Understanding of eukaryotic ribosome synthesis has been slowed by a lack of structural data for the pre-ribosomal particles. We report rRNA-binding sites for six late-acting 40S ribosome synthesis factors, three of which cluster around the 3' end of the 18S rRNA in model 3D structures. Enp1 and Ltv1 were previously implicated in 'beak' structure formation during 40S maturation--and their binding sites indicate direct functions. The kinase Rio2, putative GTPase Tsr1 and dimethylase Dim1 bind sequences involved in tRNA interactions and mRNA decoding, indicating that their presence is incompatible with translation. The Dim1- and Tsr1-binding sites overlap with those of homologous Escherichia coli proteins, revealing conservation in assembly pathways. The primary binding sites for the 18S 3'-endonuclease Nob1 are distinct from its cleavage site and were unaltered by mutation of the catalytic PIN domain. Structure probing indicated that at steady state the cleavage site is likely unbound by Nob1 and flexible in the pre-rRNA. Nob1 binds before pre-rRNA cleavage, and we conclude that structural reorganization is needed to bring together the catalytic PIN domain and its target.