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Mechanism of premature translation termination on a sense codon

Open AccessPublished:June 25, 2018DOI:https://doi.org/10.1074/jbc.AW118.003232
      Accurate translation termination by release factors (RFs) is critical for the integrity of cellular proteomes. Premature termination on sense codons, for example, results in truncated proteins, whose accumulation could be detrimental to the cell. Nevertheless, some sense codons are prone to triggering premature termination, but the structural basis for this is unclear. To investigate premature termination, we determined a cryo-EM structure of the Escherichia coli 70S ribosome bound with RF1 in response to a UAU (Tyr) sense codon. The structure reveals that RF1 recognizes a UAU codon similarly to a UAG stop codon, suggesting that sense codons induce premature termination because they structurally mimic a stop codon. Hydrophobic interaction between the nucleobase of U3 (the third position of the UAU codon) and conserved Ile-196 in RF1 is important for misreading the UAU codon. Analyses of RNA binding in ribonucleoprotein complexes or by amino acids reveal that Ile–U packing is a frequent protein–RNA-binding motif with key functional implications. We discuss parallels with eukaryotic translation termination by the release factor eRF1.

      Introduction

      Translation termination defines the lengths of all cellular proteins. Three stop codons–UAA, UAG, and UGA–signal the end of the mRNA open reading frame (ORF). A stop codon in the ribosomal A site is recognized by a bifunctional protein called a release factor (RF),
      The abbreviations used are: RF
      release factor
      FSC
      Fourier shell correlation
      PDB
      Protein Data Bank
      βME
      β-mercaptoethanol
      aa
      amino acid.
      which (i) recognizes stop codons and discriminates against sense codons, and (ii) catalyzes peptidyl-tRNA hydrolysis, releasing the peptide from the ribosome. Bacteria express two release factors: RF1 recognizes UAA/UAG codons, and RF2 recognizes UAA/UGA codons. A single eukaryotic release factor eRF1 recognizes all three stop codons.
      Accurate termination on stop codons is crucial for the cell and organism. Premature termination, either on sense codons or on premature stop codons arising from nonsense mutations, would result in accumulation of truncated proteins with compromised or toxic activities. Many genetic diseases are caused by premature stop codons (
      • Frischmeyer P.A.
      • Dietz H.C.
      Nonsense-mediated mRNA decay in health and disease.
      ,
      • Keeling K.M.
      • Bedwell D.M.
      Suppression of nonsense mutations as a therapeutic approach to treat genetic diseases.
      ), highlighting the deleterious effects of premature termination. In an intact ORF (i.e. no premature stop codon), however, near-stop codons may cause premature termination (
      • Jørgensen F.
      • Adamski F.M.
      • Tate W.P.
      • Kurland C.G.
      Release factor-dependent false stops are infrequent in Escherichia coli.
      ). Sense codons differing from the stop codon in the third-nucleotide (wobble) position are most promiscuous (
      • Freistroffer D.V.
      • Kwiatkowski M.
      • Buckingham R.H.
      • Ehrenberg M.
      The accuracy of codon recognition by polypeptide release factors.
      ). A study measuring the activity of bacterial release factors on near-stop codons identified the UAU sense codon as a “hot spot” for RF1 (
      • Freistroffer D.V.
      • Kwiatkowski M.
      • Buckingham R.H.
      • Ehrenberg M.
      The accuracy of codon recognition by polypeptide release factors.
      ). We recently confirmed these findings (
      • Svidritskiy E.
      • Korostelev A.A.
      Conformational control of translation termination on the 70S ribosome.
      ), showing that RF1 can catalyze release on a UAU sense codon almost as efficiently as on a UAA codon when RF1 is in excess (Fig. 1A). Affinity of release factors to some sense codons does not lead to predominant premature termination in vivo due to efficient decoding of sense codons by aminoacyl-tRNAs, but it is likely responsible for background levels of premature termination (
      • Jørgensen F.
      • Adamski F.M.
      • Tate W.P.
      • Kurland C.G.
      Release factor-dependent false stops are infrequent in Escherichia coli.
      ).
      Figure thumbnail gr1
      Figure 1A, Michaelis-Menten curves for in vitro formyl-methionine release from fMet-tRNA by RF1 on the stop codon UAA and sense codons UAU, UGG, and AAA (adapted from Refs.
      • Svidritskiy E.
      • Korostelev A.A.
      Conformational control of translation termination on the 70S ribosome.
      ,
      • Svidritskiy E.
      • Madireddy R.
      • Korostelev A.A.
      Structural basis for translation termination on a pseudouridylated stop codon.
      ). B, cryo-EM structure of the 70S·RF1 complex formed on the UAU sense codon. FSC curve is shown for the 70S·RF1 cryo-EM map (lower left). C, local resolution of RF1 in the cryo-EM map, determined using Blocres (
      • Cardone G.
      • Heymann J.B.
      • Steven A.C.
      One number does not fit all: mapping local variations in resolution in cryo-EM reconstructions.
      ). RF1 is oriented similarly to the view shown in B. The map was sharpened by applying a B-factor of −100 Å2 and is shown at 2σ, colored using a resolution scale ranging from 2.8 to 5.3 Å (left). D, cryo-EM map (mesh) in the peptidyl transferase center (PTC). E, cryo-EM map (mesh) in the decoding center (DC). In structural models, the large 50S ribosomal subunit is shown in cyan, small 30S subunit in yellow, RF1 in green, mRNA in dark blue, P-site tRNA in orange, and E-site tRNA in pink. Domains of RF1 are labeled in B and C.
      The structural mechanism of stop-codon recognition was elucidated by high-resolution crystal structures (
      • Weixlbaumer A.
      • Jin H.
      • Neubauer C.
      • Voorhees R.M.
      • Petry S.
      • Kelley A.C.
      • Ramakrishnan V.
      Insights into translational termination from the structure of RF2 bound to the ribosome.
      ,
      • Laurberg M.
      • Asahara H.
      • Korostelev A.
      • Zhu J.
      • Trakhanov S.
      • Noller H.F.
      Structural basis for translation termination on the 70S ribosome.
      ,
      • Korostelev A.
      • Asahara H.
      • Lancaster L.
      • Laurberg M.
      • Hirschi A.
      • Zhu J.
      • Trakhanov S.
      • Scott W.G.
      • Noller H.F.
      Crystal structure of a translation termination complex formed with release factor RF2.
      ,
      • Korostelev A.
      • Zhu J.
      • Asahara H.
      • Noller H.F.
      Recognition of the amber UAG stop codon by release factor RF1.
      ) and further investigated by molecular dynamics simulations (
      • Sund J.
      • Andér M.
      • Aqvist J.
      Principles of stop-codon reading on the ribosome.
      ). Release factors strongly discriminate against purines in the first position, so that no product can be detected when the ribosome encounters sense codons with A1 or G1 (i.e. A or G in position 1 of the A-site codon; Fig. 1A) (
      • Freistroffer D.V.
      • Kwiatkowski M.
      • Buckingham R.H.
      • Ehrenberg M.
      The accuracy of codon recognition by polypeptide release factors.
      ,
      • Svidritskiy E.
      • Madireddy R.
      • Korostelev A.A.
      Structural basis for translation termination on a pseudouridylated stop codon.
      ,
      • Korkmaz G.
      • Sanyal S.
      R213I mutation in release factor 2 (RF2) is one step forward for engineering an omnipotent release factor in bacteria Escherichia coli.
      ). The structures showed that the Watson-Crick side of the first nucleotide faces the backbone of an α-helix of RF1 or RF2, so the strict requirement for pyrimidine in the first position is explained by base recognition by a rigid structural element of release factor. The second and third positions are limited to purines and interact with side chains of RF1 or RF2 (
      • Korostelev A.A.
      Structural aspects of translation termination on the ribosome.
      ,
      • Dunkle J.A.
      • Cate J.H.
      Ribosome structure and dynamics during translocation and termination.
      ,
      • Zaher H.S.
      • Green R.
      Fidelity at the molecular level: lessons from protein synthesis.
      ,
      • Nakamura Y.
      • Ito K.
      tRNA mimicry in translation termination and beyond.
      ,
      • Ramakrishnan V.
      ). The third nucleotide is also sandwiched between the conserved 16S rRNA nucleotide G530 and a side chain of release factor: Ile-196 of RF1 or Arg-218 of RF2 (Escherichia coli numbering, unless noted otherwise). The Hoogsteen side of the third nucleotide (at atoms N6/O6 and N7) is stabilized by a conserved threonine (Thr-194 in RF1 or Thr-216 in RF2). In the eukaryotic 80S termination complex formed with eRF1, the third position of the stop codon similarly packs between a purine and Ile-62 (Homo sapiens) of the essential NIKS motif of eRF1 (
      • Frolova L.
      • Seit-Nebi A.
      • Kisselev L.
      Highly conserved NIKS tetrapeptide is functionally essential in eukaryotic translation termination factor eRF1.
      ), and it interacts with Thr-58 (
      • Brown A.
      • Shao S.
      • Murray J.
      • Hegde R.S.
      • Ramakrishnan V.
      Structural basis for stop codon recognition in eukaryotes.
      ,
      • Matheisl S.
      • Berninghausen O.
      • Becker T.
      • Beckmann R.
      Structure of a human translation termination complex.
      ).
      In this work, we asked how a UAU sense codon is misread by RF1 as a stop codon, and why U in the third position is preferred over C, rendering UAU a more efficient “mis-terminator” than UAC. We determined a cryo-EM structure of the bacterial 70S ribosome complex that helps answer these questions. Our analyses suggest that the packing of U3 against Ile-196 of RF1 is critical for the preference of U over C. We find that the Ile–U packing is a prevalent motif in the ribosome and other protein–RNA structures, consistent with the role of Ile and other nonaromatic hydrophobic side chains in specific recognition of uridine.

      Mechanism of termination on the UAU sense codon

      We determined a cryo-EM structure of the E. coli 70S·RF1 complex formed in response to the UAU codon in the A-site at 3.7 Å average resolution (Fig. 1B and Table 1), with local resolution achieving ∼3 Å in ribosomal functional centers (Fig. 1C), allowing near-atomic interpretation (Fig. 1, D and E). The overall conformations of release factor and the UAU codon are similar to those in canonical termination complexes formed with stop codons. We note one main difference between our structure and previous high-resolution 70S·RF1 structures from Thermus thermophilus and heterologous systems. In our E. coli structure, domain 1 of RF1 binds both the large-subunit ribosomal protein L11 and 23S rRNA of the L11 stalk, consistent with observations at lower resolutions for E. coli 70S·RF1 complexes formed on a stop codon (
      • Rawat U.
      • Gao H.
      • Zavialov A.
      • Gursky R.
      • Ehrenberg M.
      • Frank J.
      Interactions of the release factor RF1 with the ribosome as revealed by cryo-EM.
      ). Specifically, hydrophobic Ile-29 of RF1 docks at the proline-rich region of L11 (aa Pro-21 to Pro-25), and Phe-35 binds near A1095 of 23S rRNA, similar to that seen for RF2 (
      • Weixlbaumer A.
      • Jin H.
      • Neubauer C.
      • Voorhees R.M.
      • Petry S.
      • Kelley A.C.
      • Ramakrishnan V.
      Insights into translational termination from the structure of RF2 bound to the ribosome.
      ,
      • Korostelev A.
      • Asahara H.
      • Lancaster L.
      • Laurberg M.
      • Hirschi A.
      • Zhu J.
      • Trakhanov S.
      • Scott W.G.
      • Noller H.F.
      Crystal structure of a translation termination complex formed with release factor RF2.
      ,
      • Jin H.
      • Kelley A.C.
      • Loakes D.
      • Ramakrishnan V.
      Structure of the 70S ribosome bound to release factor 2 and a substrate analog provides insights into catalysis of peptide release.
      ,
      • Petry S.
      • Brodersen D.E.
      • Murphy 4th, F.V.
      • Dunham C.M.
      • Selmer M.
      • Tarry M.J.
      • Kelley A.C.
      • Ramakrishnan V.
      Crystal structures of the ribosome in complex with release factors RF1 and RF2 bound to a cognate stop codon.
      ). In other high-resolution structures, however, domain 1 of RF1 is either unresolved (
      • Svidritskiy E.
      • Madireddy R.
      • Korostelev A.A.
      Structural basis for translation termination on a pseudouridylated stop codon.
      ,
      • Svidritskiy E.
      • Korostelev A.A.
      Mechanism of inhibition of translation termination by blasticidin S.
      ,
      • Pierson W.E.
      • Hoffer E.D.
      • Keedy H.E.
      • Simms C.L.
      • Dunham C.M.
      • Zaher H.S.
      Uniformity of peptide release is maintained by methylation of release factors.
      ,
      • Florin T.
      • Maracci C.
      • Graf M.
      • Karki P.
      • Klepacki D.
      • Berninghausen O.
      • Beckmann R.
      • Vázquez-Laslop N.
      • Wilson D.N.
      • Rodnina M.V.
      • Mankin A.S.
      An antimicrobial peptide that inhibits translation by trapping release factors on the ribosome.
      ) or binds near the L11 stalk without contacting it (
      • Laurberg M.
      • Asahara H.
      • Korostelev A.
      • Zhu J.
      • Trakhanov S.
      • Noller H.F.
      Structural basis for translation termination on the 70S ribosome.
      ,
      • Korostelev A.
      • Zhu J.
      • Asahara H.
      • Noller H.F.
      Recognition of the amber UAG stop codon by release factor RF1.
      ,
      • Petry S.
      • Brodersen D.E.
      • Murphy 4th, F.V.
      • Dunham C.M.
      • Selmer M.
      • Tarry M.J.
      • Kelley A.C.
      • Ramakrishnan V.
      Crystal structures of the ribosome in complex with release factors RF1 and RF2 bound to a cognate stop codon.
      ). In our structure, domain 1 is not as well resolved as other domains of RF1 (Fig. 1C), consistent with the idea that it is dynamic. The different positions of domain 1 in different structures highlight that interactions between domain 1 and the ribosome are dynamic and are likely important at early stages of RF1 binding or during RF1 dissociation, in keeping with functional interaction between RF1 and L11 (
      • Van Dyke N.
      • Murgola E.J.
      Site of functional interaction of release factor 1 with the ribosome.
      ,
      • Tate W.P.
      • Dognin M.J.
      • Noah M.
      • Stöffler-Meilicke M.
      • Stöffler G.
      The NH2-terminal domain of Escherichia coli ribosomal protein L11. Its three-dimensional location and its role in the binding of release factors 1 and 2.
      ,
      • Bouakaz L.
      • Bouakaz E.
      • Murgola E.J.
      • Ehrenberg M.
      • Sanyal S.
      The role of ribosomal protein L11 in class I release factor-mediated translation termination and translational accuracy.
      ).
      Table 1Cryo-EM data collection and refinement statistics
      Data collection
       EM equipmentFEI Talos Arctica
       Voltage (kV)200
       DetectorGatan K2 Summit
       SoftwareSerialEM
       Pixel size (Å)0.944
       Electron dose (e2)37.7 (used 19)
       Defocus range (μm)−0.7 to −1.7
       Data set size (no. of particles)1,065,147
      Reconstruction
       SoftwarecisTEM and Frealign versions 9.10 and 9.11
       No. of particles used639,088
       Final resolution (Å, FSC = 0.143)3.7
      Model composition
       Nonhydrogen atoms152,439
       Protein residues6520
       RNA bases4728
      Refinement
       SoftwareRSRef and Phenix
       Correlation coefficient0.86
      R-factor0.186
      Ramachandran-plot statistics (%)
       Favored (overall)86.8
       Allowed (overall)12.4
       Outlier (overall)0.9
       Rotamer outliers (%)0.02
       C-β deviations0
      Root mean square deviations
       Bond length (Å)0.005
       Bond angle (°)0.84
      Density in the peptidyl transferase and decoding centers (Fig. 1, D and E) showed interactions similar to those found in canonical termination complexes with RF1 (
      • Laurberg M.
      • Asahara H.
      • Korostelev A.
      • Zhu J.
      • Trakhanov S.
      • Noller H.F.
      Structural basis for translation termination on the 70S ribosome.
      ,
      • Korostelev A.
      • Zhu J.
      • Asahara H.
      • Noller H.F.
      Recognition of the amber UAG stop codon by release factor RF1.
      ). Consistent with the catalytic activity of RF1 on the UAU codon, the catalytic 233GGQ235 loop is positioned next to the terminal nucleotide A76 of the P-site tRNA and is stabilized by interactions with A2602, which is essential for termination (Fig. 1D) (
      • Polacek N.
      • Gomez M.J.
      • Ito K.
      • Xiong L.
      • Nakamura Y.
      • Mankin A.
      The critical role of the universally conserved A2602 of 23S ribosomal RNA in the release of the nascent peptide during translation termination.
      ,
      • Youngman E.M.
      • Brunelle J.L.
      • Kochaniak A.B.
      • Green R.
      The active site of the ribosome is composed of two layers of conserved nucleotides with distinct roles in peptide bond formation and peptide release.
      ,
      • Amort M.
      • Wotzel B.
      • Bakowska-Zywicka K.
      • Erlacher M.D.
      • Micura R.
      • Polacek N.
      An intact ribose moiety at A2602 of 23S rRNA is key to trigger peptidyl-tRNA hydrolysis during translation termination.
      ). In the decoding center, the first two nucleotides of the UAU codon interact with the codon-recognition residues of RF1 similarly to stop-codon nucleotides. The sense-codon U3 residue is sandwiched between Ile-194 of RF1 and G530 of 16S rRNA. Its carbonyl oxygen O4 faces the hydrogen-bond donors OH of Thr-196 and NH of Gln-185 (Figs. 1E and 2A), similar to the carbonyl oxygen of G3 in the UAG stop codon (Fig. 2B) (
      • Korostelev A.
      • Zhu J.
      • Asahara H.
      • Noller H.F.
      Recognition of the amber UAG stop codon by release factor RF1.
      ). However, whereas Thr-196 can form a hydrogen bond with one of two possible hydrogen-bond acceptors within G3 (at N7 and O6), the threonine can interact with a single acceptor within U3 of the UAU sense codon. Moreover, the sense codon U3–G530 pyrimidine–purine stacking is expected to be less stable than the stop codon G3–G530 purine-purine stacking (
      • Jafilan S.
      • Klein L.
      • Hyun C.
      • Florián J.
      Intramolecular base stacking of dinucleoside monophosphate anions in aqueous solution.
      ,
      • Friedman R.A.
      • Honig B.
      A free energy analysis of nucleic acid base stacking in aqueous solution.
      ,
      • Brown R.F.
      • Andrews C.T.
      • Elcock A.H.
      Stacking free energies of all DNA and RNA nucleoside pairs and dinucleoside-monophosphates computed using recently revised AMBER parameters and compared with experiment.
      ,
      • Florián J.
      • Šponer J.
      • Warshel A.
      Thermodynamic parameters for stacking and hydrogen bonding of nucleic acid bases in aqueous solution: ab initio/Langevin dipoles study.
      ). Thus, the interactions between RF1 and UAU sense codon resemble those between RF1 and UAG stop codon and suggest why RF1 recognizes UAU less efficiently than stop codons (Fig. 1A and Ref.
      • Freistroffer D.V.
      • Kwiatkowski M.
      • Buckingham R.H.
      • Ehrenberg M.
      The accuracy of codon recognition by polypeptide release factors.
      ).
      Figure thumbnail gr2
      Figure 2Recognition of the third nucleotide of the UAU and UAG codons by RF1 and eRF1. A, interactions of E. coli RF1 with U3 of the UAU sense codon in the 70S ribosome (this work). B, interactions of T. thermophilus RF1 with G3 of the UAG stop codon in the 70S ribosome (PDB code 4V7P (
      • Korostelev A.
      • Zhu J.
      • Asahara H.
      • Noller H.F.
      Recognition of the amber UAG stop codon by release factor RF1.
      )). Thr-198 forms one of two possible hydrogen bonds with G3 (shown with the dashed and dotted lines). C, interactions of Oryctolagus cuniculus eRF1 with G3 of the UAG stop codon in the 80S ribosome (PDB code 3JAH (
      • Brown A.
      • Shao S.
      • Murray J.
      • Hegde R.S.
      • Ramakrishnan V.
      Structural basis for stop codon recognition in eukaryotes.
      )).
      Unlike a UAU codon, a UAC sense codon inefficiently triggers peptide release, similar to sense codons that differ from the stop codon at their second position, e.g. UCA or UGG (
      • Freistroffer D.V.
      • Kwiatkowski M.
      • Buckingham R.H.
      • Ehrenberg M.
      The accuracy of codon recognition by polypeptide release factors.
      ). This might appear surprising because the hydrogen-bonding valences of the amino group N4 of cytosine, placed similarly to the carbonyl oxygen O4 of uracil, could be satisfied by interactions with Thr-196 (its OH group becoming the H-bond acceptor) and the amide group of Gln-185 (
      • Korostelev A.
      • Zhu J.
      • Asahara H.
      • Noller H.F.
      Recognition of the amber UAG stop codon by release factor RF1.
      ) or with an ordered water molecule (
      • Sund J.
      • Andér M.
      • Aqvist J.
      Principles of stop-codon reading on the ribosome.
      ). In fact, just as U3 in UAU mimics a G3 in the UAG stop codon, a C3 in UAC could mimic A3 in the UAA stop codon. We propose that the difference in hydrophobicity between the cytidine and uridine makes the termination on UAC less efficient than on UAU. Because C is substantially less hydrophobic than U (
      • Tsukada K.
      • Ueda S.
      • Okada R.
      Preparation of liposome-encapsulated anti-tumor drugs; relationship between lipophilicity of drugs and in vitro drug release.
      ,
      • Cullis P.M.
      • Wolfenden R.
      Affinities of nucleic acid bases for solvent water.
      ,
      • Shih P.
      • Pedersen L.G.
      • Gibbs P.R.
      • Wolfenden R.
      Hydrophobicities of the nucleic acid bases: distribution coefficients from water to cyclohexane.
      ), packing of C between Ile-169 and G530 would be less energetically favorable than the packing of U. Base-stacking energy of cytosine on guanosine is similar to that of uracil on guanosine (
      • Jafilan S.
      • Klein L.
      • Hyun C.
      • Florián J.
      Intramolecular base stacking of dinucleoside monophosphate anions in aqueous solution.
      ,
      • Friedman R.A.
      • Honig B.
      A free energy analysis of nucleic acid base stacking in aqueous solution.
      ,
      • Brown R.F.
      • Andrews C.T.
      • Elcock A.H.
      Stacking free energies of all DNA and RNA nucleoside pairs and dinucleoside-monophosphates computed using recently revised AMBER parameters and compared with experiment.
      ,
      • Florián J.
      • Šponer J.
      • Warshel A.
      Thermodynamic parameters for stacking and hydrogen bonding of nucleic acid bases in aqueous solution: ab initio/Langevin dipoles study.
      ), suggesting that the major discrimination between U and C in position 3 results from favorable hydrophobic packing of Ile-169 on U rather than on the less hydrophobic C.
      In summary, our structure shows that recognition of the sense codon UAU by RF1 is similar to that of stop codon UAG. This suggests that other hot-spot sense codons likely undergo similar conformational rearrangements and are recognized by release factors similarly to stop codons, with which they have partial stereochemical resemblance. Structural analysis points at Ile–U packing being critical for making UAU a hot spot for mis-termination by RF1.

      Ile–U interactions in protein·RNA complexes

      The role of Ile–U packing in mis-termination by RF1 prompted us to investigate whether Ile–U is a common interaction employed in protein–RNA recognition. Nucleotide stacking is the major stabilizing interaction in secondary and tertiary structures of nucleic acids. In protein·RNA complexes, the energy of unstacking a nucleotide from its stacking partner(s) is usually compensated by interaction between the aromatic base of the nucleotide and protein side chain(s). The best-characterized interactions include stacking of nucleotides on aromatic side chains and on positively charged side chains (Ref.
      • Wilson K.A.
      • Holland D.J.
      • Wetmore S.D.
      Topology of RNA-protein nucleobase-amino acid π-π interactions and comparison to analogous DNA-protein π-π contacts.
      and references therein). Because stacking interactions involve a hydrophobic energy contribution (nonpolar, nonelectrostatic, and solvent entropy) (
      • Friedman R.A.
      • Honig B.
      A free energy analysis of nucleic acid base stacking in aqueous solution.
      ,
      • Norberg J.
      • Nilsson L.
      Solvent influence on base stacking.
      ,
      • Luo R.
      • Gilson H.S.
      • Potter M.J.
      • Gilson M.K.
      The physical basis of nucleic acid base stacking in water.
      ), nucleotides also stack on aliphatic hydrophobic side chains. Isoleucine is the most hydrophobic side chain, according to many hydrophobicity scales (
      • Zhu C.
      • Gao Y.
      • Li H.
      • Meng S.
      • Li L.
      • Francisco J.S.
      • Zeng X.C.
      Characterizing hydrophobicity of amino acid side chains in a protein environment via measuring contact angle of a water nanodroplet on planar peptide network.
      ,
      • Woese C.R.
      • Dugre D.H.
      • Saxinger W.C.
      • Dugre S.A.
      The molecular basis for the genetic code.
      ,
      • Kyte J.
      • Doolittle R.F.
      A simple method for displaying the hydropathic character of a protein.
      ,
      • Hessa T.
      • Kim H.
      • Bihlmaier K.
      • Lundin C.
      • Boekel J.
      • Andersson H.
      • Nilsson I.
      • White S.H.
      • von Heijne G.
      Recognition of transmembrane helices by the endoplasmic reticulum translocon.
      ), and the uracil and adenine nucleobases are more hydrophobic than cytosine and guanine (
      • Tsukada K.
      • Ueda S.
      • Okada R.
      Preparation of liposome-encapsulated anti-tumor drugs; relationship between lipophilicity of drugs and in vitro drug release.
      ,
      • Cullis P.M.
      • Wolfenden R.
      Affinities of nucleic acid bases for solvent water.
      ,
      • Shih P.
      • Pedersen L.G.
      • Gibbs P.R.
      • Wolfenden R.
      Hydrophobicities of the nucleic acid bases: distribution coefficients from water to cyclohexane.
      ). Thus, uridine and adenosine are more likely than cytidine and guanosine to interact with isoleucine and other aliphatic side chains. Indeed, in computational simulations, U was the only nucleotide with a favorable free energy of binding to Ile (i.e. negative ΔΔG) in methanol, which is thought to represent the environment for nucleic acid–protein interfaces more accurately than water (
      • de Ruiter A.
      • Zagrovic B.
      Absolute binding-free energies between standard RNA/DNA nucleobases and amino-acid sidechain analogs in different environments.
      ).
      To test whether packing of isoleucine on uracil is among the preferred protein–RNA interactions, we calculated the number of stacking interactions between RNA nucleotides and aliphatic, aromatic, or charged protein side chains in high-resolution crystal structures of ribosomes, including the 2.4-Å resolution E. coli 70S ribosome (
      • Noeske J.
      • Wasserman M.R.
      • Terry D.S.
      • Altman R.B.
      • Blanchard S.C.
      • Cate J.H.
      High-resolution structure of the Escherichia coli ribosome.
      ), the 2.5-Å resolution T. thermophilus 70S ribosome (
      • Polikanov Y.S.
      • Melnikov S.V.
      • Söll D.
      • Steitz T.A.
      Structural insights into the role of rRNA modifications in protein synthesis and ribosome assembly.
      ), and the 3.0-Å resolution S. cerevisiae 80S ribosome (
      • vBen-Shem A.
      • Garreau de Loubresse N.
      • Melnikov S.
      • Jenner L.
      • Yusupova G.
      • Yusupov M.
      The structure of the eukaryotic ribosome at 3.0 A resolution.
      ). Collectively, these structures provide a large pool of protein–RNA interactions comprising ∼29,000 amino acids and ∼19,000 nucleotides. We normalized each type of stacking to the number of nucleotides of each type (i.e. number of amino acids per thousand nucleotides).
      These data show that Ile does indeed prefer to pack on uridine (Fig. 3). Ile–A packing is also well represented, whereas Ile packed the least on less hydrophobic C and G nucleotides. As expected, most of packing interactions of nucleotides occur with the positively charged Arg. Aliphatic, aromatic, and positively charged side chains pack more frequently on U or A (collectively >50%) than on the less hydrophobic C or G. This preference is notable for Ile (72%), Pro (85%), Phe (70%), and Tyr (65%). Negatively charged Asp and Glu are the least represented among protein side chains packing on aromatic bases, as expected. Interestingly, however, 70S rescue complexes formed with ArfA and RF2 on truncated mRNAs employ the packing of Glu-30 of ArfA on G530 of 16S rRNA (
      • James N.R.
      • Brown A.
      • Gordiyenko Y.
      • Ramakrishnan V.
      Translational termination without a stop codon.
      ,
      • Zeng F.
      • Chen Y.
      • Remis J.
      • Shekhar M.
      • Phillips J.C.
      • Tajkhorshid E.
      • Jin H.
      Structural basis of co-translational quality control by ArfA and RF2 bound to ribosome.
      ,
      • Ma C.
      • Kurita D.
      • Li N.
      • Chen Y.
      • Himeno H.
      • Gao N.
      Mechanistic insights into the alternative translation termination by ArfA and RF2.
      ,
      • Huter P.
      • Müller C.
      • Beckert B.
      • Arenz S.
      • Berninghausen O.
      • Beckmann R.
      • Wilson D.N.
      Structural basis for ArfA-RF2-mediated translation termination on mRNAs lacking stop codons.
      ,
      • Demo G.
      • Svidritskiy E.
      • Madireddy R.
      • Diaz-Avalos R.
      • Grant T.
      • Grigorieff N.
      • Sousa D.
      • Korostelev A.A.
      Mechanism of ribosome rescue by ArfA and RF2.
      ), suggesting functional roles for this underrepresented group of interactions. The carboxyl group of Glu-30 is stabilized by interactions with the side chain of Arg-213 of RF2 and with 2′-OH of G530.
      Figure thumbnail gr3
      Figure 3Distribution of amino acid side chains packing on the four types of RNA nucleotide bases (the more hydrophobic bases U and A are shown in gray and C and G are shown in purple) in three high-resolution ribosome structures from E. coli (PDB code 4YBB (
      • Noeske J.
      • Wasserman M.R.
      • Terry D.S.
      • Altman R.B.
      • Blanchard S.C.
      • Cate J.H.
      High-resolution structure of the Escherichia coli ribosome.
      )), T. thermophilus (PDB code 4Y4P (
      • Polikanov Y.S.
      • Melnikov S.V.
      • Söll D.
      • Steitz T.A.
      Structural insights into the role of rRNA modifications in protein synthesis and ribosome assembly.
      )), and S. cerevisiae (PDB code 4V88 (
      • vBen-Shem A.
      • Garreau de Loubresse N.
      • Melnikov S.
      • Jenner L.
      • Yusupova G.
      • Yusupov M.
      The structure of the eukaryotic ribosome at 3.0 A resolution.
      )).
      The specific affinity between U and Ile is also emphasized by amino acid–RNA affinity selection experiments, in which isoleucine selectively bound to UAU-containing RNA motifs (
      • Lozupone C.
      • Changayil S.
      • Majerfeld I.
      • Yarus M.
      Selection of the simplest RNA that binds isoleucine.
      ,
      • Majerfeld I.
      • Yarus M.
      Isoleucine:RNA sites with associated coding sequences.
      ). This specificity plays key functional roles in protein·RNA complexes. For example, isoleucine–U interaction is critical for specific and efficient recognition of bovine immunodeficiency virus transactivation-response element by the Tat protein (
      • Chen L.
      • Frankel A.D.
      A peptide interaction in the major groove of RNA resembles protein interactions in the minor groove of DNA.
      ). Crystal structure revealed that the side chain of Ile-79 of Tat packs on the aromatic ring of U10 and stabilizes the U10–A13:U24 base triplet (
      • Moras D.
      • Poterszman A.
      Getting into the major groove. Protein-RNA interactions.
      ). In Drosophila, Sxl regulates alternative splicing by specific recognition of a U-rich sequence in pre-mRNA, involving a uridine sandwiched between two isoleucines (Ile–U–Ile) (
      • Handa N.
      • Nureki O.
      • Kurimoto K.
      • Kim I.
      • Sakamoto H.
      • Shimura Y.
      • Muto Y.
      • Yokoyama S.
      Structural basis for recognition of the tra mRNA precursor by the sex-lethal protein.
      ). In archaeal RNase P, protein Rpp38 provides Ile-63 to stabilize bulged U19 of the enzyme's RNA (
      • Oshima K.
      • Gao X.
      • Hayashi S.
      • Ueda T.
      • Nakashima T.
      • Kimura M.
      Crystal structures of the archaeal RNase P protein Rpp38 in complex with RNA fragments containing a K-turn motif.
      ,
      • Oshima K.
      • Kakiuchi Y.
      • Tanaka Y.
      • Ueda T.
      • Nakashima T.
      • Kimura M.
      • Yao M.
      Structural basis for recognition of a kink-turn motif by an archaeal homologue of human RNase P protein Rpp38.
      ).

      Implications for eukaryotic termination

      Structural and biochemical work has yielded detailed insights into the mechanism of eukaryotic termination, but the mechanism of premature termination on sense codons in eukaryotes remains poorly understood. The UGG codon binds eRF1 (
      • Chavatte L.
      • Seit-Nebi A.
      • Dubovaya V.
      • Favre A.
      The invariant uridine of stop codons contacts the conserved NIKSR loop of human eRF1 in the ribosome.
      ,
      • Kryuchkova P.
      • Grishin A.
      • Eliseev B.
      • Karyagina A.
      • Frolova L.
      • Alkalaeva E.
      Two-step model of stop codon recognition by eukaryotic release factor eRF1.
      ,
      • Chavatte L.
      • Frolova L.
      • Laugâa P.
      • Kisselev L.
      • Favre A.
      Stop codons and UGG promote efficient binding of the polypeptide release factor eRF1 to the ribosomal A site.
      ), but termination activity was not detected in vitro (
      • Frolova L.
      • Le Goff X.
      • Rasmussen H.H.
      • Cheperegin S.
      • Drugeon G.
      • Kress M.
      • Arman I.
      • Haenni A.L.
      • Celis J.E.
      • Philippe M.
      A highly conserved eukaryotic protein family possessing properties of polypeptide chain release factor.
      ). The sequences and structures of codon-recognition domains of eukaryotic and bacterial release factors are very different. Therefore, perhaps not surprisingly, structural recognition of stop codons by eukaryotic eRF1 also differs from that by bacterial release factors. For example, eRF1 recognizes the U-turn–like geometry of the stop codon (
      • Brown A.
      • Shao S.
      • Murray J.
      • Hegde R.S.
      • Ramakrishnan V.
      Structural basis for stop codon recognition in eukaryotes.
      ,
      • Matheisl S.
      • Berninghausen O.
      • Becker T.
      • Beckmann R.
      Structure of a human translation termination complex.
      ,
      • Shao S.
      • Murray J.
      • Brown A.
      • Taunton J.
      • Ramakrishnan V.
      • Hegde R.S.
      Decoding mammalian ribosome-mRNA states by translational GTPase complexes.
      ) and a nucleotide downstream of the stop codon (
      • McCaughan K.K.
      • Brown C.M.
      • Dalphin M.E.
      • Berry M.J.
      • Tate W.P.
      Translational termination efficiency in mammals is influenced by the base following the stop codon.
      ). However, recognition of the third nucleotide by eRF1 is remarkably similar to that by RF1. In the structures of 80S·eRF1 complexes (
      • Brown A.
      • Shao S.
      • Murray J.
      • Hegde R.S.
      • Ramakrishnan V.
      Structural basis for stop codon recognition in eukaryotes.
      ,
      • Matheisl S.
      • Berninghausen O.
      • Becker T.
      • Beckmann R.
      Structure of a human translation termination complex.
      ,
      • Shao S.
      • Murray J.
      • Brown A.
      • Taunton J.
      • Ramakrishnan V.
      • Hegde R.S.
      Decoding mammalian ribosome-mRNA states by translational GTPase complexes.
      ), the third nucleotide is sandwiched between a purine (second base of stop codon) and Ile-62 of the universally conserved NIKS motif of eRF1 (Fig. 2C). Furthermore, the O6 atom of the guanosine of the UAG stop codon (
      • Brown A.
      • Shao S.
      • Murray J.
      • Hegde R.S.
      • Ramakrishnan V.
      Structural basis for stop codon recognition in eukaryotes.
      ) hydrogen bonds with Thr-58. The similar structural environment of the third nucleotide in the eukaryotic and bacterial complexes suggests that UAU might also be a hot spot for termination in eukaryotes. Premature termination on UAU, however, is likely less pronounced than on bacterial ribosomes, due to stringent codon discrimination facilitated by GTPase eRF3 (
      • Salas-Marco J.
      • Bedwell D.M.
      GTP hydrolysis by eRF3 facilitates stop codon decoding during eukaryotic translation termination.
      ,
      • Frolova L.
      • Le Goff X.
      • Zhouravleva G.
      • Davydova E.
      • Philippe M.
      • Kisselev L.
      Eukaryotic polypeptide chain release factor eRF3 is an eRF1- and ribosome-dependent guanosine triphosphatase.
      ).

      Materials and methods

      Preparation of the 70S·mRNA(UAU)·tRNAfMet·RF1 complex

      C-terminally His-tagged E. coli RF1 was purified as described previously (
      • Svidritskiy E.
      • Korostelev A.A.
      Conformational control of translation termination on the 70S ribosome.
      ). 70S ribosomes were prepared from E. coli (MRE600), as described previously (
      • Svidritskiy E.
      • Korostelev A.A.
      Conformational control of translation termination on the 70S ribosome.
      ), and stored in the ribosome-storage buffer (20 mm Tris-HCl (pH 7.0), 100 mm NH4Cl, 12.5 mm MgCl2, 0.5 mm EDTA, 6 mm βME) at −80 °C. Ribosomal 30S and 50S subunits were purified using sucrose gradient (10–35%) in a ribosome-dissociation buffer (20 mm Tris-HCl (pH 7.0), 300 mm NH4Cl, 1.5 mm MgCl2, 0.5 mm EDTA, 6 mm βME). The fractions containing 30S and 50S subunits were collected separately, concentrated, and stored in the ribosome-storage buffer at −80 °C. E. coli tRNAfMet was purchased from Chemical Block. RNA, containing the Shine-Dalgarno sequence and a linker to place the AUG codon in the P-site and the UAU codon in the A-site (GGC AAG GAG GUA AAA AUG UAU AAAAAA) was synthesized by IDT.
      The 70S·mRNA·tRNAfMet·RF1 complex was prepared by reconstitution in vitro. 1 μm 30S subunit (all concentrations are specified for the final solution) were pre-activated at 42 °C for 5 min in the ribosome-reconstitution buffer (20 mm Tris-HCl (pH 7.0), 100 mm NH4Cl, 20.5 mm MgCl2, 0.5 mm EDTA, 6 mm βME). After pre-activation, 0.9 μm 50S subunit with 12 μm mRNA and 5 μm tRNAfMet were added to the 30S solution and incubated for 15 min at 37 °C. An equal volume of 40 μm RF1 was then added resulting in the following final concentrations: ∼0.45 μm 70S, 6 μm mRNA, 2.5 μm tRNAfMet, and 20 μm RF1. The solution was incubated for 15 min at 37 °C and applied on cryo-EM grids at room temperature.

      Cryo-EM and image processing

      Holey-carbon grids (C-flat 1.2–1.3, Protochips) were glow-discharged with 20 mA with negative polarity for 30 s in a PELCO easiGlow glow-discharge unit. 1.5 μl of the 70S·mRNA·tRNAfMet·RF1 complex was applied to the grids. The grids were blotted for 3.5 s at blotting power 8 at 4 °C and ∼95% humidity and plunged into liquid ethane, using an FEI Vitrobot MK4. The grids were stored in liquid nitrogen.
      A dataset of 1,065,147 particles was collected as follows. 3963 movies were collected using SerialEM (
      • Mastronarde D.N.
      Automated electron microscope tomography using robust prediction of specimen movements.
      ) on a Talos Arctica (FEI) microscope operating at 200 kV equipped with a K2 Summit camera system (Gatan) with −0.7- to −1.7-μm defocus. Each exposure was acquired with continuous frame streaming with the exposure length of 80 frames per movie yielding a total dose of 37.7 e/Å2. The nominal magnification was 22,000, and the corrected super-resolution pixel size at the specimen level was 0.944 Å. The frames for each movie were processed using IMOD (
      • Kremer J.R.
      • Mastronarde D.N.
      • McIntosh J.R.
      Computer visualization of three-dimensional image data using IMOD.
      ). The movies were motion-corrected, and frame averages were calculated using frames 3–42 within each movie, using alignframes (IMOD), after multiplication with the corresponding gain reference. cisTEM (
      • Grant T.
      • Rohou A.
      • Grigorieff N.
      cisTEM, user-friendly software for single-particle image processing.
      ) was used to determine defocus values for each resulting frame average and for particle picking. The stack and FREALIGN parameter file were assembled in via cisTEM with the binning of 1, 3×, and 6× (box size of 480 for a nonbinned stack).
      Data processing was performed essentially as described previously (
      • Loveland A.B.
      • Demo G.
      • Grigorieff N.
      • Korostelev A.A.
      Ensemble cryo-EM elucidates the mechanism of translation fidelity.
      ). FrealignX version 9.11 in FrealignX mode was used for all steps of refinement and reconstruction (
      • Grigorieff N.
      Frealign: an exploratory tool for single-particle cryo-EM.
      ). The 6×-binned image stack (1,065,147 particles) was initially aligned to a ribosome reference (PDB code 5J4D (
      • Svidritskiy E.
      • Madireddy R.
      • Korostelev A.A.
      Structural basis for translation termination on a pseudouridylated stop codon.
      )) without RF1 and E-tRNA, using three cycles of mode 3 (global search) alignment, including data in the resolution range from 30 to 300 Å. Subsequently, the 6× binned stack was refined using mode 1 (refine) in the resolution ranges (sequentially): 30–300, 24–300, 18–300, and 15–300 Å (three cycles for each range). Using the 3× binned image stack, the particles were successively aligned in mode 1 (refine) by gradually increasing the high resolution limit to 12, 10, 9, 8, and 7 Å (three cycles for each resolution limit). In the last step, the unbinned (full-resolution) image stack was used to successively align particles against the common reference using mode 1 (refine; three cycles) at the resolution limit of 6 Å. 3D density reconstruction was obtained using 60% of particles with highest scores. The map contained density for the P- and E-site tRNAs, mRNA and RF1. The resolution of the resulting reconstruction was ∼3.7 Å (Fourier shell correlation (FSC) = 0.143); local resolution for the codon-recognition domain of RF1 in the decoding center and the catalytic domain in the peptidyl transferase center achieves ∼3 Å resolution, allowing near-atomic resolution interpretation of nucleotide and side-chain interactions (Fig. 1, C–E). Additional classification into 16 classes yielded three classes (87% of all particles) with the occupancy of RF1 similar to that in the initial map. The initial reconstruction was B-factor sharpened using B-factors of −150, −200, and −225 Å2 in bfactor.exe (part of the FrealignX distribution) and used for model building and structural refinements. The B-factor of −100 Å2 was also used to visualize lower-resolution details. FSC curve was calculated by FrealignX for even and odd particle half-sets (Fig. 1B). Blocres was used to assess local resolution of the unfiltered and unmasked volume using a box size of 56 pixels, step size of 10 pixels, and resolution criterion of FSC value at 0.143 (
      • Cardone G.
      • Heymann J.B.
      • Steven A.C.
      One number does not fit all: mapping local variations in resolution in cryo-EM reconstructions.
      ).

      Model building and refinement

      Recently reported cryo-EM structure of E. coli 70S·ArfA·RF2 complex (
      • Demo G.
      • Svidritskiy E.
      • Madireddy R.
      • Diaz-Avalos R.
      • Grant T.
      • Grigorieff N.
      • Sousa D.
      • Korostelev A.A.
      Mechanism of ribosome rescue by ArfA and RF2.
      ), excluding ArfA and RF2, was used as a starting model for structural refinement. The initial model of E. coli RF1 (domains 2–4) was extracted from the crystal structure of the 70S·RF1 complex (
      • Svidritskiy E.
      • Madireddy R.
      • Korostelev A.A.
      Structural basis for translation termination on a pseudouridylated stop codon.
      ), and domain 1 was obtained by homology modeling from T. thermophilus RF1 (
      • Korostelev A.
      • Zhu J.
      • Asahara H.
      • Noller H.F.
      Recognition of the amber UAG stop codon by release factor RF1.
      ) using SWISS-PROT (
      • Bairoch A.
      • Boeckmann B.
      • Ferro S.
      • Gasteiger E.
      Swiss-Prot: juggling between evolution and stability.
      ). Initial protein and ribosome domain fitting into cryo-EM maps was performed using Chimera (
      • Pettersen E.F.
      • Goddard T.D.
      • Huang C.C.
      • Couch G.S.
      • Greenblatt D.M.
      • Meng E.C.
      • Ferrin T.E.
      UCSF Chimera–a visualization system for exploratory research and analysis.
      ), followed by manual modeling using PyMOL (
      • DeLano W.L.
      ). The linker between domain 1 and domain 2 (aa 99–105) was not defined in the cryo-EM map and was not modeled.
      The structural model was refined by real-space simulated-annealing refinement using atomic electron scattering factors in RSRef (
      • Chapman M.S.
      Restrained real-space macromolecular atomic refinement using a new resolution-dependent electron density function.
      ,
      • Korostelev A.
      • Bertram R.
      • Chapman M.S.
      Simulated-annealing real-space refinement as a tool in model building.
      ), as described previously (
      • Svidritskiy E.
      • Brilot A.F.
      • Koh C.S.
      • Grigorieff N.
      • Korostelev A.A.
      Structures of yeast 80S ribosome-tRNA complexes in the rotated and nonrotated conformations.
      ). Secondary-structure restraints, comprising hydrogen-bonding restraints for ribosomal proteins and base-pairing (distance and co-planarity) restraints for RNA nucleotides, were implemented in CNS format (
      • Brünger A.T.
      • Adams P.D.
      • Clore G.M.
      • DeLano W.L.
      • Gros P.
      • Grosse-Kunstleve R.W.
      • Jiang J.S.
      • Kuszewski J.
      • Nilges M.
      • Pannu N.S.
      • Read R.J.
      • Rice L.M.
      • Simonson T.
      • Warren G.L.
      Crystallography and NMR system: a new software suite for macromolecular structure determination.
      ). Refinement parameters in RSRef, such as the relative weighting of stereochemical restraints and experimental energy term, were optimized to produce the stereochemically optimal models that closely agree with the corresponding maps. Refinement was performed using B-factor sharpened maps: all-atom refinement (−200 Å2), then local refinement of P-tRNA, mRNA, RF1, and neighboring residues (−225 Å2) at the starting annealing temperature 1000 K. In the final stages, the structures were refined using phenix.real_space_refine (
      • Adams P.D.
      • Afonine P.V.
      • Bunkóczi G.
      • Chen V.B.
      • Davis I.W.
      • Echols N.
      • Headd J.J.
      • Hung L.W.
      • Kapral G.J.
      • Grosse-Kunstleve R.W.
      • McCoy A.J.
      • Moriarty N.W.
      • Oeffner R.
      • Read R.J.
      • Richardson D.C.
      • et al.
      PHENIX: a comprehensive Python-based system for macromolecular structure solution.
      ) against a B-sharpened map (−150 Å2) at 300 K, followed by a round of refinement in RSRef applying harmonic restraints to preserve protein backbone geometry (−150 Å2 and 300 K). The refined structural model closely agrees with the corresponding maps, as indicated by the low real-space R-factor of ∼0.19 (RSRef) and high correlation coefficient of 0.86 (PHENIX, CC around atoms). The resulting models have good stereochemical parameters, characterized by low deviation from ideal bond lengths and angles, low number of protein–backbone and rotamer outliers, as shown in Table 1. Structure superpositions and comparisons were performed in PyMOL.

      Structural analyses of protein side chain interactions with RNA nucleotides

      The following ribosome structures were downloaded from RCSB for the analyses of amino acid–nucleotide interactions: E. coli 70S ribosome (PDB code 4YBB (
      • Noeske J.
      • Wasserman M.R.
      • Terry D.S.
      • Altman R.B.
      • Blanchard S.C.
      • Cate J.H.
      High-resolution structure of the Escherichia coli ribosome.
      )), T. thermophilus 70S ribosome (PDB code 4Y4P (
      • Polikanov Y.S.
      • Melnikov S.V.
      • Söll D.
      • Steitz T.A.
      Structural insights into the role of rRNA modifications in protein synthesis and ribosome assembly.
      ), and S. cerevisiae 80S ribosome (PDB code 4V88 (
      • vBen-Shem A.
      • Garreau de Loubresse N.
      • Melnikov S.
      • Jenner L.
      • Yusupova G.
      • Yusupov M.
      The structure of the eukaryotic ribosome at 3.0 A resolution.
      ). PyMOL was used to calculate the number of side chains packed on RNA nucleotides (number of aa per 1000 nucleotides, Fig. 3). The following distance cutoff criterion was used: at least one side-chain nonhydrogen atom (i.e. any atom excluding the backbone atoms) within 3.7 Å from the following carbon atoms of the aromatic base of a nucleotide: C2, C4, or C5 (U or C); C4, C5, or C6 (A); and C2, C4, C5, or C6 (G). Selected amino acids were confirmed by visual inspection of the PDB structures in PyMOL, supporting the stringency of the selection criterion.

      Structure accession codes

      The cryo-EM map and PDB coordinates have been deposited in EMDB and the Protein Data Bank with accession codes EMD-7970 and 6DNC.

      Acknowledgments

      We thank Chen Xu for help with screening cryo-EM grids and for assistance with data collection at the Cryo-EM Facility, University of Massachusetts Medical School, Anna B. Loveland for assistance with data processing, and Darryl Conte, Jr., for helpful comments on the manuscript.

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