Their projection structures. It might be argued that further important details could be obtained from 3D analysis of the data. However, it is apparent that although we have selected over 12 000 AZD2171 particles from the autophosphorylated sample, it is very unlikely that these all derive from a single autophosphorylation state of the DNA PKcs. DNA PKcs has over 30 autophosphorylation sites, which can be modified in different combinations. This is likely to result in a multiplicity of conformations. A correct 3D analysis would require the recovery of individual 3D maps for each such conformation, and we therefore restricted ourselves to a 2D analysis. This allowed observing a number of interesting consequences of autophosphorylation.
Autophosphorylation generates heterogeneity The previous studies of DNA PKcs, Ku and dephosphorylated DNA PK by single particle analysis dealt with samples characterized by Droxinostat compositional and conformational homogeneity. In the present study, the incubation of DNA PK with ATP and MgCl2 led to incorporation of in the DNA PKcs and probably the Ku70 subunits. This gives rise to a heterogeneous population of single particles on the electron microscopy grids, as expected from previous biochemical studies. Visual inspection of the micrographs showed different appearances between the dephosphorylated and autophosphorylated samples, although classification was required to clearly identify the constituent complexes.
Importantly, because we have previously resolved the 3D structures of Ku, DNA PKcs and DNA PK, we can rule out that the different averages we now found could just arise from different orientations on the grid of the phosphorylated DNA PK molecule. We can rather conclude that phosphorylation induces disassembly and conformational changes in the DNA PK complex. Partitioning a heterogeneous data set We used classification and alignment procedures in order to identify the various types of complexes present in our preparation upon autophosphorylation. The procedure is described in the,Materials and Methods, and,Results, sections. This partition could in principle be done visually, but this would result in a tedious and very time consuming procedure, which is difficult because of the high level of noise in individual single particles and the high level of heterogeneity of the sample.
We found that in the DNA PK autophosphorylated sample the heterogeneity lies on two different levels. The heterogeneity is first based on the oligomeric state and therefore a considerable variation in particle size. In this case, the partition would be feasible by visual inspection of raw particles, as in. The second degree of heterogeneity is based on a more subtle difference in size, which is more difficult to be identified,by eye, at the single image level. Characterization of autophosphorylated DNA PK by classification In order to reveal all the information present in the three subsets of images, we classified each subgroup using a full set of 69 eigenimages. We interpreted our analysis by comparison of the projection images of the autophosphorylated DNA PK sample with projections from the 3D reconstructions of the species calculated in previous studies. Moreover, we performed a quantitative comparis.