Particle picking and crude classification

Tilt-pairs were collected from the 1:2 dilution of TatE in GF buffer + 0.02% DDM at ~ 86,000x magnification, with a tilt angle of 45, as shown in Figure 4.4.1. This tilt angle was used to avoid the contrast-fringe artefact seen in the 50 tilted TatAd data- set (see Chapter 3). The average defocus across the selected tilt pairs was 1.8 µm, a higher defocus was used as the particles are very small and do not present much contrast at lower defocus levels making them very difficult to distinguish from the carbon background.

Chapter 4: Single-particle EM analysis of TatE complexes

129 Figure 4.4.1. Micrographs of TatE-strep 45 tilt-pair

Sections of two micrographs covering the same area of the grid are shown. A. microscope stage untilted (0). B. Micrograph stage tilted to 45. Corresponding particles are circled across both images. Micrographs were taken at 85878x magnification under ~1.8 µm defocus. The grid was stained with 2% uranyl acetate.

100 nm

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Chapter 4: Single-particle EM analysis of TatE complexes

130 The micrographs were assessed for image quality with reference to their 2D power spectra in the same way as the TatAd data-set (see Chapter 3). This resulted in the selection of 10 tilt-pairs of images. From these, 1012 initial particles were windowed out into 128x128 pixel boxes using the interactive particle picking tool in Jweb.

Initial analysis of these particles revealed the majority of them to form ring-shaped structures with a maximal diameter of ~ 8 nm and a dark central stain pool indicating a potential pore or cavity. However, after multiple attempts it became clear that too much heterogeneity was present within the data-set to follow the eigenimage based size-separation method (White et al., 2004) as used before for TatAd (see Chapter 3). Figure 4.4.2 shows the total centred average (T) and class averages (1-7) generated upon applying this classification method to the entire TatE particle set. Although size variance can be seen, most clearly by comparing 1 and 6, the averages appear blurred, off centre and badly defined.A number of factors contributed towards this issue: both different sized particles and potentially different views are present, the magnification and defocus are both increased compared to the TatAd sample and the particles appear on average to be smaller.

Figure 4.4.2. Initial attempt at size-based classification on entire TatE data-set Centred average of total TatE particle set (T) and class averages (1-7) generated by HAC on eigenfactors showing a concentric circular pattern indicative of size variance.

To overcome these problems a step-wise method of cross-correlation was used to sequentially separate out different sub-populations prior to any size-based classification. First, only images showing a clear top down view of a ring-shaped structure were selected for analysis as these represent the vast majority of TatE complexes. In this way 190 particles were manually picked, examples of which are shown in Figure 4.4.3. These selected particles were then translationally aligned as

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10 nm T

Chapter 4: Single-particle EM analysis of TatE complexes

131 shown in Figure 4.4.4, and the centred average used as a cross-correlation reference for the rest of the data-set. Using this method for each of the 3 crude initial populations resulted in 259 ringed particles, 95 rod-shaped particles, and 132 multi- ringed structures, with the remaining 437 particles unclassified.

Figure 4.4.3. Initially selected TatE-strep complexes with clear ringed shape Examples of manually selected TatE-strep complexes displaying a clear ‘top down’ view of a ring-shaped particle. The top panel shows raw, unprocessed images. The lower panel show the same particles processed with the despeckle tool of ImageJ (ver. 1.44p) to reduce the background noise.

Figure 4.4.4. Centring of manually selected TatE-strep complexes

Averaged images of the 190 manually selected TatE particles displayed a clear ringed-structure. From left to right: Initial summed and variance images prior to alignment, translationally aligned average image and rotational average used as cross-correlation reference, variance image of aligned average showing reduced pixel variance. Aligned Average Aligned Average Variance Image Rotational Average Sum Image Variance Image 10 nm

Chapter 4: Single-particle EM analysis of TatE complexes

132 4.4.2. Assignment of the unclassified particles

As the initial class assignment was very conservative, to simply separate the clearly disparate particles, a large number of particles remained unassigned. A reference-free translational alignment was applied to these filtered particles with no centring reference. To look for more ringed particles these centred particles were cross- correlated against the clear top-down average. The average cross-correlation value appeared low so a manual cut-off selection was set at 0.3 CCC (cross-correlation coefficient) resulting in only 70 selected particles. Reference-free alignment was run on the selected particles using the top-down view as a centring reference. Running the same procedure on the 367 rejected particles resulted in an average that resembled the multi-ringed set, indicating the population was still heterogeneous. The rejected particles were manually assessed again and some much larger particles were observed and removed (49 particles in total); upon reference-free alignment the average of these shows a large ~ 11 nm wide, particle with no central pore. In contrast the remaining 317 particles produce a small, ~ 7 nm wide, average with a central stain pool but lacking a clear particle boundary. As such, these particles were cross-correlated against the original top-down average and the bottom 10% were removed, resulting in 291 additional small, round particles. After this process the crude separation of particles was distributed as shown in Figure 4.4.5.

Class Top Side Multi-ringed Big

# Particles 620 95 132 49

Figure 4.4.5. Crude classification of TatE complexes based on gross-morphology The class averages shown have been rotationally and translationally aligned using a reference-free method. Class names were selected based on the suspected particle orientation or relevant structural feature. The numbers of particles per class are provided in the accompanying table.

Top Side Multi-ringed Big

Chapter 4: Single-particle EM analysis of TatE complexes

133 4.4.3. Refinement of the crude classification

First an iterative centring method was applied to the corresponding original filtered particles, so as not to induce any bias from the prior alignments. A total of 3 iterations were performed with the size of the mask applied to the particles reduced after each round, as shown in Figure 4.4.5. The centred particles were then cross- correlated back to their centred average and the bottom 10% were removed, this edited set was then re-centred.

MSA (multi-variate statistical analysis) was then performed on the centred particles with the second eigenimage showing signs of size variation, as shown in Figure 4.4.6. HAC (Hierarchical ascendant classification) using this eigenfactor shows a very steep step after the first branching point and a great deal of divergence beyond the 4th branching point, indicating that only a small number of significant classes are present. Using a cut-off to produce 4 classes shows only 2 distinct sizes of average each with an apparent subclass. Therefore, a cut-off was selected generating only the 2 significant classes and the particles were re-centred accordingly. The bigger class measures ~ 7.7 nm across and the smaller class measures only ~ 6.0 nm. As shown in Table 4.4.3, the spread of particles between these classes is quite even suggesting that both represent significant and stable TatE complexes.

Figure 4.4.5. Iterative centring of crude small, ringed class of TatE complexes The 620 selected TatE particles were centred via 3 iterative rounds (r1-3) of reference-free translational alignment. Resulting in a final centre of gravity of -0.02, -0.02 pixel units.

r1

r2

r3

Chapter 4: Single-particle EM analysis of TatE complexes

134 Figure 4.4.6. Size classification of small, ringed TatE complexes

A. MSA of the TatE particles revealed a concentric ring pattern indicative of size variation in eigenfactor 2 (EIG2) highlighted in red. B. HAC based on EIG2 revealed 4 potential size classes based on Ward’s clustering method. A final cut-off value was selected generating 2 stable size classes.

A