Supervised classification of the projections representing the 30S:YjeQ complex. Projections of 30S:YjeQ complexes were classified using two references: the control cryo-EM structure obtained from free 30S subunit low-pass filtered to 25 Å resolution (reference #1) and the 3D reconstruction obtained from all the particle images in the data set (reference #2). Reference #2 already showed an extra mass of density attached to the upper domain of helix 44 and the nearby region of the platform. All images in the data set were aligned to the projections generated from each of the references, and each image was assigned to the projection view from each reference that yielded the highest cross-correlation coefficient, obtaining two cross-correlation coefficients per particle image (CC1 and CC2). Then, we plotted the distribution of particles against the difference between the two cross-correlation coefficients (ΔCC = CC2 − CC1). The particles were classified following a unimodal distribution. This histogram was divided into four classes (from SC 1 to SC 4) using the P_x values indicated by the dashed lines. The 3D map for each subset of particles was calculated using projection matching. The number of particles classified in each group is indicated in the figure. The reference map used to refine the four structures was the X-ray structure of the E. coli 30S subunit (PDB ID: 2AVY) (Schuwirth et al. 2005) low-pass filtered to 25 Å resolution. The estimated resolution for each map is noted in the figure below the number of particles assigned to each class. The group of particles most similar to reference #2 (SC 4) formed the 3D cryo-EM map of the 30S:YjeQ complex.

Pseudo-atomic model of the 30S:YjeQ complex and displacement of the upper domain of helix 44 in the cryo-EM map of the 30S:YjeQ complex. (A) Pseudo-atomic model of the 30S:YjeQ complex. The backbone of the 16S rRNA (ribbon in cyan); ribosomal proteins (dark blue); the YjeQ protein (black). (B) Back view of the 30S:YjeQ complex (see orientation aid in panel above) after a clipping plane orthogonal to the direction of view was applied to the cryo-EM map removing the blocking densities on the back. The panel shows the density map of the 30S:YjeQ complex as a mesh. The OB-fold (gray), GTPase (yellow), and zinc-finger (green) domains of the YjeQ structure are shown fitted into this density. (Purple) The nucleotides in helices 44 and 45 for which a corresponding density in the 30S:YjeQ complex EM map does not exist. (C) Top view of a slab of the cryo-EM map of the 30S:YjeQ complex. The slab was cut orthogonal to the longitudinal axis of helix 44 (see orientation aid in panel above). Binding of YjeQ to the 30S subunit causes a displacement of the upper domain of helix 44. The location of this motif in the free 30S subunit (purple) and in the 30S:YjeQ complex (light purple) is shown. (Black arrow) The direction of the shift.

Comparison of the interaction model of YjeQ with the 30S subunit from cryo-EM and footprinting studies. The backbone of the 16S rRNA of the 30S subunit (a ribbon in cyan). Nucleotides whose chemical reactivity was decreased (black) or increased (red) by the addition of YjeQ with a nonhydrolyzable GTP analog (Kimura et al. 2008). The area covered by YjeQ according to the 30S:YjeQ cryo-EM structure presented here is shown as a pink semitransparent density. The 30S:YjeQ map suggests that a region (dark blue) of helix 44 is displaced upon YjeQ binding.

Assembly of the 30S:YjeQ complex. Purified YjeQ was incubated with 30S subunits in ratios of 1:1 (lanes 6,7) and 5:1 (lanes 8,9) in the presence of 1 mM GMP-PNP. The concentration of 30S subunits in these reactions was 1 μM. Following incubation, samples were overlaid onto a sucrose cushion and pelleted by ultracentrifugation. Pelleted material was resuspended, and both the supernatant (S) and pellet (P) were resolved by SDS-PAGE and stained with Coomassie brilliant blue. The lanes are as follows: (lane 1) molecular weight marker; (lanes 2,3) YjeQ + GMP-PNP; (lanes 4,5) 30S + GMP-PNP; (lanes 6,7) YjeQ + GMP-PNP + 30S (1:1); (lanes 8,9) YjeQ + GMP-PNP + 30S (5:1); (lanes 10–13) known quantities of YjeQ (1–12.5 pmol). The position of YjeQ in the gel is indicated. The occupancy of the YjeQ–30S complex was assessed by quantifying the amount of YjeQ that co-pelleted with ribosomes (2.5 pmol) in each reaction using the lanes with known quantities of YjeQ (lanes 10–13). For this calculation the fraction of YjeQ that pellets in the absence of ribosomes (25%) (lane 3) was subtracted from that quantified in the reaction pellet (lanes 7,9). Using the previously determined binding ratio of 1:1, occupancy for both 1:1 and 5:1 incubations was found to be ∼70%.

Cryo-EM structure of the 30S:YjeQ complex and docking of the YjeQ structure. (A) Cryo-EM map of the 30S:YjeQ complex side by side with a control reconstruction obtained from free 30S subunits. The head, platform, body, and helix 44, which are main landmarks in the 30S subunit, are indicated as well as the inter-subunit bridges B2a, B3, and B7A covered upon YjeQ binding. The additional density (red) represents the YjeQ protein binding site. (B) The four panels provide different views of the additional density covering the upper domain of helix 44 and nearby region in the platform in the cryo-EM map of the 30S:YjeQ complex. The additional density is represented as a mesh with the docked X-ray structure of the YjeQ protein. The X-ray structure of the YjeQ protein from S. typhimurium (PDB ID 2RCN) is shown with the OB-fold domain (dark gray), the GTPase domain (yellow), and the zinc-finger domain (green). As an aid for orientation, each panel shows the cryo-EM map of the 30S:YjeQ complex in a matching view. The first residue described in the YjeQ structure (L43) and residues N245 and T259 flanking a sequence not described by the atomic coordinates are labeled. Residues H285 and A345 are indicated to mark the first and last α-helices at the C-terminal zinc-finger domain of YjeQ. Areas of the density not filled by the X-ray structure of YjeQ are labeled with numbers from 1 to 4.

Interactions of YjeQ with the 30S subunit. (A) Interactions of the OB-fold and GTPase domain of YjeQ with the 16S rRNA helices 24a and 23b in the platform and helix 45 in the 3′ minor domain of the 30S subunit. Important loops and β-strands in YjeQ for the interaction with the 30S subunit are labeled. The OB-fold (gray), GTPase (yellow), and zinc-finger (green) domains of the YjeQ structure are shown. The extra density attributed to YjeQ is displayed as a red semitransparent surface. The rRNA (cyan) except for the nucleotides (purple) for which a corresponding density in the 30S:YjeQ complex EM map does not exist. (B) Interactions of the zinc-finger domain of YjeQ with the 16S rRNA helix 44. As an aid for orientation, each panel shows the cryo-EM map of the 30S:YjeQ complex in a matching view. (C) Electrostatic surface potential of S. typhimurium (PDB ID 2RCN) YjeQ. Zones are negatively charged (red), positively charged (blue), and uncharged (white). (Left panel) The surface of YjeQ that is not in contact with the 30S subunit; (right panel) the opposite face of the protein contacting the 16S rRNA.