T = 4 viral symmetry beautifully with its twofold, fivefold and sixfold rotation axes and after a rotation of 14.9° around the crystallographic twofold it aligned perfectly with the pattern from the EM images. As expected, ALMN gave a clear solution with a sphere radius of 60 Å using sharpened data in the resolution range 18–7.4 Å.
4.3. Translation searches
Crystal symmetry and asymmetric volume required that the HepB particle be positioned on the crystallographic twofold axis, so there was no need to perform a translation search for this case. For GP6, one copy of the assembly had to be positioned in the AMoRe using rescaled data in the range 50–15 Å. The for the centre-overlap function was 39% increasing to 61% after fitting. The next ranking was 59%, only 2% lower than the correct solution but not consistent with the self-rotation results. We repeated the search using a variety of resolution ranges and temperature-factor corrections and found that this result was robust.
The best signal was obtained fromIt must be remembered that even when a particle is correctly positioned in the cell, the phase-extension step is challenging. This has been discussed in the review by Rossmann (1995) and by Mancini & Fuller (2000).
5. Conclusions
The joint exploitation of
and crystallography will doubtless be used more extensively as X-ray data from larger and larger macromolecular assemblies becomes available. At least in the test cases reported here, which have a high degree of internal symmetry, optimal parameters for resolution limits and integration sphere radius could be found which led to successful molecular replacement.Acknowledgements
The gp6 study depended on the beautiful EM images provide by Elena Orlova and her collaborators. The data collection by Fred Antson and Margaret Krause was an epic struggle, especially to obtain excellent very low resolution terms. The molecular-replacement analysis was performed in collaboration with Dr Julie Wilson. I am very grateful to Andrew Leslie and R. A. Crowther for allowing me the opportunity to use their superb HepB images and X-ray data. Further details of their analysis will be published elsewhere. Funding is provided by Wellcome Trust (grant 062788).
References
Baker, T. S., Olson, N. H. & Fuller, S. D. (1999). Microbiol. Mol. Biol. Rev. 63, 862–922. Web of Science PubMed CAS Google Scholar
Bottcher, B., Wynne, S. A. & Crowther, R. A. (1997). Nature (London), 386, 88–91. CAS PubMed Web of Science Google Scholar
Collaborative Computational Project, Number 4 (1994). Acta Cryst. D50, 760–763. CrossRef IUCr Journals Google Scholar
Dröge, A., Santos, M. A., Stiege, A., Alonso, J. C., Lurz, R., Trautner, T. A. & Tavares, P. (2000). J. Mol. Biol. 296, 117–132. Web of Science CrossRef PubMed CAS Google Scholar
Dube, P., Tavares, P., Lurz, R. & van Heel, M. (1993). EMBO J. 12, 1303–1309. CAS PubMed Web of Science Google Scholar
Jekow, P., Schaper, S., Günther, D., Tavares, P. & Hinrichs, W. (1998). Acta Cryst. D54, 1008–1011. Web of Science CrossRef CAS IUCr Journals Google Scholar
Lattman, E. (1985). Methods Enzymol. 115, 55–77. CrossRef CAS PubMed Google Scholar
Lurz, R., Trautner, T. A. & Alonso, J. (1997). J. Mol. Biol. 268, 822–839. CrossRef PubMed Web of Science Google Scholar
Mancini, E. J. & Fuller, S. D. (2000). Acta Cryst. D56, 1278–1287. Web of Science CrossRef CAS IUCr Journals Google Scholar
Navaza, J. (2001). Acta Cryst. D57, 1367–1372. Web of Science CrossRef CAS IUCr Journals Google Scholar
Orlova, E., Dube, P., Beckmann, E., Zemlin, F., Lurz, R., Trautner, T. A., Tavares, P. & van Heel, M. (1999). Nature Struct. Biol. 6, 842–846. Web of Science PubMed CAS Google Scholar
Rossmann, M. G. (1995). Curr. Opin. Struct. Biol. 5, 650–655. CrossRef CAS PubMed Web of Science Google Scholar
Tavares, P., Santos, M. A., Lurz, R., Morelli, G., Lencastre, H. & Trautner, T. A. (1992). J. Mol. Biol. 225, 81–92. CrossRef PubMed CAS Web of Science Google Scholar
Wynne, S. A., Crowther, R. A. & Leslie, A. G. W. (1999). Mol. Cell, 3, 771–780. Web of Science CrossRef PubMed CAS Google Scholar
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