Author Summary Translation is the process by which a ribosome converts the sequence of a messenger RNA (mRNA)—produced from a gene—into the sequence of amino acids that comprise a protein. Bacterial ribosomes each have one large and one small subunit: the 50S and 30S subunits. Initiation of translation entails selection of an mRNA, identification of the correct starting point from which to read its code, and engagement of the initial amino acid carrier (tRNA). These events take place in the 30S subunit and require the presence of three initiation factors (IF1, IF2, IF3). Formation of this 30S initiation complex precedes joining with the 50S subunit to assemble the functional ribosome. By using a cryo-electron microscopy approach to visualize the structures without fixation or staining, we have determined the structure of a complete 30S initiation complex and identified the positions and orientations of the tRNA and all three initiation factors. We found that the presence of the initiation factors and tRNA induces rotation of the head relative to the body of the 30S subunit, which may be essential for rapid binding to the 50S subunit and for regulating selection of the mRNA. IF3 had not been seen previously in the context of the 30S structure and its visualization gives insight into a potential role in preventing association of the two ribosomal subunits. These findings are important for understanding how the interplay of elements during the early stages of translation selects the mRNA and regulates formation of functional ribosomes.