(A) Agarose gels (stained with ethidium bromide) showing results for affinity purification for ΔrimP sucrose gradient fractions 2–3, ΔrimP lysate, and purified 30S subunits. 16S rRNA is not visible in later washes, but is visible in elution fractions for ΔrimP samples. (B) Class average of 3′-domain degradation product versus a forward projection of the 3′-domain filtered to 30 Å resolution. (C) Class averages from negative stain and cryoEM data sets with helix 44 density clearly visible, compared with a similar forward projection of the 3′-domain model. (D) Comparison of particle distribution between two affinity purification samples. In sample 1, the input 16S rRNA was already heavily degraded, and the 3′-domain was preferentially enriched based on agarose gel analysis. In sample 2, degradation was limited by the addition of RNasin (Promega) and reducing the amount of time for sample preparation. 5000 particles from negative stain data sets for each sample were combined into a single stack (10,000 particles), and subjected to reference-free maximum likelihood classification. The fraction of particles from each data set contributing to various conformations is plotted in the histogram. Putative 3′-domain classes are enriched in the degraded sample 1, while Group II classes are enriched in the intact sample 2.