Negamycin induces translational stalling and miscoding by binding to the small subunit head domain of the Escherichia coli ribosome

Significance

The identification of negamycin’s binding site within helix 34 of the small subunit head domain and the elucidation of its mechanism of action during messenger RNA decoding provide a physical framework for exploring structure–activity relationships of this largely unexplored antibiotic class. These findings lay the foundation for the rational design of improved negamycin analogs that may one day serve as potent antibacterial agents in the clinic.

Abstract

Negamycin is a natural product with broad-spectrum antibacterial activity and efficacy in animal models of infection. Although its precise mechanism of action has yet to be delineated, negamycin inhibits cellular protein synthesis and causes cell death. Here, we show that single point mutations within 16S rRNA that confer resistance to negamycin are in close proximity of the tetracycline binding site within helix 34 of the small subunit head domain. As expected from its direct interaction with this region of the ribosome, negamycin was shown to displace tetracycline. However, in contrast to tetracycline-class antibiotics, which serve to prevent cognate tRNA from entering the translating ribosome, single-molecule fluorescence resonance energy transfer investigations revealed that negamycin specifically stabilizes near-cognate ternary complexes within the A site during the normally transient initial selection process to promote miscoding. The crystal structure of the 70S ribosome in complex with negamycin, determined at 3.1 Å resolution, sheds light on this finding by showing that negamycin occupies a site that partially overlaps that of tetracycline-class antibiotics. Collectively, these data suggest that the small subunit head domain contributes to the decoding mechanism and that small-molecule binding to this domain may either prevent or promote tRNA entry by altering the initial selection mechanism after codon recognition and before GTPase activation.

Negamycin, a natural product originally isolated from cultures of Streptomyces purpeofuscus, exhibits broad-spectrum antibacterial activity against key pathogens for which clinical treatment options are dwindling (1, 2). The chemical structure of negamycin, [2-[(3R, 5R)-3,6-diamino-5-hydroxyhexanoyl]-1-methylhydrazino]acetic acid, and synthetic routes for its synthesis were elucidated in the early 1970s (Fig. 1A) (3, 4). Early toxicity studies in dogs revealed that daily administration of negamycin led to the formation of N-methylhydrazinoacetic acid, an inhibitor of glutamate pyruvate transaminase, an outcome that caused reversible hepatic coma. Consequently, further clinical studies were not pursued (5). Although the antimicrobial activity and efficacy of negamycin have since been confirmed, analogs exhibiting an improved therapeutic window have yet to be found (6, 7). Progress on this front has been hampered, at least in part, by the fact that the molecular mechanisms of negamycin-induced cell growth inhibition have yet to be discerned conclusively.

Fig. 1.

Negamycin competes with tetracycline and tigecycline for binding to the small ribosomal subunit. (A) Chemical structures, from left to right, of negamycin, tetracycline, and tigecycline. (B) [3H] Tetracycline displacement assay with unlabeled competitors. Tigecycline (red, IC₅₀ = 1.6 μM), negamycin (green, IC₅₀ = 64 μM). (C) Assessment of TetM effect on tetracycline, tigecycline, and negamycin. Results of coupled in vitro transcription–translation assay with TetM at 0 μM (▪), 0.01 μM (♦), and 0.1 μM (●) in the presence of tetracycline (blue), tigecycline (red), or negamycin (green). Error bars represent the SD of at least two independent experiments.

Negamycin decreases the viability of Escherichia coli by preferentially targeting protein synthesis (8). Early mechanistic investigations proposed that negamycin may act by inhibiting translation initiation (8), decreasing translational fidelity during the elongation phase of protein synthesis (9, 10), and disrupting proper translation termination (9, 11–13). Uncertainties regarding negamycin’s inhibition mechanism, its highly polar physicochemical properties, and the lack of streptomycin cross-resistance led some to propose that it may act through multiple binding sites (9). Crystallographic data led to the speculation that negamycin binds in the peptide exit tunnel of the Haloarcula marismortui large ribosomal subunit (14). This site of interaction, however, was difficult to reconcile with its inhibitory activities given its distance (∼100 Å) from known functional centers of the ribosome located at the interface of the large (50S) and small (30S) subunits. Experiments showing that negamycin interacts with model oligonucleotide analogs of the decoding site (15) similarly have fallen short of clarifying its mechanism of action. Although the molecular basis of negamycin’s activities remains elusive, a mode of action involving the process of messenger RNA (mRNA) decoding is supported by studies showing that negamycin can suppress premature translation termination of the dystrophin gene, mdx, in a mouse model for Duchenne muscular dystrophy (16). As ∼30% of human disease mutations stem from the introduction of stop codons that cause premature translation termination (17), such investigations suggest that a deeper understanding of negamycin’s mode of action may one day be harnessed for therapeutic purpose (reviewed in ref. 18).

Here, through genetic, biophysical, and crystallographic investigations of the bacterial translation apparatus, we show that negamycin binds and operates through a site distinct from, but proximal to, the decoding site region at the base of the small subunit head domain. This site overlaps that of tetracycline and tigecycline (Fig. 1A), structurally distinct compounds that specifically inhibit translation by disrupting the process of aminoacyl-tRNA (aa-tRNA) selection during decoding (19–21). Through this site, negamycin stabilizes near-cognate aa-tRNAs on the ribosome to promote translational stalling and the loss of translation fidelity.

Results

Negamycin (Fig. 1A) was synthesized in a novel sequence (Fig. S1) from an intermediate previously used by Nishiguchi et al. (22). Biochemical and microbiological activity was indistinguishable from negamycin that was purified as a fermentation product (Table 1). This reagent was used for all subsequent experiments.

Table 1.

Antimicrobial and biochemical activity of negamycin (NMY), tetracycline (TET), tigecycline (TIG), and gentamicin (GEN)

Strain	NMY	TET	TIG	GEN
MRE600 (S30 extracts)	1.9 (1.8)	1.4	0.35	0.01
ATCC25922	32 (32)	1	0.25	0.5
SQ110 (WT)	32	2	0.5	1
SQ110 rrnE (U1052G)	1,024	0.12	0.5	1
SQ171 pHK-rrnC (WT)	16	2	2	1
SQ171 pHK-rrnC (U1052G)	512	0.5	2	0.5
SQ171 pHK-rrnC (U1060A)	256	8	8	1

Biochemical activity (IC₅₀, in micromolar; n ≥ 3) was determined by using a coupled in vitro transcription–translation assay using S30 extracts of strain MRE600. Antimicrobial activity (MIC, in micrograms per milliliter; n ≥ 3) was determined against a wild-type E. coli strain (ATCC25922) and E. coli strains SQ110 and SQ171 containing only a single copy of the ribosomal DNA operon, including negamycin-resistant derivatives of these strains. Data obtained with NMY fermentation product are indicated in parentheses.

Resistance Mutations Map to the Head Domain of the 30S Subunit of the Ribosome.

In an effort to identify the negamycin binding site on the E. coli ribosome, mutant cells resistant to negamycin were isolated from an E. coli strain containing only a single genome-encoded rrn operon (SQ110) (22). Spontaneous resistant mutants were identified at a frequency of 10⁻⁹ when selected at 128 µg/mL, fourfold the minimum inhibitory concentration (MIC) of 32 µg/mL (∼128 µM) (Table 1) (23). Whole-genome sequencing of eight colonies obtained in four independent experiments revealed a unique mutation at position U1052G of 16S rRNA, in immediate proximity of helix 34 (h34) (Fig. S2). Similar findings were obtained when analogous experiments were performed using E. coli SQ171 (23), a strain lacking genomically encoded rRNA and harboring a single rrnC operon on a low copy number plasmid (pHK-rrnC). Such experiments led to the identification of a second, distinct resistance mutation, U1060A (Fig. S2) that also is proximal to h34. The U1052G and U1060A mutations conferred an ∼30-fold and ∼16-fold increase in the negamycin MICs, respectively (Table 1). Consistent with the significance of this region to the binding of tetracycline-class antibiotics (24, 25), the U1052G mutation resulted in hypersusceptibility to tetracycline (Table 1), whereas the U1060A mutation caused cross-resistance to both tetracycline and tigecycline (Table 1).

Collectively, these findings suggest that negamycin resistance may be conferred by local distortions in the vicinity of h34 that influence the binding of negamycin as well as the tetracycline-class antibiotics (26). In the native, apo-ribosome structure, U1052 forms a wobble base pair with G1206 and U1060 forms canonical base-pairing interactions with residue A1197 (Fig. S2) (27). Correspondingly, transversion mutations at either position may distort helical geometries in this region to affect critical drug interactions.

Negamycin Displaces Tetracycline from the Ribosome but Is Not Susceptible to TetM-mediated Resistance.

To validate the h34 site of negamycin binding inferred from these studies, filter binding displacement assays were performed to ascertain whether negamycin directly or indirectly competes for radiolabeled tetracycline binding to the E. coli 70S ribosome (21, 28). Consistent with the notion that negamycin and tetracycline-class antibiotics share overlapping binding sites, these displacement studies showed that negamycin, like tigecycline, could efficiently displace tetracycline from the ribosome (Fig. 1B). In line with their observed biochemical and antimicrobial potencies (Table 1), in this assay negamycin and tigecycline exhibited IC₅₀ values of 64 ± 1.2 µM and 1.6 ± 1.1 µM, respectively.

TetM is a ribosome protection protein that confers resistance to tetracycline by binding to the A site and displacing the drug from h34 via a GTP hydrolysis-dependent reaction (29, 30). Although a complete understanding presently is lacking, TetM must dissociate quickly, allowing ternary complex to bind before drug rebinding, thus enabling the translation elongation cycle to proceed. Notably, TetM does not confer resistance to tigecycline (21). Given that both drugs bind and recognize overlapping regions within h34 (27, 30, 31), this finding suggests that TetM may be unable to displace tigecycline effectively because of steric or affinity considerations. In a coupled in vitro transcription–translation assay using S30 extract, we observed negamycin’s inhibition of translation persisting in the presence of up to 0.1 µM TetM (see Table S1 for IC₅₀ values) (Fig. 1C). Given negamycin’s comparatively high IC₅₀ in tetracycline binding competition assays (64 µM) (Fig. 1B), from which a reduced affinity interaction is inferred, this finding suggests that negamycin binds the ribosome at a site near h34 that is physically distinct from that of tetracycline and beyond the reach of the C-terminal extension on domain IV of TetM that is believed to enter the A site to displace the drug from the ribosome (29).

Negamycin Specifically Targets the Process of Near-Cognate tRNA Decoding.

In line with the biochemical potency estimated using S30 extracts (Table 1) and the inhibitory concentrations reported by Umezawa and coworkers (10), coupled in vitro transcription–translation assays using recombinant, purified translation components from E. coli (Materials and Methods) revealed that negamycin inhibited luciferase production with an IC₅₀ of ∼1.5 µM. To ascertain negamycin’s impact on elongation cycle reactions, we set out to investigate its molecular basis of action using purified translation components and single-molecule fluorescence resonance energy transfer (smFRET) methods by (i) imaging ribosome dynamics under equilibrium conditions and (ii) imaging function during single-turnover pre–steady-state reactions of aa-tRNA selection and translocation (19, 20, 32). Such experiments have the benefit of affording intuitive structural perspectives that facilitate insights into the mechanisms of antibiotic action (31, 33, 34). Whereas chemically diverse antibiotics have been shown to substantially alter both tRNA motions between their classical and hybrid positions within the aminoacyl (A) and peptidyl (P) sites (34), as well as the process of subunit rotation, negamycin had no detectable impact on either of these structural processes. This observation suggests that the site of negamycin binding may be remote from the intersubunit bridge elements that coordinate spontaneous and reversible tRNA motions at the subunit interface (33, 35, 36).

Direct single-molecule imaging methods enable the process of tRNA selection to be tracked by time-dependent changes in FRET (19, 20, 37). In such experiments, wide-field total internal reflection fluorescence microscopy is used to monitor the binding of acceptor (Cy5)-labeled Phe-tRNA^Phe in ternary complex with elongation factor tu (EF-Tu) and GTP to surface-immobilized 70S initiation complexes bearing a donor (Cy3)-labeled initiator tRNA^fMet within the P site (Fig. 2A). In this system, changes in FRET reflect ternary complex binding to the A site and reversible conformational events and thermal fluctuations that ultimately drive the incoming aa-tRNA between codon recognition (CR) and GTPase-activated (GA) states during initial selection (a low- to intermediate-FRET transition) and between the GA and accommodated (AC) states during proofreading (an intermediate- to high-FRET transition) (Fig. 2B). In the presence of 200 µM negamycin—i.e., >100-fold the IC₅₀ for translation inhibition—the mechanism of cognate aa-tRNA selection was unaffected in terms of both the bimolecular rate constant of ternary complex binding and the efficiency and manner with which FRET evolved during the selection process (Fig. 2 C and D, Left). Thus, unlike tetracycline and tigecycline (19, 21), negamycin’s interactions with the ribosome do not sterically impinge on the entry of cognate aa-tRNA as it enters the A site.

Fig. 2.

Pre–steady-state tRNA selection experiments. (A) Schematic of the tRNA selection process imaged using smFRET, in which surface-immobilized initiation complexes bearing tRNA^fMet in the P site are monitored as the ternary complex of Phe-tRNA^Phe enters the A site. 50S subunits (gray), 30S subunits (light blue), tRNA^fMet (orange), Phe-tRNA^Phe (green), EF-Tu (blue), Cy3 (green circle), and Cy5 (red circle) are shown. From left to right: a zero FRET state is observed before ternary complex binding; during initial selection, a FRET transition occurs between a low-FRET, codon recognition (CR) state and an intermediate FRET, GTPase-activated (GA) state, in which GTP (yellow circle) is hydrolyzed to GDP (maroon). During proofreading, a FRET transition occurs between the intermediate-FRET, GA state and the fully accommodated AC state. (B) Representative smFRET trace recording (blue) and idealization (red) of the cognate tRNA selection process, in which each colored rectangle highlights the FRET values corresponding to CR, GA, and AC states. Rectangle placements approximate a 2-SD width from the mean FRET value assigned to each state. (C) Postsynchronized FRET histograms representing all molecules observed to undergo tRNA selection under pre–steady-state measurements of cognate (UUC)- (Left) and near-cognate (UCU)-programmed ribosomes (Right). (D) Postsynchronized FRET histograms showing productive tRNA selection events: those reaching a stable (>120 ms) AC state in the presence of 200 μM negamycin for cognate (UUC)- (Left) and near-cognate (UCU)-programmed ribosomes (Right).

To assess negamycin’s impact on the fidelity mechanism, identical experiments were performed using ribosomes programmed with a near-cognate mRNA codon in the A site (UCU). Although ribosomes efficiently reject near-cognate Phe-tRNA^Phe ternary complex (Fig. 2C, Right), near-cognate ternary complexes were observed to enter the A site in the presence of negamycin (Fig. 2D, Right), in line with results obtained with synthetic polynucleotides (10). Comparison of population histograms representing the subset of productive binding events observed in the absence and presence of negamycin revealed that near-cognate tRNAs are stabilized on the ribosome by negamycin in the transition between CR and GA states (Fig. 2D). At saturating negamycin concentrations (200 µM), the GA state exhibited an ∼170 ± 30-ms lifetime, roughly sixfold longer than observed in the absence of drug (Fig. 3A). We therefore infer that the additional time that near-cognate ternary complex persists on the ribosome serves to increase the probability that productive GA states may be achieved and irreversible GTP hydrolysis may occur (19, 20). In so doing, the miscoding frequency is increased commensurately (Fig. 3B). Here, the effective concentrations at which a 50% amplitude was observed (EC₅₀s) for both effects on the selection mechanism, estimated by fitting the observed data to Hill functions, were ∼10 µM (Fig. 3). By contrast, tRNA dynamics within pretranslocation ribosome complexes (both cognate and near-cognate) were indistinguishable in both the absence and presence of negamycin (data not shown). As expected from our resistance studies (Table 1), negamycin’s impact on the tRNA selection process was significantly suppressed by the U1052G resistance mutation (Fig. 3 A and B). Hence, these data suggest that negamycin specifically affects the tRNA selection mechanism on the E. coli ribosome, acting to steer the near-cognate ternary complex toward the productive GTPase-activation corridor and preventing off-pathway excursions that normally lead to the rejection of near-cognate ternary complexes during initial selection. The combined effects of translational stalling and miscoding at each step of the elongation cycle likely explain the increased potency of negamycin observed in coupled in vitro transcription–translation reactions (Table 1). In investigations using a Ser199 (TCT) to Phe199 (TTT) luciferase mutant with the goal of showing gain of function caused by negamycin-induced miscoding, we have found no evidence of such effect. We interpret the absence of gain-of-function effects in our studies to suggest that both translational stalling-induced inhibition of luciferase expression and concomitant miscoding of bona fide codons elsewhere in the transcript are functionally dominant to the negamycin-induced miscoding effects at the single, mutated codon.

Fig. 3.

Negamycin-induced translational stalling and miscoding. (A) The lifetime of the CR and GA states observed for wild-type (blue ▲ and red ●, respectively) and U1052G-mutant E. coli ribosomes (gray ▼ and black ▪, respectively) observed when tRNA selection experiments were performed on near-cognate (UCU) programmed ribosomes in the presence of a Cy5-labeled Phe-tRNA^Phe ternary complex (10 nM) over a range of negamycin concentrations. (B) The fraction of wild-type (red ▪) and U1052G-mutant ribosomes programmed with the near-cognate (UCU) codon in the A site observed to successfully complete the selection process after incubation of surface-immobilized 70S initiation complexes bearing Cy3-labeled tRNA^fMet in the P site with 10 nM Cy5-labeled Phe-tRNA^Phe ternary complex for 2 min over a range of negamycin concentrations. Data were fit using the Hill equation, where n, the number of binding sites, was set to unity. Error bars represent the SDs obtained from three independent experiments.

Consistent with the notion that negamycin, tetracycline, and tigecycline share structurally related sites of interaction with the ribosome, both tetracycline and tigecycline operate by preventing cognate and near-cognate tRNA from making the CR-to-GA transition during the tRNA selection process (19, 21). The smaller tetracycline molecule has been shown to be somewhat less effective at blocking this step of tRNA entry while modestly increasing the lifetime of both cognate and near-cognate tRNA within the CR state (19). Tigecycline’s strict prevention of aa-tRNA progression beyond the low-FRET, CR state has been attributed to its extended, ring D structure, which serves to sterically occlude tRNA entry and formation of the GA state (21). Thus, although negamycin likely occupies a site that partially overlaps that of tetracycline and tigecycline, where it directly or indirectly influences the fidelity of initial selection, the observation that later events in the selection process appear unaffected by negamycin suggests that its position is compatible with A site occupancy. In line with this notion, no perceptible impact was observed on tRNA motions or subunit rotation processes within the pretranslocation complex or the process of elongation factor G (EF-G)-catalyzed translocation in the presence of 200 µM negamycin (data not shown). Collectively, these data suggest that negamycin acts to specifically influence structural and/or conformational processes within the head domain of the small subunit that facilitate productive tRNA selection, which normally are achieved inefficiently when the A site is occupied by a near-cognate ternary complex.

Crystal Structure of E. coli 70S Ribosome in Complex with Negamycin.

To obtain direct structural insight on the nature of the negamycin–ribosome interaction, crystals containing two apo E. coli 70S ribosomes per asymmetric unit (27) were transferred sequentially in stabilization solutions with 1 mM negamycin. Diffraction data from these crystals were collected to ∼3.1 Å, and the structure was determined by molecular replacement (Table S2). Globally, the architecture of both 70S ribosomes within the asymmetric unit was unaltered by the presence of negamycin (27, 38).

In contrast to the structure of the archaeal 50S subunit in complex with negamycin (14), no evidence was found for difference electron density in the vicinity of the exit tunnel, even at a signal-to-noise cutoff of 2.5σ. Positive difference density, however, was identified through a broader search of the “classical”-state ribosome in the crystallographic asymmetric unit (Fig. 4A) (27). This unexplained difference density was positioned next to the phosphate backbone of nucleotides C1054, A1197, and G1198 proximal to h34 (Fig. 4B) at the base of the small subunit head domain (Fig. 4C). This binding site directly overlaps the previously described binding sites for tetracycline and tigecycline (Fig. S3) (21, 26, 39, 40). Although weak positive difference density was observed in the “intermediate”-state ribosome in the crystallographic asymmetric unit (27), negamycin could be placed only in the classical-state ribosome.

Fig. 4.

Negamycin binds the small subunit at the base of helix 34. (A) Difference electron density (F_obs-F_calc) map contoured at 3σ to negamycin in the 30S ribosomal subunit of E. coli. (B) Binding site of negamycin near helix 34 of the 30S ribosome of E. coli. The mesh represents a 2Fobs-Fcalc electron density map contoured at 1σ. (C) Global view of the 30S subunit of the ribosome showing negamycin’s site of interaction immediately proximal to h34 at the base of the head domain. (D) Schematic model showing the position of negamycin with respect to the mRNA codon–tRNA anticodon interaction within the A site. The model was created by superposition of the negamycin-bound ribosome with Protein Data Bank (PDB) ID code 2wrq, which contains ternary complex stalled in an intermediate (A/T) state of tRNA selection by the nonhydrolyzable GTP analog, GDPNP. The anticodon stem loop of the A/T tRNA (yellow), the A and P site codons of mRNA (blue), and the decoding site residues A1492, A1493 within 16S rRNA (red) are shown.

Given the present resolution of our crystallographic data, unambiguous placement of negamycin into the electron density map was not possible, and a variety of binding modes were considered carefully. Placement was complicated by the lower quality of the electron density map of the classical-state ribosome in this crystal form compared with the other ribosome in the asymmetric unit, which is in an intermediate state of rotation (27). The chemical structure of negamycin inherently allows this molecule to assume a large degree of rotational freedom. However, in the most plausible model of negamycin binding based on structure–activity relationships (Table S3), the presence of two Mg²⁺ ions within the binding site restricts the bound conformation of negamycin and serves to increase the number of interactions with the phosphate backbone of the ribosome (Fig. 4A). The carboxylate moiety of negamycin is positioned near the 2′ hydroxyl of A1196 and a phosphate oxygen of C1054, thereby placing the base of C1054 above the carboxylate group. The C6 carbonyl group is directed to a bound Mg²⁺ ion, and the C4 amino group extends toward the phosphate backbone of A1197. The C2 hydroxyl is pointed toward the phosphate backbone of G966, and the terminal amino group of negamycin is positioned in the solvent. This binding mode is supported by the structure–activity relationships of negamycin derivatives assessed through coupled in vitro transcription–translation assays, which showed that modification of the carboxylate moiety completely abolishes activity, whereas the addition of a methyl, ethyl, or benzyl group to the amino substituent has no impact on activity (Table S3).

Discussion

The data presented reveal that the broad-spectrum antibiotic negamycin interacts with the h34 element of 16S rRNA within the head domain of the small ribosomal subunit. This site of interaction partially overlaps the established tetracycline and tigecycline binding sites. Negamycin’s mode of action on the translation mechanism through this site, verified by both resistance mutations in this same region and tetracycline binding competition assays (Fig. 1), was shown through functional investigations to specifically affect the fidelity of tRNA selection. Here, negamycin was observed to stabilize near-cognate ternary complex within the A site during the initial selection process. This prolonged period of occupation increased the frequency of miscoding by approximately sixfold (Fig. 3B). These findings shed light on negamycin’s reported propensities to promote ribosome stalling, polysome stabilization, and miscoding, resulting in rapid cell death (8–13). Together with the known effects of both tetracycline and tigecycline, these findings provide compelling evidence that the corridor between the head domain of the small subunit and the aa-tRNA anticodon contribute to the steering of ternary complex into the A site during initial selection (Fig. 4D).

These insights reveal the chimeric nature of the h34 binding site for negamycin and the tetracycline-class antibiotics on the ribosome. Although substantial improvements in tetracycline have been achieved in this class that increase binding affinity and reduce the effectiveness of resistance mechanisms such as TetM, presently little is known regarding the now-apparent potential of small peptides to affect the fidelity mechanism. As negamycin does not appear to affect the process of cognate tRNA selection, the inhibitory potency of negamycin derivatives or other compounds operating through this site might be improved substantially. The development of negamycin derivatives also may provide a therapeutic avenue for specifically targeting tRNA misincorporation at stop codons on the human ribosome. The crystallographic system described here, in conjunction with mechanistic smFRET studies, will allow monitoring of the establishment of new small-molecule–ribosome interactions and thus support an iterative, rational chemistry program.

Materials and Methods

Ribosome Purification and Crystallization.

E. coli 70S ribosomes lacking S1 protein, used in all crystallization experiments, were derived from MRE600 and purified as previously described (41) with the exception of a combination of 2 mM DTT and 2 mM tris(2-carboxyethyl)phosphine (TCEP) instead of 4 mM 2-mercaptoethanol as the reducing agent. Ribosomes were crystallized as previously described (27). Trays of ribosome crystals were transferred to 4 °C for incubation overnight, with all subsequent harvesting steps conducted at 4 °C. Ribosome crystals then were stabilized by brief soaks in crystallization solutions supplemented with 6–18% (vol/vol) PEG 400 in a total of seven steps. To form the negamycin-bound complex, an additional soaking step was added by using crystallization solution supplemented with 24% (vol/vol) PEG 400, 0.5–1 mM ZnCl₂, and 1–2 mM negamycin, and the crystals remained in this solution for 16–72 h before rapid cooling by direct immersion into liquid nitrogen.

X-Ray Data Collection, Reduction, Structure Determination, and Refinement.

Diffraction data were measured at the Advanced Photon Source on beamline 17-ID (IMCA-CAT) using the Pilatus 6M detector from vitrified crystals under cryogenic conditions using 0.1° oscillations and exposure times of 0.15 s per frame with beam attenuation of 40%. Data were indexed with XDS (42) and scaled using Aimless (43) as scripted in autoPROC from Global Phasing (44). Structures were determined by molecular replacement using coordinates of the E. coli 70S ribosome in the apo state (27) as the starting model. Model building and compound placement were conducted with Coot (45), and structure refinement was carried out with autoBUSTER (46) or PHENIX (47). See SI Materials and Methods for a more detailed description.