X-ray crystallography studies on bacterial ribosomes with and without tRNAs have revealed that tRNAs occupy the cleft between the two subunits. They interact with the 30S subunit through their anticodon ends and with the 50S subunit through their acceptor stems. The binding sites for tRNAs primarily consist of rRNA. The anticodons of tRNAs in the A and P sites come into close proximity, allowing base-pairing with adjacent codons in the mRNA bound to the 30S subunit, as the mRNA bends 45 degrees between the two codons. The acceptor stems of tRNAs in the A and P sites also approach each other closely—within just 5 Å—within the peptidyl transferase pocket of the 50S subunit, where twelve contacts between ribosomal subunits are visible.

The crystal structure of the E. coli ribosome reveals two conformations that differ due to rigid body motions of ribosomal domains relative to each other. Specifically, the head of the 30S particle rotates by 6 degrees and by 12 degrees when compared to the T. thermophilus ribosome. This rotation is likely part of the ratchet-like motion of the ribosome during translocation.

The E. coli 30S subunit comprises a 16S rRNA and 21 proteins (S1–S21), while the 50S subunit contains a 5S rRNA, a 23S rRNA, and 34 proteins (L1–L34). Eukaryotic cytoplasmic ribosomes are larger and include more RNAs and proteins than their prokaryotic counterparts. Sequence studies of 16S rRNA proposed its secondary structure (intramolecular base pairing), which has been confirmed by X-ray crystallography studies. These studies reveal a 30S subunit with extensively base-paired 16S rRNA, whose shape essentially defines the particle's overall structure. Additionally, X-ray crystallography studies have identified the locations of most 30S ribosomal proteins.

The 30S ribosomal subunit serves two primary roles. It facilitates accurate decoding of mRNA and contributes to the overall function of the ribosome during translation.