Resource for the Visualization of Biological Complexity (RVBC)

Single-particle Approaches

Three-dimensional reconstruction of single (i.e., isolated, freestanding) macromolecules from low-dose electron micrographs is a technique pioneered in Albany (Frank et al., 1978; 2006; Radermacher et al., 1987; Frank, 1996; 2006). Briefly, molecules are flash-frozen in a thin (<1000 Å) layer of water, which becomes vitrified. The single-particle projections visible in the electron micrograph show the molecule in many different views. In the single-particle reconstruction approach, the relative angles between these views are determined, either by setting up a defined data collection geometry (as in the random-conical reconstruction approach; see Radermacher et al., 1987) or by making use of intrinsic mathematical relationships among different projections of the same object (common lines, or angular reconstitution approach; van Heel, 1987; Penczek et al., 1996). Based on this initial estimate of angles, a first, coarse 3D reconstruction is computed.

Next, this 3D density map is refined by an iterative technique that consists of the following steps: (i) compute a set of equispaced projections from the density map (the reference), covering the entire half-sphere of orientations; (ii) compare each of the experimental images with each of the computed (predicted) projections, to find the angle of best match; (iii) with the new angle assignments, compute a new, refined 3D map; (iv) compare the new set of angles with the previous set of angles. If the discrepancy is below a certain threshold, stop, else go to (i) for a new iteration. This approach of refinement is termed 3D projection matching (Penczek et al., 1994).

After refinement, the density maps are corrected for the effects of the contrast transfer function, which causes the Fourier components sitting in different zones of the Fourier transform to have alternate phases. Another correction concerns the falloff of the Fourier amplitudes, which is caused by instabilities of the stage and charging. This correction is guided by experimental average amplitude curves obtained by low-angle X-ray scattering (Gabashvili et al., 2000).

Density maps representing molecules at 8-15 Å resolution can be interpreted by docking X-ray coordinates of known components. For resolutions of 12 Å or better, 3D motif search based on locally normalized cross-correlation can be employed (Rath et al., 2003).

The single-particle reconstruction approach makes use of the assumption that all molecules visible on the micrograph have the same structure and conformation. If this assumption is not met, then classification methods must be used to extract the homogeneous sub-populations. The most straightforward way to achieve this is by using two or more different 3D references that represent the different structures or conformations (Valle et al., 2002; Gao et al., 2004). Each particle image is then compared with the set of the references to find the one that it most closely resembles. This method obviously fails if the alternative reference structures are not available.

All operations that are required for single-particle reconstruction are contained in the package SPIDER (Frank et al., 1981; 1996; 2006), which was developed in Albany, is being distributed for a nominal license fee, and maintained and further developed as part of the RVBC.

Single-particle reconstruction has been applied to a large number of macromolecular complexes, and has enabled the study of molecular machines engaged in many functions pivotal for the maintenance of the cell, such as DNA repair, transcription, splicing, translation, protein folding, and protein decay. In-house projects at the Wadsworth Center benefiting from this technology are centered around the structure and function of the ribosome (for recent articles, see Gao et al., 2003; Valle et al., 2003; Spahn et al., 2004), the ryanodine receptor (Sharma et al., 2000; Wagenknecht et al., 2002), and cytoplasmic dynein (Samso and Koonce, 2004).

References

Frank, J. (ed.) (2006) Three-dimensional Electron Microscopy of Macromolecular Assemblies - Visualization of Biological Molecules in Their Native State. Oxford University Press, New York.

Frank, J., Goldfarb, W., Eisenberg, D., and Baker, T.S. (1978). Reconstruction of glutamine synthetase using computer averaging. Ultramicroscopy 3: 283-290.

Frank, J., Shimkin, B., and Dowse, H. (1981). SPIDER - A modular software system for image processing. Ultramicroscopy 6: 343.

Frank, J., Radermacher, M., Penczek, P., Zhu, J., Li, Y., Ladjadj, M., and Leith, A. (1996). SPIDER and WEB: Processing and visualization of images in 3D electron microscopy and related fields. J. Struct. Biol. 116: 190-199.

Gabashvili, I.S., Agrawal, R.K., Spahn, C.M.T., Grassucci, R.A., Svergun, D.I., Frank, J., and Penczek, P. (2000). Solution structure of the E. coli ribosome at 11.5 Å resolution. Cell 100: 51-63.

Gao, H., Sengupta, J., Valle, M., Korostelev, A., Eswar, N., Stagg, S.M., Van Roey, P., Agrawal, R.K., Harvey, S.C., Sali, A., Chapman, M.S., and Frank, J. (2003). Study of the structural dynamics of the E. coli 70S ribosome using real space refinement. Cell 113: 789-801.

Gao, H., Valle, M., Ehrenberg, M. and Frank, J. (2004). Dynamics of EF-G interaction with the ribosome explored by classification of a heterogeneous cryo-EM dataset. J. Struct. Biol. 147: 283-290.

Penczek, P., Grassucci, R. and Frank, J. (1994). The ribosome at improved resolution: new techniques for merging and orientation refinement in 3D cryo electron microscopy of biological particles. Ultramicroscopy 53: 251-270.

Penczek, P., Zhu, J. and Frank, J. (1996). A common-lines based method for determining orientations for N>3 particle projections simultaneously. Ultramicroscopy 63: 205-218.

Radermacher, M., Wagenknecht,T., Verschoor, A. and Frank, J. (1987). Three-dimensional reconstruction from a single-exposure random conical tilt series applied to the 50S ribosomal subunit of Escherichia coli. J. Microscopy 146: 113-136.

Rath, B.K., Hegerl, R., Leith, A., Shaikh, T.R., Wagenknecht, T. and Frank, J. (2003) Fast 3D motif search of EM density maps using a locally normalized cross-correlation function. J. Struct. Biol. 144: 95-103.

Samso, M. and Koonce, M.P. (2004). 25 Å resolution structure of a cytoplasmic dynein motor reveals a seven-number planar ring. J. Mol. Biol. 340: 1059-1072.

Sharma, M.R., Jeyakumar, L.H., Fleischer, S., Wagenknecht, T. (2000). Three-dimensional structure of yanodine receptor isoform three in two conformational states as visualized by cryo-electron microscopy. J. Biol. Chem. 275: 9485-9491.

Spahn, C.M.T., Jan, E., Mulder, A., Grassucci, R.A., Sarnow, P., and Frank, J. (2004) Cryo-EM visualization of a viral internal ribosome entry site (IRES) bound to human 40S and 80S ribosomes: the IRES functions as an RNA-based translation factor. Cell 118: 465-475.

Valle, M., Sengupta, J., Swami, N.K., Grassucci, R.A., Burkhardt, N., Nierhaus, K.H., Agrawal, R.K., and Frank, J. (2002). Cryo-EM reveals an active role for the aminoacyl-tRNA in the accomodation process. EMBL J. 21: 3557-3567.

Valle, M., Zavialov, A., Sengupta, J., Rawat, U., Ehrenberg, and Frank, J. (2003b). Locking and unlocking of ribosomal motions. Cell 114, 123-134.

van Heel, M. (1987) Angular reconstitution: a posteriori assignment of projection directions for 3D reconstruction. Ultramicroscopy 21: 111-124.

Wagenknecht, T., Hsieh, C.-Ee, Rath, B.K., Fleischer, S., Marko, M. (2002). Electron Tomography of Frozen-Hydrated Isolated Triad Junctions. Biophys. J. 83: 2491-2501.