Single particle electron microscopy

Abstract

Electron microscopy (EM) in combination with image analysis is a powerful technique to study protein structures at low, medium, and high resolution. Since electron micrographs of biological objects are very noisy, improvement of the signal-to-noise ratio by image processing is an integral part of EM, and this is performed by averaging large numbers of individual projections. Averaging procedures can be divided into crystallographic and non-crystallographic methods. The crystallographic averaging method, based on two-dimensional (2D) crystals of (membrane) proteins, yielded in solving atomic protein structures in the last century. More recently, single particle analysis could be extended to solve atomic structures as well. It is a suitable method for large proteins, viruses, and proteins that are difficult to crystallize. Because it is also a fast method to reveal the low-to-medium resolution structures, the impact of its application is growing rapidly. Technical aspects, results, and possibilities are presented.

Fig. 1

An example of the footprint effect of negative staining. a A part of a double-layered two-dimensional crystal containing about 1500 photosystem I monomers from a cyanobacterium (Böttcher et al. 1992). b, c Filtered images resulting from a crystallographic analysis in which the two layers could be separated. The crystal is composed of rows of monomers. Within the rows, the monomers are either up- or down-oriented, and there is a substantial difference in overall contrast between individual rows of monomers in the upper layer with respect to the lower layer. d Scheme explaining how the uneven stain density (brown) causes the difference between the two layers, which are identical in protein arrangement

Fig. 2

The basics of single particle EM, explained from an analysis of the photosystem I–IsiA supercomplex from the cyanobacterium Synechococcus 7942, extracted from negatively stained EM specimens (Boekema et al. 2001). After translational and rotational alignment of a data set of about 5000 single particle projections showing the complex in a position as in the membrane plane, sums with increasing numbers of copies in equivalent positions show the gradual improvement in the signal-to-noise ratio (upper part of the picture). However, these particle projections may not all be identical, because small tilt variations on the membrane plane may lead to different positions. Indeed, after multivariate statistical analysis and classification, it became clear that only a small number of projections show threefold rotational symmetry which is indicative for a position parallel to the membrane (lower row, left). The other two classes (middle and right) show the supercomplex in tilted positions

Fig. 3

Example of single particle analysis on a large water-soluble protein, the 180-subunit hemoglobin of the earth worm Lumbricus terrestris. a (Boekema and van Heel 1989). b Sum of 1024 particles at 11 Å resolution in negative stain (R. Kouřil unpublished). c, d Two views of a 3D reconstruction at 13 Å resolution (W. Keegstra and G.T. Oostergetel, unpublished). e, f Model of the high-resolution (3.5 Å) X-ray structure (Royer et al. 2006)

Fig. 4

Supercomplexes of photosystem I–IsiA (PSI–IsiA) with variable amount of flexibility. a The supercomplex consisting of trimeric PSI and a ring of 18 IsiA copies, see Fig. 1. b, c Monomeric PSI with rings of 14 and 21 IsiA copies, respectively. The difference in detail between the two rings is related to the alignment procedure, see text. d–e Monomeric PSI complexes associated with an incomplete inner ring and outer ring. The inner ring is composed of six IsiA copies in register. f Monomeric PSI complex with a flexible attachment of incomplete inner and outer rings with a larger number of IsiA copies. Space bar for all frames equals 100 Å

Fig. 5

Analysis of the C₂S₂M₂ supercomplex of photosystem II. a A projection map at about 13 Å shows the exact positions of S-trimers and M-trimer of the LHCII; the triangles indicate the position of the threefold symmetry axis in the center of the trimer. b A projection map, focused on improving the centre of the supercomplex plus the S-trimer region. In this map, these areas have been slightly sharpened, but at the cost of the M-trimer. Note: no symmetry was imposed during or after the analysis. Space bar equals 100 Å

Fig. 6

Exploring transient membrane complexes by applying single particle EM without purification steps. A gallery of 2D projection maps of solubilized membrane complexes from the cyanobacteria Thermosynechoccus elongatus and Synechocystis PCC 6803. a NDH-1 side view from T. elongatus b NDH-1 top view from T. elongatus. c Purified NDH-1 from Synechocystis (reproduced from Arteni et al. 2006). d Photosystem II dimeric complex from Synechocystis. e Photosystem II double dimer complex from Synechocystis. f Rod-like protein complex of unknown origin/function with a variable extension at the base, which could be detergent and lipid, from T. elongatus. g, h A water-soluble hexagonal particle, tentatively assigned to glutamine synthetase in top- and side-view position, respectively. i Cyanobacterial fragment with trimeric symmetry assigned to allophycocyanin. j Trimeric photosystem I complex. k Proton ATP synthase complex. l Structure assigned to the GroEL-GroES supercomplex. Space bar for all frames equals 100 Å