Seeing antibiotics in action inside a pathogenic bacterium nature
To further refine the ribosome–ribosome interface in polysomes, RELION classification was performed with sub-tomograms extracted with a large box size that can accommodate two ribosomes. After refinement on the preceding ribosome , classification without refinement was performed with a local mask focusing on the following ribosome . Only ribosome pairs within tightly packed polysomes resulted in average densities with both ribosomes well resolved.
Permutation analysis was performed to test the significance of differences between occurrence frequencies of the experimental pairs compared to the theoretical pairs. The ribosome pair sequences in polysomes were represented as a matrix with single polysomes as rows and ribosome positions as columns, in which each matrix cell contains the ribosome class representing its elongation state. Columns with positions beyond each ribosome’s length were assigned to NaN .
For polysome distance threshold analysis, matrices were created from the full ribosome dataset by varying the distance threshold to the nearest neighbour in the range from 3 to 10 nm. The distances for polysome definition were calculated between the mRNA exit site of one ribosome and the mRNA entry site of another ribosome as described above. The fractions of elongation states in ribosome pairs were calculated as for the original ribosome set .
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