In spite of its many achievements during recent years, the technique of single particle cryoEM has not been widely used to study proteins smaller than 100kDa, because it is generally difficult to align images of small particles embedded in vitreous ice accurately and even harder to validate the 3D reconstruction. For large macromolecular assemblies, each single particle image contains sufficient information for accurate alignment to near-atomic resolution (Armache et al., 2010
; Chen et al., 2009
; Wolf et al., 2010
; Yu et al., 2008
; Zhang et al., 2010
). However, small particles often do not have easily recognizable structural features and appear as a single dot in images of vitrified samples (Figure S1C
). These images simply do not contain sufficient structural information for accurate image alignment (Henderson, 1995
), even when they can be visualized with high contrast. Thus, the limitation inherent to aligning such images to each other is a major obstacle for high-resolution structural analysis of small proteins by single particle cryoEM. As pointed out many years ago by Henderson (Henderson, 1995
), overcoming this limitation requires some kind crystals or geometrically ordered aggregates such as a large assembly with a icosahedral symmetry. Following this suggestion, many efforts have been invested in developing novel approaches to enable structure determination of small proteins by single particle cryoEM. One of the methods proposed was to fuse a small protein to the nucleocapsid of hepatitis B virus (HBV) particles so that the highly symmetrical HBV particle facilitates the alignment of the fused small particles (Kratz et al., 1999
). However, such an approach has very limited applicability, and is not suitable for studying small protein complexes. Also, the linker between the large assembly and the small target protein is often flexible and limits the achievable resolution.
The method we presented here uses a Fab to overcome these limitations by increasing the effective particle size and providing a recognizable structural feature required for accurate image alignment. The molecular weights of PCSK9 and IN dimer by themselves are each less than 70 kDa, but when bound to one or two Fabs, the complexes are more than 100 kDa. More importantly, clearly recognizable Fab feature in the complexes allow accurate image alignment. Also, when there is no previous knowledge about the overall architecture of a small protein, it is difficult to generate a reliable 3D initial model and, given the low resolution, there are no good means to validate the correctness of the final 3D reconstruction. However, when a Fab forms a complex with the protein, its presence in the 3D density map strongly supports the correctness of the reconstruction. In addition, there are other practical benefits of forming a protein-Fab complex for single particle cryoEM studies. For instance, sample heterogeneity could be overcome by identifying and selecting correct particles facilitated by the presence of Fabs in the complex.
Our 3D reconstruction of the frozen hydrated IN-Fab5 complex, with a molecular weight of less than 160kDa, has a resolution of close to 10 Å. This demonstrates the feasibility of generating reliable 3D reconstructions of small proteins by single particle cryoEM. We did not pursue a higher resolution reconstruction of this particular complex by collecting and averaging more images because the HIV-1 IN dimer is not a physiologically active form of the integrase. However, the quality of the current map, obtained only with a limited amount of data, suggests that it should be possible to improve the resolution further.
One concern of using a Fab as a fiducial marker is the potential flexibility between the Fab and its target, which would impair image alignment. In the past, Fabs were not used as fiducial markers for image alignment in single particle cryoEM because of such flexibility (Harris et al., 2011; Jiang et al., 2004
). There are two types of Fab epitope: linear and conformational (Van Regenmortel, 1992
). The flexibility could result from Fab binding to a linear epitope, which tends to be more flexible than a conformational epitope, or binding to a conformational epitope but located in a flexible domain. Previous cryoEM studies have shown that the densities of Fabs were not well defined in the 3D reconstruction when Fabs were bound to linear epitopes (Stewart et al., 1997
). However, Fabs bound to conformational epitopes forming rigid complexes displayed well-defined Fab densities in the 3D reconstruction (Conway et al., 2003
). Our current studies also demonstrated that once a suitable Fab is identified to form rigid complex with its target protein it can be used as fiducial marker. For the same concern, we prefer to use Fabs generated directly against the target proteins, but not the Fabs recognizing a genetically introduced protein tag, because a linker between a tag and a target protein is often flexible.
Another concern is the potential internal flexibility within a Fab between its variable and constant domains. Fabs are thought to be flexible because there is a wide range of variation in angles between variable and constant domains, the so-called “elbow” angle, among the known crystal structures of Fabs. It was found, however, that the “elbow” angle variation of the same Fab crystallized in different conditions is in general less than 15° (Sotriffer et al., 2000
). And it has been pointed out that some Fabs may be less flexible in the elbow region than others and the “elbow” angle fluctuations of Fabs in solution is not fully understood (Stanfield et al., 2006
). Some earlier studies also support that Fabs may be sufficiently rigid to be visualized in single particle cryoEM. For examples, 3D reconstruction of Fab decorated icosahedral hepatitis B capsids showed well-defined densities of tightly bound Fabs at a resolution of near 10 Å and produced excellent fitting of Fab atomic structures (Conway et al., 1998
; Conway et al., 2003
; Kandiah et al., 2011
). Furthermore, in our current study, the two 3D reconstructions of the PCSK9-J16 and IN-Fab5 complexes also produced well-defined densities of both variable and constant domains of the bound Fabs ( and ). It is possible that a small fluctuation in the elbow region may not be detectable at the resolutions achieved in the current and previous studies. Nevertheless, these results suggest that the Fabs used in these studies were sufficiently rigid to produce 3D reconstructions at near 10 Å resolutions, and strongly support that a Fab forming a rigid complex with its target protein can serve as a fiducial marker to facilitate image alignment and as internal control to validate the correctness of the final 3D reconstruction.
To make our method generally applicable, various Fab candidates need to be readily generated against a wide range of target proteins. This can be achieved by method based on highly diverse phage-display Fab libraries (Fellouse et al., 2007
). The phage display method is of particular interest because the selected Fabs often recognize conformational epitopes in their target proteins (Duriseti et al., 2010
; Kim et al., 2011
). Thus, this method increases the chances of identifying a Fab that forms a rigid complex with its target protein. As demonstrated here, a suitable Fab can easily be identified by simple inspection of 2D images of the complex in negative stain and their class averages, in which a well resolved Fab would indicate the formation of a stable and rigid complex.
Similar as shown before (Conway et al., 2003
), single particle cryoEM can also be used for epitope mapping of small proteins without high symmetry. In the case of PCSK9, the density of the Fab in the 3D reconstruction clearly indicates where the antibody binds so that one can firmly narrow down the location of the epitope to a single domain (). Given the high correlation between the map and the fitted atomic structures, one can suggest which residues are more likely to be involved in the interaction. In view of the several studies that indicate that the antibody fragment has antagonistic effects on the binding of PCSK9 to the LDL-R (Horton et al., 2007
; Kwon et al., 2008
; Liang et al., 2011
; Zhang et al., 2007
), these observations are relevant in the context of the search for drugs with potential use in the treatment of hypercholesterolemia.
In summary, we have established a new approach that enables single particle cryoEM studies of proteins or protein complexes smaller than 100 kDa. In conjunction with other developments in the field of cryoEM, this method has the potential of determining correct 3D reconstructions of small proteins, including integral membrane proteins, as demonstrated here by frozen hydrated HIV-1 IN-Fab complex.