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An attempt was made to improve overall contrast over the grid whilst demonstrating that the extent of nanogold labelling is dose-dependent. In this case a 1:50 dilution of Ni-NTA-nanogold® was used, and the grid washed only two times with detergent- free GF buffer. The grid was then stained in the same way as before, but using 1% uranyl acetate rather than the 2% solution, as suggested by the Nanogold manufacturer. As such, this grid represents the same washing conditions as the first

Chapter 3: Structural analysis of TatAd complexes

77 labelled sample (Figure 3.4.1) but with a 5x reduced nanogold concentration and 2x reduced stain concentration. An image of this grid is shown in Figure 3.4.2. The number of labelled complexes appears significantly reduced (see arrow heads) whilst the particle boundaries of all complexes are clearly resolved. The morphology of the labelled particles also appears distinct; they are small and round. From these data it can be concluded that the Nanogold is specifically labelling TatAd-his complexes however, this dilution does not appear sufficient for optimal labelling of the sample.

Figure 3.4.2. Micrograph of 1:8 TatAd-his + 1:50 Nanogold + 2x wash

The micrograph was taken at 57252x magnification under ~1.5 µm defocus. The grid was stained with 1% uranyl acetate. 1.8 nm Ni-NTA-Nanogold® was applied to the grid at ~ 0.2 nmol/ml, diluted in GF buffer + 0.02% DDM, for 15 minutes, following the 1 minute TatAd sample on-grid incubation. Nanogold labelled particle are indicated by white arrow heads. Scale bar = 100nm

Chapter 3: Structural analysis of TatAd complexes

78 3.4.3. Effect of extra washing on TatAd-his + 1:10 Ni-NTA-Nanogold®

All sample concentrations were kept the same as the first experiment (3.4.1) but the grid was washed an additional two times with detergent-free GF buffer to remove excess Nanogold that may be binding non-specifically. As shown in Figure 3.4.3, the morphology of the particle population has now altered significantly. The grid is now covered in a continuous series of large amorphous aggregates (resembling those shown in Figure 3.3.2) and smaller discrete particles are almost entirely absent. Nanogold particles can still be seen bound to these aggregates indicating that they consist of TatAd-his.This suggests that the additional washing of the grid has pushed the DDM concentration below the CMC, forcing the sample to aggregate. This drastic change in particle morphology again demonstrates the effect of the protein to detergent ratio and the minimal contribution of detergent to the normally observed PDCs (as in Figure 3.4.2).

Chapter 3: Structural analysis of TatAd complexes

79 Figure 3.4.3. Micrograph of 1:8 TatAd-his + 1:10 Nanogold + 4x wash

The micrograph was taken at 57252x magnification under ~1.5 µm defocus. The grid was stained with 2% uranyl acetate. 1.8 nm Ni-NTA-Nanogold® was applied to the grid at ~ 1 nmol/ml, diluted in GF buffer + 0.02% DDM, for 15 minutes, following the 1 minute TatAd sample on-grid incubation. The grid was washed a total of 4 times in detergent- free GF buffer before staining. Nanogold labelled particle are visible as intense black dots. Scale bar = 100 nm

3.4.4. Optimised wash method for TatAd-his + 1:20 Ni-NTA-Nanogold®

Due to the observed drastic effects of reducing the detergent concentration, a different approach was applied to reduce non-specific Nanogold binding, whilst maintaining TatAd-his complex stability, and keeping on-grid detergent levels as low as possible. A 1:20 dilution of Nanogold in GF buffer + 0.02% DDM was used (0.5 nmol/ml), and incubated on the grid following sample addition as before.

Chapter 3: Structural analysis of TatAd complexes

80 Subsequently the grid was washed three times in GF buffer + 0.02% DDM before three washes in the same buffer minus detergent. In this way the grid is being washed a total of 6 times with the washes with detergent compensating for those without. As can be seen in Figure 3.4.4 the particle morphology under these conditions appears much more stable compared to Figure 3.4.3. Distinct, well separated and homogenous particles dominate the grid and overall imaging conditions are much improved compared to Figure 3.4.1. Many labelled complexes are visible, these appear to vary in diameter and in some cases double ringed structures are seen. However, for many of the labelled TatAd-his complexes, the Nanogold is visible but the particle boundary with the background is obscured (see selected particles in Figure 3.4.4). This is due to the large difference in contrast between the Nanogold and the protein, compared to the protein and carbon background.

In images taken of more heavily stained areas, the particle boundary for the labelled complexes is much more apparent whereas the resolution of internal density variations in unlabelled complexes is reduced. These features are demonstrated in Figure 3.4.5. Nanogold can be seen binding to small round particles that display a slight size range of ~ 7-9 nm. The Nanogold is bound to the centre of these particles, and the size of the Nanogold cluster increases in proportion to the diameter of the particle, as highlighted in Table 3.4.4.

This suggests that the His-tag, and therefore the C-terminus, of the TatAd-his subunits are arranged so as to point towards the middle of the complex. The proportional binding of Nanogold indicates that more His-tags are present in the larger complexes and provides direct visual evidence for the modular assembly of TatAd-his complexes. These data support the accepted model of subunit arrangement in TatA-type complex organisation; in which the ‘lid’ of the ring is formed by multiple copies of the amphipathic helices.

Chapter 3: Structural analysis of TatAd complexes

81 Figure 3.4.4. 1:8 TatAd-his + 1:20 Nanogold, optimised wash, low contrast The micrograph was taken at 57252x magnification under ~1.5 µm defocus. The grid was stained with 2% uranyl acetate. 1.8 nm Ni-NTA-Nanogold® was applied to the grid at ~ 0.5 nmol/ml, diluted in GF buffer + 0.02% DDM, for 15 minutes, following the 1 minute TatAd sample on-grid incubation. The grid was washed a total of 6 times; 3 in GF buffer + 0.02% DDM followed by 3 in detergent- free GF buffer, before staining. Nanogold labelled particle are visible as intense black dots Particles shown below were windowed from the micrograph shown. The Scale bar for the micrograph = 100 nm.

Chapter 3: Structural analysis of TatAd complexes

82 Figure 3.4.5. 1:8 TatAd-his + 1:20 Nanogold, optimised wash, high contrast The micrograph was taken at 57252x magnification under ~1.5 µm defocus. The grid was stained with 2% uranyl acetate. 1.8 nm Ni-NTA-Nanogold® was applied to the grid at ~ 0.5 nmol/ml, diluted in GF buffer + 0.02% DDM, for 15 minutes, following the 1 minute TatAd sample on-grid incubation. The grid was washed a total of 6 times; 3 in GF buffer + 0.02% DDM followed by 3 in detergent- free GF buffer, before staining. Nanogold labelled particle are visible as intense black dots Particles shown below were windowed from the micrograph shown. The Scale bar for the micrograph = 100 nm.

Chapter 3: Structural analysis of TatAd complexes

83 Complex diameter (nm) Nanogold size (nm)

6.0 2.5

7.5 3.6

8.0 4.2

9.0 5.0

Table 3.4.4. TatAd-his complex diameter vs. size of bound Nanogold cluster The size of the bound Nanogold cluster was seen to increase in proportion with the diameter of the TatAd-his complex to which it was bound.