The low-resolution cryo-EM map of γ-secretase has whetted our appetite for a higher resolution structure. However, a crystal structure of the full γ-complex may not be on the horizon just yet. The complex maturation pathway (Spasic and Annaert, 2008
), the general requirement of certain lipids for activity (Osenkowski et al., 2008
), the multiple glycosylations of nicastrin, the tendency to break into subcomplexes in detergents that are more appropriate for structural studies (Fraering et al., 2004a
), the existence of multiple isoforms of Aph-1 and presenilin resulting in at least 6 different γ-complexes (Wakabayashi and De Strooper, 2008
), and the relatively small quantity of the γ-complex that can be produced for crystallization trials (Cacquevel et al., 2008
) are all formidable barriers to achieving the crystal structure of this 19-TMD complex. However, much progress has been made in recent years in over-expressing mammalian membrane proteins (Tate, 2001
), in robot-based nano-crystallography that significantly reduces the required amount of highly purified specimen (Bodenstaff et al., 2002
), and in the availability of synchrotron-based x-ray diffraction equipment enabling the use of ever-smaller crystals (Jiang and Sweet, 2004
). Considering how seemingly impossible an atomic structure of an ion channel or a transporter was twenty years ago and how many such crystal structures are available now (Gouaux and Mackinnon, 2005
), it might be reasonable to expect that the atomic structure of γ-secretase will be achieved in the not-so-distant future.
Besides attempting crystallization of the entire γ-complex, another route for the X-ray crystallography approach would be to solve the structures of individual components or domains of complex. In this regard, signal peptide peptidase (SPP), a homolog of presenilin (Narayanan et al., 2007
), the nicastrin ectodomain (Shah et al., 2005
), Aph1, and PEN-2 might each be a good target for crystallography or NMR structure determination. The presenilin holoprotein has been efficiently expressed alone in Sf9 insect cells and purified from microsomes (Tu et al., 2006
). This provides a unique opportunity for solving the structure of the catalytic subunit of γ-secretase. When the high-resolution structures of these γ-secretase components become available, interpretation of the lower resolution EM structure of the intact γ-complex will be more meaningful.
In recent years, the methodology of single particle cryo-EM has advanced dramatically, to a point that near-atomic resolution structures for a number of very large and highly symmetrical biological assemblies such as the icosahedral or helical virus have been achieved (Jiang et al., 2008
; Yu et al., 2008
; Zhang et al., 2008
). The attainable resolution for several very large protein complexes without any symmetry has also been significantly advanced to well within the nanometer resolution range (Agirrezabala et al., 2008
; Chandramouli et al., 2008
; Matadeen et al., 1999
). It might thus be possible for the atomic resolution cryo-EM structure of a very large asymmetrical complex to be achieved. However, large and tightly packed protein complexes with masses well over 1 MDa are a minority in biology. The vast majority of the known protein complexes are in the size range of 100 – 400 kDa, a range to which γ-secretase belongs. From a structural biologist’s point of view, it is a difficult middle ground, since this range is somewhat too big for x-ray crystallography and too small for single particle cryo-EM.
Cryo-EM likely will continue to play an important role in our understanding of the structure and function of the γ-secretase complex. In the absence of atomic structure of the γ-secretase by crystallography, more work by cryo-EM might further improve the resolution of the existing structure of the γ-secretase complex; it might be possible to determine the EM structure at better than 10 Å resolution. A structure at this level of resolution is far more desirable or informative than the 12 Å structure we have obtained, because many of the predicted transmembrane helices would be resolved, so that homology-based modeling or docking of the component atomic structures, once available, into the EM structure could be carried out. Furthermore, the potential substrate docking site and the catalytic site could also be visualized by determining the cryo-EM structures of a catalytically inactive (aspartate-mutant) γ-secretase in complex with substrates or helical peptide inhibitors (Bihel et al., 2004
). When an atomic structure is finally achieved by x-ray crystallography, the EM structure may serve to cross-validate the physiological relevance of crystal structures that could be influenced by the crystalline packing. An interesting case in point is the role cryo-EM played in supporting the protein-lipid interface movement model of the voltage sensor paddle as proposed from the crystal structures of potassium channels (Jiang et al., 2004
Within the cryo-EM single particle approach, despite its theoretical potential for determining relatively small protein structures down to 40 kDa (Henderson, 1995
), several practical issues, including beam-induced specimen charging and specimen movement, have so far hindered its realization. Another problem in single particle cryo-EM is the reliance on phase contrast generated by deeply under-focusing the objective lens. A large defocus value not only scrambles the higher frequency information in the micrographs that makes accurate information recovery difficult but also significantly cuts off the very low frequency information that is essential for image alignment. The emerging technique of phase plate enables in-focus electron imaging while still providing high image contrast (Cambie et al., 2007
; Danev and Nagayama, 2001
). This technique will likely make a significant difference in cryo-EM structural determination of relatively small protein complexes such as γ-secretase.
The cryo-EM method also encompasses another approach that is called electron crystallography (Fujiyoshi and Unwin, 2008
). With this approach, detergent-purified membrane proteins are reconstituted back into a lipid bilayer, forming two-dimensional crystalline sheets. The structures then are determined by extracting the amplitudes from electron diffraction patterns and the phases from high-resolution electron images of these crystalline sheets (Glaeser et al., 2007
). Membrane protein structures solved by this method are invaluable in evaluating the x-ray crystal structures that are necessarily solved in detergents (Fujiyoshi and Unwin, 2008
). For γ-secretase, the lipid environment appears to be particularly important, since its proteolytic function occurs within the lipid bilayer, and this function is regulated by the lipid composition (Osenkowski et al., 2008
). Many 2D crystals of membrane proteins reported so far do not diffract to a high resolution; nevertheless, there are several examples where atomic resolution structures have been solved by electron crystallography (Fujiyoshi and Unwin, 2008
; Gonen et al., 2005
). Therefore, we hope that electron crystallography will also contribute to the structural assault on the γ-secretase complex.