Two size classes of small, ringed TatE complexes

3D reconstructions of the TatE complexes were generated and refined using the same methods described for TatAd (chapter 3). Figure 4.5.1 shows several stages in the projection matching refinement procedure for both the small class (A) and the large class (B). As can be seen the initial models appear more rotationally averaged with a rounder more featureless appearance. Over the course of the projection matching the models become more structurally defined and the globular densities forming the ring become apparent, matching well with those seen in the 2D averages (Figure 4.4.7). Importantly the presence of the central cavity occluded on one face can be seen in the initial models and is conserved throughout the refinement.

The final 3D density maps (C) clearly show the small variation in overall size between the 2 classes of TatE complexes, less than 2 nm. The models are shown contoured to ~ 4.8 σ (SD of mean density) and filtered to 29 Å. The total height of both the complexes is ~ 5 nm, sufficient to span the plasma membrane. Interestingly, the ‘pore’ size for either complex appears approximately the same at 2.5 nm. This is far smaller than the ~ 7 nm wide pore recorded for the largest size class of TatA complex (Gohlke et al., 2005); a size that was deemed sufficient to accommodate the large E. coli tat substrate TorA. Overall these data indicate that the TatE complexes would not be large enough to accommodate TorA via their central cavity. The small central cavity and dramatically reduced size range of TatE complexes casts doubt on the proposed ‘size-fitting pore’ model of Tat mediated translocation

The molecular weights of the complexes were estimated at 68 kDa and 90 kDa respectively, based on the enclosed density contoured at the ~ 4.8 level σ, using an alpha-helical protein packing of 0.844 Da/Å3. These size estimates fit very well with the small, discrete bands observed by BN-PAGE (Figure 4.2.2) and together provide good evidence that these models are a reliable estimation of the purified TatE complexes.

Chapter 4: Single-particle EM analysis of TatE complexes 140 Initial Model 1 st_round Projection Matching Final round Projection Matching Final filtered Model Top View Side View Cross- section

A

B

Chapter 4: Single-particle EM analysis of TatE complexes

141 Figure 4.5.1. Final 3D density maps of TatE-strep complex assemblies

A+B. The 2 size classes of single-ringed complexes were refined by projection

matching as shown. 3D maps are shown over a number of stages during refinement to show the effects. The presence of a central cavity occluded on one side can be seen in the cross-sectional views. C. The final models are shown filtered to 29 Å. and contoured to ~ 5 σ (standard deviations above the mean density). From top to bottom the dimensions shown are: complex diameter, complex height, and ‘pore’ width. Molecular weights were estimated based on a helical protein packing density of 0.844 Da/Å3. All models were displayed using UCSF Chimera (Pettersen et al., 2004). Class 1 Class 2 68kDa 90kDa 5.9 nm 7.6 nm 5.0 nm 5.0 nm 2.5 nm 2.5 nm

C

Chapter 4: Single-particle EM analysis of TatE complexes

142 4.5.2. 3D reconstruction of TatE complex ‘Side view’

Finally an attempt was made to produce a relevant 3D reconstruction of the proposed side-view class of TatE complexes. These density maps were generated and refined using the same method used for the top-down reconstruction and the results are shown below in Figure 4.5.2. More particle variance was present in this class compared to the top-down size classes, and therefore the 3D models appear noisier. Throughout the refinement process the presence of a central cavity became clear. A range of low-pass filters were applied to the refined model to accentuate this feature, as shown in Figure 4.5.2.B; multiple cavities, that would link this class to the multi- ringed structures, could not be resolved. These data therefore suggest the rod-shaped structures could represent a flattened population of single ringed structures or a mixed population of single and multi-ringed particles. Although these particles are a structurally heterogeneous minority population they still represent a significant proportion of the TatE particle-set and warrant further investigation in the future.

Initial Model 1 st_round Projection Matching Final round Projection Matching Top View Side View Cross- section

A

Chapter 4: Single-particle EM analysis of TatE complexes

143 Figure 4.5.2. 3D reconstructions of TatE complex ‘Side’ views

A. 3D density maps were generated by projection matching refinement of the side view class of TatE complexes. B. Final density maps were generated for the side class by applying a range of low-pass Butterworth filters. In each case a central cleft can be seen that is occluded on one side.

Top View Side View Cross- section

Filter 1 Filter 2 Filter 3

Chapter 4: Single-particle EM analysis of TatE complexes

144 4.6. Discussion

Although the role of the Tat(A)BC complex in substrate binding has been demonstrated multiple times using a variety of experimental methods, including cross-linking studies (Alami et al., 2003) and direct EM imaging (Tarry et al., 2009), the function of the TatA complex remains inferred only. The most widely accepted theory suggests that TatA forms a translocation channel made mostly, if not entirely, from TatA subunits. This theory is based heavily on the findings of a previous EM study into E. coli TatA complexes where ring-shaped particles of varying size were observed (Gohlke et al., 2005). The data presented here, and published previously (Baglieri et al., 2012), appear inconsistent with this ‘size-fitting pore’ model.

It has been demonstrated previously that TatE can complement a ΔtatA mutant (Sargent et al., 1999) and more recently that it can translocate several Tat substrates including the large TorA protein (90 kDa) (Baglieri et al., 2012). These results indicate that TatE is capable of substituting for E. coli TatA. In the previous study of TatA the largest sized class, consisting of 86 particles, measured 13.5 nm across and had a potential channel diameter of 6.5-7.0 nm (Gohlke et al., 2005). However, unlike TatA, TatE presents only 2 major size classes measuring less than 8 nm in diameter, smaller than the smallest sizes TatA complexes. No evidence of particles with a pore 7 nm wide, and therefore potentially capable of accommodating TorA, were seen. As such the small size of these TatE complexes suggests that in this state they would not be capable of forming a pore large enough to translocate folded Tat substrates.

The complexes isolated here by single-particle analysis fit well with the size estimates given by BN-PAGE (see Figure 4.2.2) and it is important to note that larger complexes could not be observed in the whole membranes either, as seen by biochemical analysis, thereby ruling out the possibility of degradation during the purification (Baglieri et al., 2012). Purification in other detergents including digitonin and C12E9 was also not seen to alter the gel filtration elution profile of TatE

Chapter 4: Single-particle EM analysis of TatE complexes

145 The interesting multi-ringed structures suggest some kind of modular assembly intermediate of a larger complex may be present within the TatE sample. Similar structures were observed and discussed previously for TatAd (see chapter 3) and even larger formations were observed for TatAyCy (see chapter 5). These small, modular complexes lend themselves to the idea of a flexible protein conducting channel (PCC), a theory that is currently favourable within the Sec field (Frauenfeld

et al., 2011; Haider et al., 2006; Tian and Andricioaei, 2006). Experimenting with

the purification conditions to attempt to increase the number of multi-ringed complexes would be an interesting avenue for future research.

A TatBC complex with multiple substrates bound to its periphery has been previously observed by EM (Tarry et al., 2009). A central cavity was shown in this complex; however, it was deemed too small to accommodate a Tat substrate and presented no opening to the particle surface. Further analysis of TatA purified in digitonin presented in this study did not reveal the large central pore shown previously (Gohlke et al., 2005). It is important to note that partial projections of complexes can be produced by variable staining across the grid and this can alter the resolution of such internal cavities. Also, the 3D models of all these Tat complexes have been produced using a random conical tilt reconstruction method. This method results in a cone of missing structural information along the z-axis, which can obscure finer detail within the 3D models. The preferred orientation of these particles on the grid can also obscure whether these cavities are open or closed to the surrounding environment. To circumvent these technical issues related to the imaging method future work may focus on abolishing the preferred orientation. It may be possible to achieve this by experimenting with glow-discharge, staining and gridding procedures, or by using cryo-EM approaches.

Importantly, until a Tat complex can be isolated with clear evidence of a substrate stalled within a central pore or cavity it cannot be safely concluded that these ring- shaped particles produce channels.

146

Chapter 5

Structural investigation of