Alexander Golubev

Bulat Fatkhullin

Iskander Khusainov
email: khusaino@embl-heidelberg.de

Lasse Jenner

Azat Gabdulkhakov

Shamil Validov

Marat Yusupov
email: marat.yusupov@igbmc.fr

Gulnara Yusupova

Konstantin Usachev
email: k.usachev@kpfu.ru

2 Å resolution

Keywords: Cryo-EM, cryogenic electron microscopy, fMet-tRNA i Met , formylated methionine tRNA, LSU Ribosome, rRNA modifications, Staphylococcus aureus, methyltransferases, antibiotic resistance, drug targets

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Introduction

Antibiotic resistance is becoming a major threat to healthcare systems around the world. A certain subset of microorganisms (ESKAPE [START_REF] Rice | Federal funding for the study of antimicrobial resistance in nosocomial pathogens: No ESKAPE[END_REF] organisms) was highlighted for their tenacious and pathogenic behavior. They pose extreme threat due to their fast adaptation to drug treatment, host invasive techniques, severe outcomes for patients and their leading role in causing hospital-acquired infections. Amongst these organisms, S. aureus is a prominent pathogenic Gram-positive bacterium that causes acute and chronic infections with a number of highly resistant strains (such as MRSA, VRSA).

The ribosome is one of the most complex molecular machines and is one of the primary targets of antibiotics. It accomplishes the central metabolic process in living organisms -protein synthesis. The 70S bacterial ribosome is a two-subunit (large 50S and small 30S) nucleoprotein complex consisting of 3 strands of ribosomal RNA (rRNA) and 50 individual ribosomal proteins. Detailed structural data have created the foundation for mechanistic explanations of ribosome functions and the action of antibiotics.

Previously, X-ray structural data of the ribosome from the model organisms Escherichia coli [START_REF] Noeske | Highresolution structure of the Escherichia coli ribosome[END_REF][START_REF] Borovinskaya | Structural basis for aminoglycoside inhibition of bacterial ribosome recycling[END_REF], Thermus thermophilus [START_REF] Yusupov | Crystal structure of the ribosome at 5.5 A resolution[END_REF][START_REF] Selmer | Structure of the 70S ribosome complexed with mRNA and tRNA[END_REF][START_REF] Jenner | Structural rearrangements of the ribosome at the tRNA proofreading step[END_REF][START_REF] Jenner | Structural aspects of messenger RNA reading frame maintenance by the ribosome[END_REF][START_REF] Polikanov | Structural insights into the role of rRNA modifications in protein synthesis and ribosome assembly[END_REF], Deinococcus radiodurans [START_REF] Harms | High resolution structure of the large ribosomal subunit from a mesophilic eubacterium[END_REF], Saccharomyces cereviseae [START_REF] Ben-Shem | The structure of the eukaryotic ribosome at 3.0 A resolution[END_REF] were determined at high resolution. Recently, using the advantages of Cryo-EM, more organisms were added to the list such as S. aureus [START_REF] Khusainov | Structure of the 70S ribosome from human pathogen Staphylococcus aureus[END_REF], Bacillus subtilis [START_REF] Sohmen | Structure of the Bacillus subtilis 70S ribosome reveals the basis for species-specific stalling[END_REF], Plasmodium falciparum [START_REF] Wong | Cryo-EM structure of the Plasmodium falciparum 80S ribosome bound to the antiprotozoan drug emetine[END_REF][START_REF] Sun | Dynamical features of the Plasmodium falciparum ribosome during translation[END_REF], Mycobacterium tuberculosis [START_REF] Hentschel | The Complete Structure of the Mycobacterium smegmatis 70S Ribosome[END_REF],

Pseudomonas aeruginosa [START_REF] Halfon | Structure of Pseudomonas aeruginosa ribosomes from an aminoglycoside-resistant clinical isolate[END_REF].

Complete and high resolution structures of ribosomes from pathogenic bacteria, especially the species exhibiting increasing antibiotic resistance, are necessary research objects in the never-ending battle against antibiotic resistance. A series of S. aureus ribosome structures were investigated in the last decadevacant 70S [START_REF] Khusainov | Structure of the 70S ribosome from human pathogen Staphylococcus aureus[END_REF], hibernating ribosome dimers [START_REF] Khusainov | Structures and dynamics of hibernating ribosomes from Staphylococcus aureus mediated by intermolecular interactions of HPF[END_REF], RsfS-inactivated 50S subunit [START_REF] Khusainov | Mechanism of ribosome shutdown by RsfS in Staphylococcus aureus revealed by integrative structural biology approach[END_REF] with antibiotics [START_REF] Belousoff | Structural Basis for Linezolid Binding Site Rearrangement in the Staphylococcus aureus Ribosome[END_REF].

Main species-specific features such as rRNA and protein extensions were described [START_REF] Khusainov | Structure of the 70S ribosome from human pathogen Staphylococcus aureus[END_REF]. However, the accuracy of the models and the level of detail that was achieved were limiting the interpretation of antibiotic binding sites, and better models will give new implications for structure-based drug design. Furthermore, small S. aureus species-specific details can be discovered using a higher resolution map.

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This article is protected by copyright. All rights reserved One of the important features of ribosomes are ribosomal RNA modifications. Ribosomal RNA contains many modified nucleotides in addition to the standard nucleotides. In bacteria, there are three major types of rRNA modifications: methylation of bases, methylation of ribose and pseudouridylation. rRNA modifications broaden the chemical and structural properties of nucleotides. The specific function of a modified nucleotide does not solely stem from the chemical structure but is also based on the particular structural environment. Modifications can create a wide range of changes such as increases in structural rigidity, thermodynamics, bonding potential changes, local base stacking enhancement etc., so the effects can propagate beyond the modification site [START_REF] Decatur | rRNA modifications and ribosome function[END_REF]. rRNA modifications (see [START_REF] Sergiev | Modifications of ribosomal RNA: From enzymes to function[END_REF][START_REF] Sergiev | Structural and evolutionary insights into ribosomal RNA methylation[END_REF] for review) are not merely randomly distributed throughout the ribosome, but often found clustered in functional centers: the aminoacyl-(A-), peptidyl-(P-) and exit-(E-) tRNA binding sites, intersubunit bridges and the peptidyl transferase center (PTC) [START_REF] Noeske | Highresolution structure of the Escherichia coli ribosome[END_REF][START_REF] Polikanov | Structural insights into the role of rRNA modifications in protein synthesis and ribosome assembly[END_REF][START_REF] Brimacombe | Clustering of modified nucleotides at the functional center of bacterial ribosomal RNA[END_REF][START_REF] Sergeeva | What do we know about ribosomal RNA methylation in Escherichia coli?[END_REF]. While the exact role of each modification is not yet uncovered, the general view is that modifications are involved in proper construction of functional centers where they finetune ribosome functions [START_REF] Sergiev | Structural and evolutionary insights into ribosomal RNA methylation[END_REF]. Even though the rRNA modifications do not seem to be directly involved in the chemistry of translation, they most likely participate by stabilization of rRNA secondary and tertiary structure [START_REF] Sloan | Tuning the ribosome: The influence of rRNA modification on eukaryotic ribosome biogenesis and function[END_REF]. Modifications may stabilize otherwise unfavorable rRNA folds either by acting on the local electrostatic environment or hydrogen-bonding capacity [START_REF] Chawla | An atlas of RNA base pairs involving modified nucleobases with optimal geometries and accurate energies[END_REF]. rRNA modifications have also been implicated in ribosome assembly [START_REF] Sergiev | Modifications of ribosomal RNA: From enzymes to function[END_REF] where they may function as folding chaperones or quality control markings [START_REF] Song | Modification of rRNA as a 'quality control mechanism' in ribosome biogenesis[END_REF]. In bacteria, almost all rRNA modifications are made by site specific enzymes [START_REF] Sergiev | Structural and evolutionary insights into ribosomal RNA methylation[END_REF]. Studies of methylation enzymes showed that deletion of a modification enzyme is rarely accompanied by a noticeable fitness-loss for bacteria in non-native environments [START_REF] Lovgren | The rlmB gene is essential for formation of Gm2251 in 23S rRNA but not for ribosome maturation in Escherichia coli[END_REF][START_REF] Pletnev | Comprehensive Functional Analysis of[END_REF]. However, several studies have also shown that loss of some particular rRNA modifications lead to diverse negative effects: severe growth retardation [START_REF] Huang | Identification of two Escherichia coli pseudouridine synthases that show multisite specificity for 23S RNA[END_REF], slower rates and accuracy of translation [START_REF] Baudin-Baillieu | Nucleotide modifications in three functionally important regions of the Saccharomyces cerevisiae ribosome affect translation accuracy[END_REF], alterations of active sites [START_REF] Demirci | Modification of 16S ribosomal RNA by the KsgA methyltransferase restructures the 30S subunit to optimize ribosome function[END_REF] and a decrease in the fidelity of translation initiation [START_REF] Vanbuul | Increased Translational Fidelity Caused by the Antibiotic Kasugamycin and Ribosomal Ambiguity in Mutants Harboring the Ksga Gene[END_REF]. Due to recent advances in X-ray crystallography [START_REF] Noeske | Highresolution structure of the Escherichia coli ribosome[END_REF][START_REF] Polikanov | Structural insights into the role of rRNA modifications in protein synthesis and ribosome assembly[END_REF] and single-particle electron cryo-electron microscopy [START_REF] Fischer | Structure of the E. coli ribosome-EF-Tu complex at <3 A resolution by Cs-corrected cryo-EM[END_REF][START_REF] Coureux | Cryo-EM study of an archaeal 30S initiation complex gives insights into evolution of translation initiation[END_REF] (Cryo-EM), structural biology techniques have become efficient methods for verification rRNA modifications. Exceptional scientific rigor should be taken to cross-validate with biochemical and bioinformatical data if available to ensure that errors do not occur.

Particular attention has to be given to rRNA modifications in bacteria due to their role in antibiotic resistance [START_REF] Cundliffe | Ribosomal modification and resistance in antibiotic-producing organisms[END_REF]. Antibiotics targeting the ribosome often bind near or in functional centers of the ribosome where they disrupt normal ribosome function [START_REF] Arenz | Bacterial Protein Synthesis as a Target for Antibiotic Inhibition[END_REF].

Having detailed species-specific structural data of the S. aureus ribosome and locations of the rRNA modifications is useful for understanding the resistance mechanisms of S. aureus and for the design of new antibiotics that can help in the fight against this pathogen. The data on S. aureus ribosomal RNA modifications and corresponding modification enzymes are very limited, and the structural data is missing.

We aimed to improve the current models of the S. aureus ribosome with functional ligands by determining its structure at a much higher resolution than what is currently available and, thereby, create a basis that can

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This article is protected by copyright. All rights reserved be used in structure-based drug design. Also, we wanted to reveal the rRNA modification pattern of the S. aureus ribosome and correlate them to the existing data of other bacterial species.

In this study, we present the Cryo-EM structure of the S. aureus 70S ribosome with P-tRNA and mRNA and assign 10 rRNA modifications. We correlate this information with how the modifying enzymes responsible for said modifications are conserved and possibly exploitable as potential drug targets.

Results and Discussion

We determined the single-particle Cryo-EM structure of the in vitro reconstituted complex of S. aureus 70S bound to mRNA containing AUG start-codon and Shine-Dalgarno sequence and initiator tRNA with formylated methionine residue (fMet-tRNA i Met ) (Figs S1 andS2). The general resolution of the resulting Cryo-EM map is 3.2 Å (0.143 criterion, Fig S1). In order to assess the quality of the Cryo-EM map, a local resolution map was constructed [START_REF] Kimanius | Accelerated cryo-EM structure determination with parallelisation using GPUs in RELION-2[END_REF](Fig 1a-b). Mostly, the ribosome and functionally important parts such as the PTC and decoding center are resolved at 2.8-3.1 Å. This ensures reliable building of the atomic model and interpretation of small features such as interactions in the decoding center and rRNA modifications (

Ribosomal RNA modifications

Modeling and assignment of rRNA modification were based on a thorough analysis of the Cryo-EM map. We are aware of the errors that may occur when assigning modifications solely based on Cryo-EM maps as has been pointed out earlier [START_REF] Taoka | Landscape of the complete RNA chemical modifications in the human 80S ribosome[END_REF]. To correctly assign modifications, cross correlation of data from different experimental sources is necessary. Mass spectrometry analysis of rRNA modification still comes with some limitations and the analysis process is methodically difficult [START_REF] Popova | Quantitative Analysis of rRNA Modifications Using Stable Isotope Labeling and Mass Spectrometry[END_REF], thus the structural validation of rRNA modification is an advantage. Unfortunately, no MS data for the rRNA modifications exist for S. aureus. We therefore chose to cross correlate with biochemical data available from other bacterial species.

We thoroughly inspected the Cryo-EM map to find places with unmodelled map density.

Modifications were assigned if they were supported by the Cryo-EM map and at the same time if they correlated with structurally equivalent modified positions in E. coli and T. thermophilus that have been confirmed experimentally [START_REF] Sergiev | Structural and evolutionary insights into ribosomal RNA methylation[END_REF]. We also used the availability of the corresponding homologous enzymes in S. aureus for further correlation and validation. Using this procedure, we could identify 10 sites for S. aureus rRNA modifications (Table 1, Figs 2 and3) where the density allowed the confident modeling of the modified nucleotide.

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In addition to the mapped modifications we found sites, where the density map allows us to add modification, but supporting experimental data is missing. Most probably, many of the sites correspond to Mg 2+ ions coordinated by N7 of purine nucleotides (see Fig S3 (g-h) for two examples). However, two places -G47 of 16S rRNA and G2083 of 23S rRNA -are not coordinated by Mg 2+ and could be possible modifications (Fig S3 (e-f)). Additional biochemical experiments should be carried out to corroborate the results.

Modifications of 23S rRNA

We could detect sufficient density for the methyl group at 2′-O of ribose of C1947 in Helix 69 (H69), despite the lack of density for the 2′-O itself. The density is not sufficiently large for it to be a Mg 2+ ion (Fig 3b). According to the experimental model, the methylated nucleotide C1947 resides in a location of high functional importance -at intersubunit bridge region B2a, in which the loop of 23S rRNA H69 contacts helix 44 (h44) of 16S rRNA of small 30S ribosomal subunit. It was shown that H69 of large 50S ribosomal subunit acts as a regulator of decoding nucleotides in h44 of the 16S rRNA in the decoding center of the small 30S ribosomal subunit, thus is responsable for the precise conversion of genetic information into proteins [START_REF] Rozov | New Structural Insights into Translational Miscoding[END_REF]. Many bacteria, including E. coli, do not have this modification, because they do not possess the corresponding 2′-O-methyltransferase tlyA [START_REF] Johansen | Capreomycin binds across the ribosomal subunit interface using tlyA-encoded 2 '-O-methylations in 16S and 23S rRNAs[END_REF]. It was shown that TlyA acts as virulence factor and is likely involved in ribosome biogenesis and hemolytic activity [START_REF] Rahman | Mycobacterium tuberculosis TlyA Protein Negatively Regulates T Helper (Th) 1 and Th17 Differentiation and Promotes Tuberculosis Pathogenesis[END_REF][START_REF] Rahman | Molecular characterization of tlyA gene product, Rv1694 of Mycobacterium tuberculosis: A non-conventional hemolysin and a ribosomal RNA methyl transferase[END_REF][START_REF] Arenas | Molecular modeling and in silico characterization of Mycobacterium tuberculosis TlyA: Possible misannotation of this tubercle bacilli-hemolysin[END_REF]. Despite that inactivation of TlyA results in resistance to capreomycin and viomycin antibiotics [START_REF] Johansen | Capreomycin binds across the ribosomal subunit interface using tlyA-encoded 2 '-O-methylations in 16S and 23S rRNAs[END_REF], it could be a potential target for inhibition studies in S. aureus, due to its involvement in virulence and other major biochemical processes. The biological functions of the S. aureus homolog of TlyA are still largely unknown.

At a level of 1.6 σ in the density map, there is clear density indicating a methylation of U1966 at position C5 (Fig 3c). U1966 is located on the periphery of the A-site tRNA binding pocket where it can potentially interact directly with the aminoacyl end of A-site bound tRNA. It can also indirectly influence the interactions through stabilization or destabilization of the area. In the Gram-positive bacteria B. subtilis, the methyltransferase RlmCD modifies both U747 and U1939 in the 23S rRNA [START_REF] Desmolaize | A single methyltransferase YefA (RlmCD) catalyses both m(5)U747 and m(5)U1939 modifications in Bacillus subtilis 23S rRNA[END_REF] whereas this is not the case in G-negative bacteria like E. coli. Since S. aureus resembles B. subtilis more than E. coli it is most likely that it also possesses a single methyltransferase. However, we observed only poor density for the possible modification at position U792 (corresponding to E. coli U747) and could not assign the modification at this position with high confidence (Fig S3c). The direct studies on RlmCD homolog functions in S. aureus could be the simplest method to discover the presence of U792 modification.

With an overall high local resolution in the area near G2278 we clearly see density for a methyl group at 2′-O of ribose of G2278 (Fig 3d). The modified G2278 is located in the PTC in the P-loop of the

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This article is protected by copyright. All rights reserved 23S rRNA. This nucleotide forms a direct connection with the CCA end of P-tRNA. The modification is located on the opposite side from the binding pocket for the P-tRNA CCA-end in vicinity of the three nucleotides U2476, C2092 and A2477. The modified G2278 likely plays a role in correct accommodation of P-tRNA, by supporting the conformation of nucleotides engaged in a base pairing. Despite the position of the modification in an important functional center, deletion of RlmB does not lead to a significantly different phenotype from the normal one [START_REF] Lovgren | The rlmB gene is essential for formation of Gm2251 in 23S rRNA but not for ribosome maturation in Escherichia coli[END_REF].

Very strong density indicating the presence of a methyl group at N2 was observed for the nucleotide G2472 (Fig 3e). This modified G2472 is located in H74 of the 23S rRNA near PTC. Nucleotide G2472 forms a Watson-Crick pair with C2092 that is in very close proximity to the CCA-end of P-tRNA.

The methyl group can potentially weaken hydrogen bonding between N2 of G2472 and O2 of C2092, thus indirectly influence the stability of the PTC area by prevention of base triples formation [START_REF] Lesnyak | Identification of Escherichia coli m(2) G methyltransferases: I. The ycbY gene encodes a methyltransferase specific for G2445 of the 23 S rRNA[END_REF]. On the other side, the modification also could indirectly affect the interaction with Arg74 of the uL4 protein. In E. coli enzyme RlmKL modifies both the G2445 (equivalent to S. aureus G2472) and G2069 [START_REF] Kimura | Base methylations in the doublestranded RNA by a fused methyltransferase bearing unwinding activity[END_REF]. In our data we could not detect any presence of a modification at position G2096 (corresponding to G2069 in E. coli) possibly due to the low local resolution of the Cryo-EM map in that area. Interestingly, it is possible that similar to bacteria from the genus Neisseria, S. aureus may have two separate protein homologs RlmK and RlmL corresponding to two different domains of a fusion methyltransferase RlmKL [START_REF] Kimura | Base methylations in the doublestranded RNA by a fused methyltransferase bearing unwinding activity[END_REF].

In the same region, the map clearly shows the presence of a modification at the 2′-O of C2525 ribose (Fig 3f). The modified nucleotide C2525 is buried within the peptidyl transferase loop. It interacts with A2524 that is located in proximity to protein uL16 and the CCA end of P-tRNA. Through this interaction, the modified C2525could possibly influence binding of P-tRNA. It is also likely that the modification could impact the position of the C2525 nucleotide and thus have an effect on the architecture of the peptidyl transferase loop. The possible importance of the modification in S. aureus still has to be assessed; however, deletion studies of RlmM methyltransferase that modifies C2498 in E. coli did not show significant effects on growth [START_REF] Purta | YgdE is the 2'-O-ribose methyltransferase RlmM specific for nucleotide C2498 in bacterial 23S rRNA[END_REF].

The high resolution of our data in the area around the PTC allowed us to assign the modification at A2530 (Fig 3g). This methylation even shows up in the map at very high sigma levels (2.5σ). The modified A2530 is located in the PTC, where it creates part of the A-site binding pocket for the side chain of amino acid of A-tRNA. The modification probably enhances the stacking between nucleotides A2086 and A2530, thus likely stabilize rRNA of the PTC loop, which forms the wall of the peptide exit tunnel. Deletion of the corresponding methyltransferase YfgB/RlmN modifies translation accuracy in E. coli [START_REF] Benitez-Paez | The Escherichia coli RlmN methyltransferase is a dual-specificity enzyme that modifies both rRNA and tRNA and controls translational accuracy[END_REF] and grants a minor increase in susceptibility to a number of peptidyl transferase antibiotics [START_REF] Toh | The methyltransferase YfgB/RlmN is responsible for modification of adenosine 2503 in 23S rRNA[END_REF]. In clinical isolates of E. coli, additional methylation of A2503 by methyltransferase Cfr provides resistance to a wide range of PTCtargeting antibiotics [START_REF] Arenz | Blast from the Past: Reassessing Forgotten Translation Inhibitors, Antibiotic Selectivity, and Resistance Mechanisms to Aid Drug Development[END_REF][START_REF] Giessing | Identification of 8-methyladenosine as the modification catalyzed by the radical SAM methyltransferase Cfr that confers antibiotic resistance in bacteria[END_REF]. In the study of Seok-Ming Toh [START_REF] Toh | The methyltransferase YfgB/RlmN is responsible for modification of adenosine 2503 in 23S rRNA[END_REF], it was proposed based on experimental

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data, that by analogy with E. coli, S. aureus should carry a methyl modification at the corresponding position A2530 of 23S rRNA, which our data corroborates.

S. aureus possess modifying protein

OrfX that is a homolog to E. coli YbeA that methylates 23S rRNA at position U1915 in E. coli, located at the end of hairpin of H69. It was previously shown that in S. aureus OrfX methylates 70S ribosome [START_REF] Boundy | Characterization of the Staphylococcus aureus rRNA Methyltransferase Encoded by orfX, the Gene Containing the Staphylococcal Chromosome Cassette mec (SCCmec) Insertion Site[END_REF]. According to the study, there is no effect of the loss of OrfX on bacteria fitness, growth and oxacillin susceptibility in vitro. The modification at S. aureus U1942 (equivalent to E. coli U1915) is expected to be present; however, the quality of the Cryo-EM map in this particular area is not of sufficient quality to allow us to assign the modification. Local resolution map at this region is around 3.1 Å, but the nucleotide is located at the periphery and thus highly flexible. YbeA activity requires prior conversion of nucleotide to pseudourine [START_REF] Ero | Identification of pseudouridine methyltransferase in Escherichia coli[END_REF], which in E. coli is accomplished by the pseudouridine synthase RluD that modifies three sites (Ψ1911, Ψ1915, and Ψ1917 in E. coli). These modified nucleotides are located at the subunit interface and interact with P-and A-site tRNAs. By inference these nucleotides should also be modified in S. aureus, and due to the positional importance of this cluster, the homolog of RluD could be a suitable target for inhibition studies in S. aureus.

Methylation of ribose at position U2579 (U2552 in E. coli) is amongst the most conserved modifications found throughout all three kingdoms of life. This modification is located in the A-loop of 23S RNA and can influence binding of aminoacyl-tRNA to the A-site [START_REF] Widerak | U2552 methylation at the ribosomal A-site is a negative modulator of translational accuracy[END_REF]. The deficiency of the corresponding modifying enzyme RlmE leads to one of the most severe phenotypes across methyltransferase knockouts in E. coli [START_REF] Caldas | The FtsJ/RrmJ heat shock protein of Escherichia coli is a 23 S ribosomal RNA methyltransferase[END_REF]. Since this modification is so highly conserved it is expected to be present in the S. aureus ribosome, however, even though the Cryo-EM density map is of quite high quality in this area we were not able to confidently determine its presence (Fig S3b). In any case, the S. aureus homolog of RlmE may still represent a promising potential target for binding of inhibitors.

Modifications of 16S rRNA

The resolution of the map in the area of small 30S subunit of the ribosome allowed us to detect four modification sites. The Cryo-EM map plainly shows modification at the G535 nucleotide (Fig 4b). The modified nucleotide G535 (equivalent of G527 in E. coli) is located in the loop of helix 18 near the A-site of the 30S subunit, close to the streptomycin binding site. The methylated nucleotide forms a Watson-Crick pair with C530 and the methyl group occupies a position near Asn59 of uS12 protein. In E. coli, streptomycin binds to four distinct regions of the 16S rRNA, including G526 and G527 [START_REF] Brakiergingras | The Control of Accuracy during Protein-Synthesis in Escherichia-Coli and Perturbations of This Control by Streptomycin, Neomycin, or Ribosomal Mutations[END_REF], which potentially points to a role for the S. aureus G535 modification in acquired resistance to streptomycin.

Another modified nucleotide is located in h44 of the 16S rRNA in the heart of the P-site and directly contacts the mRNA P-codon. According to the high resolution map in this region, C1412 holds two highly conserved modifications: 2′-O-methylation of the ribose and base methylation at N4 (Fig 4c). The

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This article is protected by copyright. All rights reserved methyl group at N4 directly contacts the center nucleotide of P-codon. The base methylation could also be important for the interaction of C1412 with A1511. The ribose methylation points towards the opposite site and possibly has a role in stabilization of the surrounding area. The modifications, especially the base methylation, most probably participate in the formation of the binding pocket for P-tRNA on a small subunit and could potentially stabilize the interactions with mRNA in the P-site. Not surprisingly, it was shown that methylations of C1402 in E. coli are necessary for the maintenance of the reading frame and start-codon recognition [START_REF] Kimura | Fine-tuning of the ribosomal decoding center by conserved methylmodifications in the Escherichia coli 16S rRNA[END_REF].

In vicinity of C1412 we investigated the highly conserved modified trinucleotide loop 1527-1530 (Fig 3d-f), that is in direct contact with modified C1947 of 23S rRNA that we discussed above. We found two universally conserved dimethylated nucleotides; A1529 and A1530, which are stacking with each other (Fig 3f). They are also interacting with methylated G1527 and all these methyl modifications are clustered together. The group of nucleotides is located in h45, in close proximity to the codon-anticodon duplex between P-tRNA and mRNA, and could be involved in formation of decoding area. In agreement with this, it was shown that the absence of these modifications restructures the decoding center [START_REF] Demirci | Modification of 16S ribosomal RNA by the KsgA methyltransferase restructures the 30S subunit to optimize ribosome function[END_REF].

One nucleotide G1527 (see Fig S3d) has the extra density appropriate for a modification, but we didn't find the homologous enzyme in S. aureus. It is possible that in S. aureus this position is modified by another enzyme (not RsmJ). The map shows extra density next to the N2 atom of the corresponding nucleotide. It has even more prominent features next to O6 and N7 atoms. Thus we could not confidently model the modification. Supposedly, as the modified nucleotide G1527 is not highly conserved, it may play a more species-specific fine-tuning role than the dimethylated nucleotides.

The position of the modified G1207 in E. coli (G1189 in T. thermophilus) corresponds to C1217 in our S. aureus model. This nucleotide is located in h34 that has been implicated in translocation and decoding [START_REF] Sergiev | The path of mRNA through the bacterial ribosome: A site-directed crosslinking study using new photoreactive derivatives of guanosine and uridine[END_REF][START_REF] Kubarenko | Involvement of Helix 34 of 16 S rRNA in decoding and translocation on the ribosome[END_REF]. In E. coli, G1207 pairs with C1051 (C1051 is equivalent to G1062 in S. aureus). Thus, in S. aureus the G-C pair has been switched to a C-G pair when comparing to E. coli and T. thermophilus. The Cryo-EM map clearly indicates density for possible methyl group at N2 of G1062 (Fig S3a), but since there is no other experimental nor bioinformatics data to corroborate this finding, we did not include it in the final model. E. coli mutants with conversion of G1207 to pyrimidines have a dominant lethal phenotype [START_REF] Jemiolo | Mutations in 16s Ribosomal-Rna in Escherichia-Coli at Methyl-Modified Sites -G966, C967, and G1207[END_REF], therefore the S. aureus functional homolog corresponding to the methyltransferase RsmC [START_REF] Sunita | Functional specialization of domains tandemly duplicated within 16S rRNA methyltransferase RsmC[END_REF] responsible for the modification of G1207 in E. coli could be a prospective drug target.

Analysis of the functional states of the ribosomes

The resolved structure represents the 70S initiation complex formed prior to accommodation of incoming EF-Tu*tRNA complex in the A-site, and contains its characteristic features such as Shine-

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This article is protected by copyright. All rights reserved Dalgarno and anti-Shine-Dalgarno base pairing, AUG codon-anticodon interaction at the P-tRNA binding site, and a vacant A-tRNA binding site. The present structure expands the number of resolved states of the ribosomes from S. aureus, thus prompting us to perform comparative analysis of similar translational states of the ribosomes from different bacterial species. We performed such a structural comparison of the S. aureus, E. coli, T. thermophilus and B. subtilis 70S ribosomes i) formed with P-tRNA (initiation-like state), ii) formed with P-tRNA and A-tRNA (elongation-like state), iii) HPF-bound 70S (hibernating state), and iv) ligand-free vacant 70S ribosome (Table S1). We focused our attention on the rearrangements of the small ribosomal subunit upon transition through the given states (Fig S4). The most pronounced of them happens during accommodation of the A-tRNA. Movement of the shoulder and the head towards the body (often called "shoulder locking" or "domain closure") lead to stabilization of the decoding center through a network of interactions of nucleotides A1492, A1493, G530 of 16S rRNA and Pro58, Asn59 of uS12 protein via coordination of K+ ions [START_REF] Jenner | Structural rearrangements of the ribosome at the tRNA proofreading step[END_REF][START_REF] Ogle | Recognition of cognate transfer RNA by the 30S ribosomal subunit[END_REF][START_REF] Rozov | Importance of potassium ions for ribosome structure and function revealed by longwavelength X-ray diffraction[END_REF].

In our initiation complex with the A-tRNA binding site is vacant, the shoulder is in an open conformation and nucleotide 1492 is flipped into the grove of h44 and therefore in the "in" conformation, whereas the density for nucleotide 1493 is very poor and therefore must be flexible. This is similar to what is observed for the other bacterial species in this state (Fig 2a). However, the shoulder region and more specifically the helices 16 and 18 constituting part of the shoulder region is positioned a little further away (1.5Å for h16) from the decoding center than in B. subtilis and interestingly even further away (2-3Å) than the two gram-negative bacteria T. thermophilus or E. coli. Interestingly, the nucleotide G530 is found in the anti-conformation in S. aureus and E. coli whereas it is in the syn-conformation in T. thermophilus and B.

subtilis. The preference for the "anti" conformation of G530 in the initiation state does not seem to reflect a tighter domain closure as E. coli and S. aureus have the largest difference in the distance between their shoulder region and the decoding center (Fig 2).

In the elongation state, it has been shown structurally for T. thermophilus [START_REF] Jenner | Structural rearrangements of the ribosome at the tRNA proofreading step[END_REF][START_REF] Demeshkina | A new understanding of the decoding principle on the ribosome[END_REF][START_REF] Rozov | Structural insights into the translational infidelity mechanism[END_REF][START_REF] Rozov | Novel basepairing interactions at the tRNA wobble position crucial for accurate reading of the genetic code[END_REF][START_REF] Loveland | Cryo-EM of elongating ribosome with EF-Tu*GTP elucidates tRNA proofreading[END_REF][START_REF] Loveland | Ensemble cryo-EM elucidates the mechanism of translation fidelity[END_REF] and E. coli [START_REF] Loveland | Cryo-EM of elongating ribosome with EF-Tu*GTP elucidates tRNA proofreading[END_REF][START_REF] Loveland | Ensemble cryo-EM elucidates the mechanism of translation fidelity[END_REF], that accommodation of either cognate or near-cognate tRNA in the A-site of 70S lead to domain closure around the decoding center and movement of G530 to the anti-conformation if it was not there already, and A1492 and A1493 to both be in the flipped "out" conformation. There are no structures available of the elongation state of S. aureus. In order to do this comparison, we therefore elected to use the structure of S. aureus containing the miscoding antibiotic paromomycin that known to be intercalated into the h44 and cause the flip-out conformation resulting in the stabilization of the decoding center similar to that caused by binding of A-tRNA. In all three "elongation" complexes thus analyzed and compared, the decoding center was structurally similar, while h16 of the 30S remained in open conformation in S. aureus (Fig 2b). This is most probably because paromomycin alone is not capable to induce such auxiliary distant rearrangements but causes a more local reorganization. This reorganization makes the decoding center appear as if A-tRNA is already present, and thereby facilitate binding of near-cognate tRNAs explaining the

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This article is protected by copyright. All rights reserved error frequency increase caused by this antibiotic as previously shown [START_REF] Demeshkina | A new understanding of the decoding principle on the ribosome[END_REF][START_REF] Pape | Conformational switch in the decoding region of 16S rRNA during aminoacyl-tRNA selection on the ribosome[END_REF]. In T. thermophilus, however, paromomycin was shown to favor a 30S closed conformation [START_REF] Ogle | Recognition of cognate transfer RNA by the 30S ribosomal subunit[END_REF][START_REF] Demeshkina | A new understanding of the decoding principle on the ribosome[END_REF][START_REF] Rozov | Structural insights into the translational infidelity mechanism[END_REF][START_REF] Ogle | Selection of tRNA by the ribosome requires a transition from an open to a closed form[END_REF].

The open state of the 30S shoulder was also found in the hibernating 100S complexes (Fic 3c) and vacant 70S ribosome (Fig 2d). Interestingly, the conformation of the G530 nucleotide was inconsistent between the four analyzed species as illustrated in Fig 2c-d. However, for more comprehensive analysis, more structures of the identical complexes are needed to draw a reliable conclusion whether these differences are species-specific or only a result of a particular complex formation.

Conclusion

In summary, we have determined the Cryo-EM structure of S. aureus 70S ribosome containing messenger RNA and P-tRNA at a resolution of 3.2 Å. Based on the density and existing experimental and bioinformatic data, we positively identified and assigned 10 modifications of rRNA. The present model is the highest resolution and most precise that is available at the moment. The modification sites have been thoroughly described, and possible influence on their environment and functions were proposed and correlated with existing data. Our findings indicate that at least G2278 (23S rRNA) and C1412 (16S rRNA) modifications may warrant future investigations because they are located in areas of particular interest. Due to the universal conservation of modification A2530, it also may benefit from particular scrutiny in terms of its role in antibiotic resistance. It is crucial for correct identification to perform biochemical studies of rRNA modifications in S. aureus (for example by MS analysis similar to Popova [START_REF] Popova | Quantitative Analysis of rRNA Modifications Using Stable Isotope Labeling and Mass Spectrometry[END_REF]), in order to be able to corroborate the existence of these modifications. We believe the proposed modifications may play an important role in protein synthesis and its regulation and, therefore, are worth investigating in greater detail.

With this study, we would like to contribute to future research on the role of rRNA modifications in S. aureus and their corresponding methyltranferases. Assessing the inhibition potential of the rRNA modification enzymes in S. aureus that we have proposed could be the way to commence exploitation of their potential to serve as general inhibition targets and targets against antibiotic resistance mechanisms.

Finally, with the presented structure, we hope to accelerate structure-based drug design research on new ribosome-binding antibiotics against S. aureus.

Materials and Methods

S. aureus ribosome purification

70S ribosomes from S. aureus were obtained as described previously [START_REF] Khusainov | Structure of the 70S ribosome from human pathogen Staphylococcus aureus[END_REF]. Two liters of S. aureus RN6390 culture were grown at 37°C in Brain Heart Infusion broth (BHI) media and harvested in early logarithmic

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This article is protected by copyright. All rights reserved phase. Cells were washed with buffer A (20 mM HEPES-KOH pH 7.5, 100 mM NH 4 Cl, 21 mM Mg(CH 3 COO) 2 , 1 mM DTT), pelleted and the cell pellet was frozen at -80 °C. The cell pellet was resuspended in buffer A, supplemented with the addition of protease inhibitor cocktail (EDTA free, Roche), DNase I (Roche), and lysostaphin (Sigma-Aldrich) then lysed at 37°C for 45 min. Cell debris were removed by centrifugation at 30,000×g for 90 min. The supernatant was supplemented with 2.8% w/v PEG 20,000 (Hampton Research) for fractionation. The supernatant was then recovered, and the PEG 20,000 concentration was increased to 4.2% w/v for the second fractionation. The solution was then centrifuged at 20,000×g for 10 min, and the ribosome pellet was resuspended in buffer A and layered on a sucrose cushion (10 mM Hepes-KOH pH 7.5, 500 mM KCl, 25 mM Mg(CH 3 COO) 2 , 1.1 M Sucrose, 0.5 mM EDTA, 1 mM DTT). Centrifugation was subsequently carried out at 45,000 rpm for 15 h using a 45Ti rotor (Beckman).

The pellet containing ribosomes was resuspended in buffer E (10 mM Hepes-KOH pH 7.5, 100 mM KCl, 10 mM Mg(CH 3 COO) 2 , 0.5 mM EDTA, 1 mM DTT) up to a concentration of 7 mg/mL. Diluted samples were loaded onto 7-30% sucrose density gradients and centrifuged at 17,100 rpm for 15.5h using a Beckman SW28 rotor. The fractions corresponding to 70S particles were pooled and the concentration of Mg(CH 3 COO) 2 was adjusted to 25 mM. For the sample concentrations, PEG 20,000 was added to a final concentration of 4.5% w/v. Ribosomes were pelleted by centrifugation at 20,000 ×g, the pellet was gently dissolved in buffer G (10 mM Hepes-KOH pH 7.5, 50 mM KCl, 10 mM NH 4 Cl, 10 mM Mg(CH 3 COO) 2 , 1 mM DTT) to a final concentration of 20-25 mg/mL. Aliquots of 30 µL were flash frozen in liquid nitrogen and stored at -80°C.

Preparation of tRNA i Met

tRNA i Met was prepared according to Y.Mechulam [START_REF] Mechulam | Protection-based assays to measure aminoacyl-tRNA binding to translation initiation factors[END_REF] with small adjustments. tRNA i Met was overexpressed in E. coli using the plasmid pBStRNAfMetY2. The total tRNA was isolated by phenol extraction (pH 5) and then NaCl-ethanol precipitated. Total tRNA was deacetylated in1.8 M Tris-HCl pH 8.0 at 37 o C for 90 min and then NaCl-ethanol precipitated. The pellet was dissolved in buffer T (20 mMTris-HCl pH 7.5, 8 mM MgCl 2 , 0.1 mM EDTA, 0.2 M NaCl) and tRNA i Met was purified at Q Sepharose (GE Healthcare). tRNA i Met was then NaCl-ethanol precipitated and the pellet was dissolved in mQ water. The sample was snap-frozen in liquid nitrogen and stored at -80°C.

In vitro aminoacetylation and formylation of fMet-tRNA i Met

Amynoacetylation and formylation of tRNA i Met was done according to references [START_REF] Mechulam | Crystal structure of Escherichia coli methionyl-tRNA synthetase highlights species-specific features[END_REF][START_REF] Fourmy | Identification of residues involved in the binding of methionine by Escherichia coli methionyl-tRNA synthetase[END_REF]. Briefly, tRNA i Met (30 AU 260 ) was added to 1.5mL reaction mixture (30 mM imidazole-HCl pH 7.5, 100 mM KCl, 10 mM Mg(CH 3 COO) 2 , 1 mM DTT, 0.1 μg/μL methionyl-tRNA i Met synthethase (purified according to Fourmy [START_REF] Fourmy | Identification of residues involved in the binding of methionine by Escherichia coli methionyl-tRNA synthetase[END_REF]), 5 mM ATP (Thermo-Fisher), 5 mM PEP (Sygma), 0.025 mg/ml pyruvate kinase (Sygma), 80 μM

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Cryo-EM data collection

Data collection was performed on a Cs-corrector equipped Titan Krios electron microscope (FEI Company) at 300 kV, using the Serial-EM software for automated data acquisition. Data were collected with a defocus of -1.5 to -3.3 μm at a magnification of 96,000 X, giving a nominal pixel size of 1.1 Å/px. The micrographs (7166) were recorded in super-resolution mode using Gatan K2 summit detector (Gatan, Inc.) as movie stacks resulting in pixel size 0.55 Å 2 /px. The exposure time for each movie stack was ~ 45 sec, corresponding to an electron dose per frame ~ 1.5 electrons/ Å 2 divided into 40 frames. Drift, gain corrections, and dose-weighting were performed with MotionCor2 [START_REF] Zheng | MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy[END_REF]using all frames on 1x1 patches.

Data processing and map calculation

The contrast transfer function (CTF) was calculated from motion-corrected dose-weighted images using CTF [START_REF] Zhang | Gctf: Real-time CTF determination and correction[END_REF]. After removing images having poor CTF quality (leaving a total of 6662 micrographs), ribosome particles were picked from motion-corrected and dose-weighted images in Relion 2.1 [START_REF] Kimanius | Accelerated cryo-EM structure determination with parallelisation using GPUs in RELION-2[END_REF]. After extraction and 5-fold binning (5.5 Å/px) of 555,523 particles, reference-free 2D classification using RELION 2.1 was performed with 110 classes. After the removal of non-ribosomal particles, the remaining 411,770 particles were subjected to three-dimensional (3D) classification using 5 classes. An initial reference model for 3D classification was generated using stochastic gradient descent (SGD) algorithm in RELION 2.1. The 3D classification was performed in two steps using 15° and 7.5° angular sampling, resulting in 127,598 selected particles that were extracted with the pixel size of 3.3 Å/px. For further sorting, particles were sorted into 4 classes using focused 3D classification with a mask at the P-tRNA region. The tRNA containing particles were unbinned to their original pixel size of 1.1 Å/px, and subjected to 3D autorefinement using the same reference as for the 3D classification job followed by post processing using automatic B factor estimation. The final map was estimated to be at a resolution of 3.2 Å using a FSC=0.143 gold-standard threshold. Local resolution was estimated using Relion 2.1 [START_REF] Kimanius | Accelerated cryo-EM structure determination with parallelisation using GPUs in RELION-2[END_REF].

Model building and validation

For model building, the 70S ribosome was extracted from the S. aureus vacant 70S ribosome model (PBD ID 5LI0) and P-tRNA with mRNA was extracted from the model of a T. thermophillus elongation complex (PBD ID 4V6F). The mRNA sequence was changed according to the sequence of mRNA used in this experiment. The obtained constructed model was rigid body fitted into our Gaussian filtered Cryo-EM density map. Initial coarse fitting of the flexible elements at the intersubunit interface and periphery, was performed using phenix.real_space_refinement in phenix [START_REF] Liebschner | Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix[END_REF] including simulated annealing (starting temperature = 800 K; cool rate = 100 K) and global minimization. Model and map were visually inspected in Coot [START_REF] Emsley | Coot: model-building tools for molecular graphics[END_REF]. Modeling of posttranscriptional modifications was performed in Coot and was based on

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This article is protected by copyright. All rights reserved manual analysis of the Cryo-EM map of rRNAs. The densities that can be attributed to solvent molecules have been assigned as Mg 2+ ions and few K + ions were assigned based on the map signal, the alignment with initiation complex from T. thermophilus 70S with experimentally identified K + ions [START_REF] Rozov | Importance of potassium ions for ribosome structure and function revealed by longwavelength X-ray diffraction[END_REF] and reasonable stereochemistry. Final minimization of coordinates was carried out in Phenix real-space refinement with default parameters. For model validation, we used the MolProbity webserver [START_REF] Williams | MolProbity: More and better reference data for improved all-atom structure validation[END_REF] and model-to-map correlation statistics from Phenix. Data and refinement statistics are summarized in Table S2.

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This article is protected by copyright. All rights reserved Structural comparison includes representative structures of 70S from S. aureus (first raw, orange ribbon), E. coli (second raw, grey ribbon), T. thermophilus (third raw, cyan ribbon), B. subtilus (fourth raw, orchid ribbon). Leftcorner insets depict interface of the 30S subunits with highlighted decoding center and shoulder regions, and ligands.

To measure the distances, all structures were aligned on 16S rRNA.

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  Fig 1c,d,e and Fig S2).

FIGURE CAPTIONS Figure 1 .

 CAPTIONS1 FIGURE CAPTIONSFigure 1. Local resolution map of the S. aureus Cryo-EM structure. (a) Right -general overview, left -slice-view through the core of ribosome. (b) Cryo-EM map of P-tRNA colored according to the local resolution. High resolution features of the map: (c) ССA-end of P-tRNA. Modified G2278 in direct interaction with C75 of tRNA, modification is shown by orange color and arrow; (d) map quality in decoding center, AUG codon of mRNA is in magenta and P-tRNA is in blue; (e) Highly modified cluster of nucleotides near the decoding center, modifications are colored in orange.

Figure 2 .

 2 Figure 2. Dynamics of the decoding center and shoulder position in the 70S ribosome bound to various ligands representing initiation-like state (a), elongation-like state (b), hibernation state (c) and vacant ribosome (d).

Figure 3 .

 3 Figure 3. Locations of modified nucleotides at LSU. (a) Locations of modified nucleotides at LSU and the map quality of assigned rRNA modifications of 23S rRNA (b-g). Modifications are shown by orange transparent circles. PTC -peptydyl transferase center, B2aintersubunit bridge B2a area

Figure 4 .

 4 Figure 4. Locations of modified nucleotides at SSU. (a) Locations of modified nucleotides at SSU (G1527 position is shown, but it is not modified, see Sup. Picture 2 for more information) and the map quality of assigned rRNA modifications of 16S rRNA (b-f). Modifications are shown by arrows and orange transparent circles. DC-decoding center, B2a -intersubunit bridge B2a area.

Table 1 . Modifications of rRNA found in this study.

 1 Searches for S. aureus proteins sharing similarity with known rRNA modifying enzymes were performed using BLASTp[START_REF] Altschul | Gapped BLAST and PSI-BLAST: a new generation of protein database search programs[END_REF]. ID of possible S. aureus methylases is linked to NCBI protein database.

	Positio n in E. coli 23S rRNA Positio n in S. aureus -C1947 U1939 U1966 G2251 G2278 G2445 G2472 Accepted Article	Modification type 2'-O-Methylcytidine 5-Methyluridine 2'-O-Methylguanosine N2-Methylguanosine	Location of modification H69, intersubunit bridge PTC, A-site RlmD Correspondin g modifying enzyme in E. coli -PTC, P-site RlmB PTC RlmKL	Possible aureus methylase S. with BLAST identity percent (to E. coli methylase if another is not stated) SQA11259.1 (48.36% to M. tuberculosis tlyA) WP_037590442. 1 28.82% WP_072490807 38.62% WP_037588129. 1 32.63%	Results of knockouts and information about the modification inhibition potential. M. tuberculosis methylttransferasetly A is a virulence factor and involved in hemolytic activity [43,44] The inactivation of the E. coli methyltransferase has mild growth effects [46,85] Modification is conserved throughout all kingdoms. Deletion of RlmB in E .coli causes slightly different phenotype from normal [28] Knockout of the methyltransferase gene reduces the

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Acknowledgements

The authors wish to thank IGBMC Cryo-EM platform staff for helping in data collection and especially Corinne Crucifix for extensive help in grid preparation. The work was supported by the Russian Science Foundation, Grant 17-74-20009, Russia (to A.G., B.F., I.K., K.U., S.V.) and Cryo-EM experiments were supported by "La Fondation pour la Recherche Médicale" DBF20160635745, France (to L.J, G.Y and M.Y.).

Data availability

For the Cryo-EM structure of the S. aureus 70S ribosome with P-site tRNA and messenger RNA coordinates were deposited in the Protein Data Bank, PDB ID 6YEF, while the Cryo-EM map was deposited in the electron microscopy database, EMD-10791.

methionine (Sygma), 0.1 μg/μL methionyl-tRNA i Met formyltransferase (purified according to [START_REF] Schmitt | Crystallization and preliminary X-ray analysis of Escherichia coli methionyl-tRNA(fMet) formyltransferase[END_REF][START_REF] Schmitt | Crystallization and preliminary X-ray analysis of Escherichia coli methionyl-tRNAMet(f) formyltransferase complexed with formyl-methionyl-tRNAMet(f)[END_REF]), prepared 0.3 mM folinic acid as a formyl donor).The reaction was incubated at 30°C for 50 min. Reaction was stopped by adding 1/10 of volume of 20% CH 3 CO 2 K solution at pH 5.0. fMet-tRNA i Met was phenol extracted and precipitated by NH 4 OAc-ethanol. Precipitated dry pellet was dissolved in buffer (50 mM K 3 PO 4 pH 7.0, 1.7M (NH 4 ) 2 SO 4 ) and fMet-tRNA i Met was purified by hydrophobic interaction chromatography using TSK-gel Phenyl-5PW column. Pooled peak fractions of fMet-tRNA i Met were buffer exchanged into 20 mM NH 4 CH 3 CO 2 pH 5.0 and precipitated by NH 4 CH 3 CO 2 -ethanol. The pellet of fMet-tRNA i Met was dissolved in the final buffer (20 mM NH 4 CH 3 CO 2 pH 5.0), snap-frozen in liquid nitrogen and stored at -80°C.

mRNA

The mRNA used was chemically synthesized by Dharmacon and had the sequence 5'-GGCAAGGAGGUAAAAAUGGUAAAAAAAAA-3' which contained AUG start codon (underlined bold). mRNA was deprotected according to manufacturer's protocol.

S. aureus ribosome complex preparation

All components were thawed on ice. After thawing, purified S. aureus70S ribosome sample was filtered through the 0.22 μm filter unit (Millipore). The S. aureus 70S ribosome complex was formed in the buffer (G (10 mM Hepes-KOH pH 7.5, 50 mM KCl, 10 mM NH 4 Cl, 10 mM Mg(CH 3 COO) 2 , 1 mM DTT). To form the complex S. aureus ribosomes, at a concentration of 60 nM, were first incubated with a 2.5 fold excess of mRNA for 15 min at 37°C, and further incubated with 5-fold excess of fMet-tRNA i Met for 15 min at 37°C.The complex was kept on ice until the applying to grids.

Grid preparation

4 µl of the complex (with the ribosome complex concentration of 60 nM) were applied to 400 mesh carboncoated holey carbon Quantifoil 2/2 grids (Quantifoil Micro Tools; glow discharge time =30 sec), blotted with filter paper from both sides for 1.5 s in a temperature-and humidity-controlled Vitrobot apparatus Mark IV (FEI, Eindhoven, Netherlands, T = 4°C, humidity 100%, blot force 5, blot time 4 s, blot waiting time 30 s) and vitrified in liquid ethane pre-cooled by liquid nitrogen.

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Conflict of interest

The authors declare no conflict of interest.

Author contributions

A.Gol, I.K., G.Y., M.Y. and K.U. conceived the project: A.Gol., I.K. and S.V. purified 70S ribosomes under the supervision of G.Y. and M.Y.; A.Gol, B.F., A.G., I.K. and L.J. refined the structures and processed the data.

A.Gol., B.F., I.K., L.J., A.Gab. , S.V., and K.U. wrote the manuscript with input from G.Y. and M.Y.; all authors revised the manuscript. highlighted ribosomal helices and protein uS12 -orange; large subunit -blue; mRNA -red; P-tRNA -green. (b) The 30S interface view of the complex with elements of shoulder (h16, h17), decoding center (h18, uS12) and entire h44 is highlighted in orange. (c) Close-up view at the decoding center and its respective cryo-EM density (E. coli numbering). Nucleotides C518, G530 in anti-conformation (beige square) and the backbone of the Thr54-Leu62 loop of protein uS12 (blue square) are clearly resolved. In the absence of A-tRNA, the side chains of uS12 and nucleotide A1493 are destabilized and not well resolved in our structure, while in the P-site mRNA-tRNA base-pairing is clearly visible.

Supporting Information