The Structure of The 70S E. Coli Rtoosome In Ice

We have obtained two reconstructions of the 70S ribosomal subunit embedded in ice using the random-conical method on particles showing two distinctive views.Ribosomes were applied to grids covered with thick holey carbon film whose holes were in turn covered with a second, thin carbon film. The frozen-hydrated preparation was done as described previously using GATAN cryo-transfer equipment and specimen holder on a Philips EM420. The images were taken in pairs at 2μm defocus and a dose of less than 10 e-1/Å2. The first was taken with the specimen tilted to 50°, the second untilted. A total of 838 particles from two micrograph pairs were selected for image analysis.A new reference-free alignment algorithm was used to align the 0°-images. In the subsequent multivariate statistical analysis and factor-space based hierarchical classification, several distinct groups were found, two of which (averages, Fig. 1) with memberships above 10% were selected for two separate 3D reconstructions. The averages were found to have a resolution of 3.3 nm as established by phase residual analysis. For computing the reconstruction, we used an iterative scheme that is based on a Chi-square minimization. In this way, we were able to reduce distortions caused by the missing cone as well as by large gaps in angular spacing.