Journal: Cell reports

Article Title: Structures of 9-1-1 DNA checkpoint clamp loading at gaps from start to finish and ramification on biology

doi: 10.1016/j.celrep.2023.112694

Figure Lengend Snippet: (A) DNA oligos were annealed with a longer template DNA to provide either an isolated 5′ end (primer #2 only) or an ssDNA gap of various lengths (both primers #1 and #2; see ). The 32 P-9-1-1 clamp is blocked from sliding by the biotin (attached to magnetic streptavidin beads) on one end and the DIG moiety attached to the Fab of an antibody to DIG at the other end. See for details. (B) Comparison of 9-1-1 loading at a recessed 5′ end and at a 10-nt gap in the presence of RPA, along with control reactions lacking one assay component. (C) Magnetic bead assays using nicked DNA or different-sized gaps either minus RPA (left) or plus RPA (right). (D) Comparison of DNA loading rates of 9-1-1 (by Rad24-RFC) and PCNA (by RFC) were performed under identical conditions using the 10-nt gap DNA. Each experiment using the magnetic bead assay of (B)–(D) was carried out using three independently performed experiments. The data of are shown as standard error (SD) of the mean (SEM) performed by Microsoft Excel v.16.71.

Article Snippet: Cryo-EM map of Rad24-RFC–9-1-1 clamp–10-nt gapped DNA at step 2 , This study , EMDB: EMD-29413.

Techniques: Isolation, Comparison

Journal: Cell reports

Article Title: Structures of 9-1-1 DNA checkpoint clamp loading at gaps from start to finish and ramification on biology

doi: 10.1016/j.celrep.2023.112694

Figure Lengend Snippet: (A) Domain architecture of Rad24-RFC, 9-1-1 clamp, and the 10-nt gapped DNA. The Rfc2-5 and three subunits of 9-1-1 have similar respective domain arrangements and are shown together for brevity. Dashed lines indicate unsolved regions in EM maps. LL is the long linker between the Rad24 AAA+ module and the collar domain. The N-terminal domain (NTD) and CTD in each 9-1-1 subunit are linked by the inter-domain connecting loop (IDCL). The 10-nt gapped DNA harbors both 5′ and 3′ junctions. (B) Structures of five Rad24-RFC loading intermediates of 9-1-1 clamp, arranged in a plausibly temporary sequence based on the progression of DNA binding in the central chamber of Rad24-RFC. In step 1, the 9-1-1 gate is open, and the DNA has not bound into the clamp loader chamber. In step 2, the 9-1-1 gate remains open, the DNA has entered the central chamber and passed through the 9-1-1-gate, but the DNA is partially stable, and the EM density for 4-bp DNA between the loader and the clamp is missing. In step 3, the 9-1-1 gate remains open, and the DNA in the chamber is fully stabilized. In step 4, the 9-1-1 gate is partially closed, and DNA is fully engaged. In step 5, the 9-1-1 gate is fully closed around the loaded DNA. A representative cryo-EM density map rendered at a high threshold (0.2) is shown at the bottom right corner. For clarity, the Rad24-RFC structure in all five models is in ivory. The red arrow points to the 9-1-1 gate. The DNA entry A gate in Rad24-RFC is labeled in the step 1 structure. The structures are aligned and shown in the same front view.

Article Snippet: Cryo-EM map of Rad24-RFC–9-1-1 clamp–10-nt gapped DNA at step 2 , This study , EMDB: EMD-29413.

Techniques: Sequencing, Binding Assay, Cryo-EM Sample Prep, Labeling

Journal: Cell reports

Article Title: Structures of 9-1-1 DNA checkpoint clamp loading at gaps from start to finish and ramification on biology

doi: 10.1016/j.celrep.2023.112694

Figure Lengend Snippet: Cryo-EM data collection, refinement, and atomic model validation

Article Snippet: Cryo-EM map of Rad24-RFC–9-1-1 clamp–10-nt gapped DNA at step 2 , This study , EMDB: EMD-29413.

Techniques:

Journal: Cell reports

Article Title: Structures of 9-1-1 DNA checkpoint clamp loading at gaps from start to finish and ramification on biology

doi: 10.1016/j.celrep.2023.112694

Figure Lengend Snippet: (A) The step 5 structure of Rad24-RFC–9-1-1–10-nt gapped DNA in a front (left), back (middle), and top (right) view. Subunits are individually colored. 9-1-1 is omitted in the right panel to better show the bound ATPγS and ADP. The Rad24 upper loop inside the chamber is highlighted in green. The region in the cyan box is shown enlarged in (B). (B–F) Comparison of DNA binding in the chamber of Rad24-RFC (B), RFC (C), E. coli clamp loader (D), and T4 phage clamp loader (E). (F) Sketch comparing the 3′ DNA-binding mode in the chamber of RFC and Rad24-RFC based on structures in (B) and (C). The loaders are aligned but omitted except for a few labeled key elements. The α4 and α5 helices of each loader subunit follow and wrap around the template strand in purple. In (B), Rad24 Arg-199 and Rfc5 Asn-80 H-bond with the primer phosphate backbone in blue. The Rad24 upper loop blocks the primer strand from advancing upward, in contrast to the higher reach of the primers in all other loaders. The equivalent loops in other loaders are much shorter and do not block the primer strand (C and D). The T4 loader lacks the equivalent loop (indicated by a green arrow) as the corresponding AAA+ module is highly degenerated (E) (see also and ). Notably, all clamp loaders—except for Rad24-RFC—harbor an aromatic residue (lime) at the top that functions as a separation pin to unwind DNA from the 3′ junction.

Article Snippet: Cryo-EM map of Rad24-RFC–9-1-1 clamp–10-nt gapped DNA at step 2 , This study , EMDB: EMD-29413.

Techniques: Comparison, Binding Assay, Labeling, Blocking Assay, Residue

Journal: Cell reports

Article Title: Structures of 9-1-1 DNA checkpoint clamp loading at gaps from start to finish and ramification on biology

doi: 10.1016/j.celrep.2023.112694

Figure Lengend Snippet: (A) Flexibility analysis of the five loading intermediates. The atomic models are colored by their respective local B factors in ChimeraX. Rad24-RFC is omitted for clarity. Mec3 and Ddc1 lining the DNA gate are partially mobile (yellow) in the open gate and the partially open gate in steps 1–4, but they become stable (blue) in the closed gate in step 5. Consistent with the assigned temporal sequence, the chamber DNA is absent in step 1, is present but flexibly bound (red) in step 2, and becomes more stably bound (yellow) in step 3. The partial gate closure in step 4 destabilizes the chamber DNA (red), likely due to the perturbation by Mec3 movement. The chamber DNA is better stabilized in the 9-1-1 gate fully closed step 5. The 9-1-1 gate size is labeled. Four nucleotides and three nucleotides near the top 5′ junction and the bottom 3′ junction, respectively, are stabilized in the Rad24-RFC chamber. The dashed line represents three disordered nucleotides in the gap region. Because the Rad24-RFC A gate is open in all structures, ssDNA longer than 10 nt in the gap region can be easily accommodated by looping outward through the A gate. (B) Although both Mec3 and Ddc1 line the DNA entry gate, Mec3 is the actual “gate” of the 9-1-1 clamp. The 9-1-1 structures in steps 1–4 are colored by their local RMSD Cα compared with the step 5 structure. Ddc1 is static, and gate opening and closing only involve the in-plane rotation of Mec3.

Article Snippet: Cryo-EM map of Rad24-RFC–9-1-1 clamp–10-nt gapped DNA at step 2 , This study , EMDB: EMD-29413.

Techniques: Sequencing, Stable Transfection, Labeling

Journal: Cell reports

Article Title: Structures of 9-1-1 DNA checkpoint clamp loading at gaps from start to finish and ramification on biology

doi: 10.1016/j.celrep.2023.112694

Figure Lengend Snippet: (A) A sketch of the used 5-nt gapped DNA substrate. (B) Structure of the Rad24-RFC–9-1-1–5-nt gapped DNA complex in a front view. Subunits are individually colored. Right panel shows enlarged views of the 5-nt DNA gap region. The Rad24 upper loop and the Rfc5 plug are shown at the 3′ DNA junction to block the upward movement of the chamber DNA (i.e., DNA bound in the central chamber of Rad24-RFC). (C) EM map of the 5-nt gapped DNA is shown in transparent gray surface and superimposed on the atomic model. The auto-refined map by Relion before post-processing at 3.66-Å resolution has stronger DNA density and is used here. (D) Side-by-side comparison of the Rad24 upper loop (green) and Rfc5 plug (cyan) interacting with the 5-nt gapped DNA (left) and the 10-nt gapped DNA from step 5 (right). Note that the lower 3′ dsDNA in the left structure is rotated by 180° around its helical axis such that the purple template strand is oriented to connect with the upper 5′ dsDNA with a minimum length (5 nt).

Article Snippet: Cryo-EM map of Rad24-RFC–9-1-1 clamp–10-nt gapped DNA at step 2 , This study , EMDB: EMD-29413.

Techniques: Blocking Assay, Comparison

Journal: Cell reports

Article Title: Structures of 9-1-1 DNA checkpoint clamp loading at gaps from start to finish and ramification on biology

doi: 10.1016/j.celrep.2023.112694

Figure Lengend Snippet: (A) In preparation for loading (step 0), Rad24-RFC binds the 9-1-1 clamp to form a binary complex in the presence of ATP (or ATPγS used in this study). In a mechanism similar to the mutual activation of RFC and PCNA, we suggest that binding energy between Rad24-RFC and 9-1-1 drives the DNA gate opening in Rad24-RFC (the A gate) and 9-1-1 (between Mec3 and Ddc1). This intermediate is grayed out as it is yet to be captured. In step 1, the shoulder DNA binds first to the Rad24-RFC external site. This is followed by the chamber DNA binding and passing through the 9-1-1 gate in steps 2 and 3. Once the DNA has fully entered, in steps 4 and 5, Mec3 moves toward Ddc1 to close the 9-1-1 gate, as indicated by the curved red arrow. The green Rad24 upper loop, with the help of the cyan Rfc5 plug, prevents DNA with a gap size shorter than 5 nt from entering the Rad24-RFC chamber. The consistent presence of four ATPγS and one ADP throughout steps 1 to 4 suggests that ATP hydrolysis is not required for DNA binding and clamp gate closure. Stable DNA binding and clamp gate closure likely induce a conformational change in Rad24-RFC to trigger ATP hydrolysis, leading to the dissociation of Rad24-RFC from 9-1-1 in step 6, leaving 9-1-1 alone encircling the DNA 3′ end. (B) A sketch illustrating that Rad24-RFC does not load 9-1-1 onto a nicked DNA or very short gap (1–4 nt). By virtue of its recognition to the 5′ end, Rad24-RFC loads 9-1-1 to the 3′ end of a medium-sized gap, but loading at a very long gap or a 5′ recessed DNA leads to loading onto ssDNA at a 5′ ssDNA junction, which is a very different outcome. Loading on the duplex of a 3′ end enables the 9-1-1 clamp to be used by a Pol, such as TLS Pol recruitment for gap filling at 3′ end, while loading at a 5′ end may result in ATM activation and cell-cycle arrest.

Article Snippet: Cryo-EM map of Rad24-RFC–9-1-1 clamp–10-nt gapped DNA at step 2 , This study , EMDB: EMD-29413.

Techniques: Activation Assay, Binding Assay

Journal: Cell reports

Article Title: Structures of 9-1-1 DNA checkpoint clamp loading at gaps from start to finish and ramification on biology

doi: 10.1016/j.celrep.2023.112694

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Cryo-EM map of Rad24-RFC–9-1-1 clamp–10-nt gapped DNA at step 2 , This study , EMDB: EMD-29413.

Techniques: Virus, Recombinant, Bradford Protein Assay, Isolation, Expressing, Plasmid Preparation, Software

Journal: Nature

Article Title: mRNA decoding in human is kinetically and structurally distinct from bacteria

doi: 10.1038/s41586-023-05908-w

Figure Lengend Snippet: a , Schematic of FRET donor (P-site tRNA) and acceptor (aa-tRNA) fluorophores. b , Example smFRET data (10-ms time resolution) of a decoding reaction from the perspective shown in a showing progression from IC to CR and GA to AC for a single trace (left), and a population histogram of n traces (right). c , Transition density plot showing the FRET efficiency before and after each transition detected in this population of traces using hidden Markov model (HMM) idealization. d , Population histograms as in b of decoding reactions in the presence of an mRNA displaying a near-cognate (nc) A-site codon, GTPγS, PLT (10 µM), SR-A3 (10 µM), ANS (50 µM) or HHT (50 µM), showing stalling or rejection along the reaction coordinate. e , Overview of four cryo-EM reconstructions along the decoding reaction coordinate, filtered by local resolution and contoured at 3 σ . Insets: details of eEF1A interacting with the GAC (top; 3 σ ) and the aa-tRNA (Phe-tRNA Phe ) ASL (bottom; 4 σ ) in the CR-to-GA transition. f , g , tRNA motions in the transition between the CR and GA complexes ( f ) and the GA and AC complexes ( g ) showing the positions of the FRET label attachment points and the distance between them, coloured as in a .

Article Snippet: Human polymix buffer was further supplemented with 5 mM MgCl 2 and reducing agent (1 mM DTT for cryo-EM and 1.5 mM 2-mercaptoethanol for smFRET).

Techniques: Cryo-EM Sample Prep

Journal: Nature

Article Title: mRNA decoding in human is kinetically and structurally distinct from bacteria

doi: 10.1038/s41586-023-05908-w

Figure Lengend Snippet: a – c , Cryo-EM density of complexes stalled with plitidepsin, anisomycin, lactimidomycin and GTPγS (PLT, ANS, LTM and GTPγS) highlighting the locations of visualized post-transcriptional (dark blue) and post-translational (dark red) modifications on (a) the LSU and (b) the SSU from the high-resolution consensus reconstruction and (c) aa-tRNA (Phe-tRNA Phe ), P-site tRNA (Met-tRNA fMet ), eEF1A and eIF5A from the GTPase activated reconstruction. d – n , High-resolution structural features visible within the consensus cryo-EM map, including (d) the centre of the LSU core, (e) the start codon–anticodon nucleotides in the P site (f) polyamines, (g, k) hydroxylhistidines, (h) fully hydrated Mg 2+ ions (lime green, left ) and partially hydrated and fully coordinated and Mg 2+ ions ( top , middle and right ), (i) 2′O-Me uridine (Um), (j) pseudouridine (Ψ), (l) methyl-histidine, (m) trimethyl-lysine and (n, o) methyl-lysine. Protein and nucleic acid modifications are indicated with black arrows. Cryo-EM density is from post-processed high-resolution consensus LSU map and is contoured at 3 σ for panel m and 5 σ for all remaining panels. See also , Extended Data Fig. and Extended Data Table .

Article Snippet: Human polymix buffer was further supplemented with 5 mM MgCl 2 and reducing agent (1 mM DTT for cryo-EM and 1.5 mM 2-mercaptoethanol for smFRET).

Techniques: Cryo-EM Sample Prep

Journal: Nature

Article Title: mRNA decoding in human is kinetically and structurally distinct from bacteria

doi: 10.1038/s41586-023-05908-w

Figure Lengend Snippet: a , Overview of the E site from the indicated orientation ( centre ), showing interactions between uL1, uL5, eL42, eIF5A, the L1 stalk rRNA, H74 and P-site tRNA. Lactimidomycin (LTM) shown in ball-and-stick representation. Insets running left to right show zoom-ins of the position of eIF5A relative to LTM, H74 base G3922, and eL42 ( left ), the interactions between P-site tRNA base pair G20-C57 and uL5/eL42 and P-site tRNA D-loop residues 16-21 interaction with eIF5A ( middle ) and the CCA-end of P-site tRNA ( right ). b , Overview of the P-site tRNA acceptor stem from the indicated direction showing interactions with eS25 (N terminus), uS13 (C terminus), uS19 (C terminus), uS9 (C terminus) and SSU bases of h31 (m 1 acp 3 Ψ1248, red) and h29 (1639-1642, PE loop). Insets running left to right show zoom-ins of interactions between the P-site tRNA and: eS25, uS13 and the PE loop ( left ); mRNA, uS9 and m 1 acp 3 Ψ1248 ( middle ); and uS19 and uS13 ( right ). Cryo-EM density and atomic model are from the GTPase activated complex. All cryo-EM density is filtered by local resolution and is contoured in units of σ as indicated.

Article Snippet: Human polymix buffer was further supplemented with 5 mM MgCl 2 and reducing agent (1 mM DTT for cryo-EM and 1.5 mM 2-mercaptoethanol for smFRET).

Techniques: Cryo-EM Sample Prep

Journal: Nature

Article Title: mRNA decoding in human is kinetically and structurally distinct from bacteria

doi: 10.1038/s41586-023-05908-w

Figure Lengend Snippet: a , Interactions between the tRNA CCA-ends and the peptidyl transferase centre (PTC). b – d , Overview of aa-tRNA interactions with the L11 stalk (H42 and H44), H89 and the LSU A-site finger (ASF) in (b) the CR, (c) GA and (d) AC complexes stalled with plitidepsin, anisomycin, lactimidomycin and GTPγS. The relative position of aa-tRNA and P-site tRNA and the ASF, H89 and the GTPase activating centre (GAC, left ). A zoom in on the region within the dotted line is provided with ( right ) and without ( middle ) experimental density, showing the interaction with aa-tRNA (Phe-tRNA Phe ) bases G19 and C56. The vertical green fields show the overall position of the tRNA bases G19 and C56 relative to the LSU. These show the stacking between G19 and C56 of the aa-tRNA elbow with G1981 of H44 and A2009 of H42, respectively, in the CR complex. In the GA complex the aa-tRNA has moved past H89 further towards the P site and the SSU, shifting the stacking interaction between the elbow and H42 and H44 such that C56 of the elbow now stacks on G1981 of H44 while G19 no longer makes any stacking interaction. In the AC complex, G19 and C56 interact weakly with the ASF. e , View of aa-tRNA accommodation showing the spatial relationship between the aa-tRNA, the P-site tRNA and the accommodation corridor. The inset shows the movement of the aa-tRNA CCA end due to subunit rolling, the white tRNA is a model of an accommodated tRNA with its anticodon stem loop (ASL) aligned onto that of an aa-tRNA on an unrolled ribosome in the GA complex. The dashed green line shows the likely path of the aa-tRNA CCA end. f , g , Zoom-ins of the accommodation corridor corresponding to the dashed rectangle in (e) showing (f) experimental cryo-EM density and (g) atomic model showing the additional crowding due to the eukaryote extension of uL3. All cryo-EM density is filtered by local resolution and is contoured at 3 σ.

Article Snippet: Human polymix buffer was further supplemented with 5 mM MgCl 2 and reducing agent (1 mM DTT for cryo-EM and 1.5 mM 2-mercaptoethanol for smFRET).

Techniques: Cryo-EM Sample Prep

Journal: Nature

Article Title: mRNA decoding in human is kinetically and structurally distinct from bacteria

doi: 10.1038/s41586-023-05908-w

Figure Lengend Snippet: a , Cryo-EM density of the SSU from the GA complex coloured by backbone root-mean-squared deviation (r.m.s.d.) compared with the CR complex, contoured at 3 σ . b , Overview of the SSU from the GA complex, showing the positions of the ternary complex, P-site tRNA and eIF5A and illustrating the size of the mobile shoulder domain (surface representation). Inset: solvent-exposed post-translationally modified C-terminal helix of eS6; known phosphorylation sites on the structured part of the C terminus are indicated in yellow. c , Magnification of eEF1A contacts with the SSU (surface representation) in the CR complex. The PLT-binding site is indicated. d , Overview of missing intersubunit bridges in the CR complex. Known phosphorylation and acetylation (Ac, orange) locations on eL24 are shown as spheres. SSU is shown in surface representation. Inset: atomic model and cryo-EM density illustrating the separated elements of bridge B8 and the α2 helix of eEF1A in the CR complex. Cryo-EM density is contoured at 3 σ . Alignment is on the LSU core.

Article Snippet: Human polymix buffer was further supplemented with 5 mM MgCl 2 and reducing agent (1 mM DTT for cryo-EM and 1.5 mM 2-mercaptoethanol for smFRET).

Techniques: Cryo-EM Sample Prep, Modification, Binding Assay

Journal: Nature

Article Title: mRNA decoding in human is kinetically and structurally distinct from bacteria

doi: 10.1038/s41586-023-05908-w

Figure Lengend Snippet: a , Remodelling of the decoding centre to recognize the codon–anticodon helix in the CR (top) to GA (bottom) transition, highlighting the monitoring bases (red) and post-transcriptionally and post-translationally modified residues (yellow outline). b , Ternary complex contacts at the subunit interface near bridge B8 in the CR (top) and GA (bottom) complexes. Inset (left): coordination of a catalytic water in the eEF1A G domain in the GA complex; eEF1A-focused refinement. Insets (right): formation of the temporary bridge B8 through the α2 helix of eEF1A. c , Overlay of CR (grey) and GA (coloured) complexes showing SSU domain closure and ternary complex movements (top), combined movement of the SSU shoulder and ternary complex (middle; LSU alignment), and ternary complex movements in addition to those induced by SSU shoulder domain closure (bottom; SSU-shoulder alignment). Cryo-EM density is contoured at 3 σ . Alignment is on the LSU core, unless otherwise noted.

Article Snippet: Human polymix buffer was further supplemented with 5 mM MgCl 2 and reducing agent (1 mM DTT for cryo-EM and 1.5 mM 2-mercaptoethanol for smFRET).

Techniques: Modification, Cryo-EM Sample Prep

Journal: Nature

Article Title: mRNA decoding in human is kinetically and structurally distinct from bacteria

doi: 10.1038/s41586-023-05908-w

Figure Lengend Snippet: a – c , Views of the decoding centre of IC, CR, GA and AC reconstructions, from left to right. Overview of aa-tRNA (Phe-tRNA Phe ) in the decoding centre as seen from the leading edge of the SSU (a). The monitoring bases (red) G626, A1824 and A1825 (530, 1492 and 1493 in E. coli , respectively) are disengaged in the IC and CR complexes. A1824 resides inside h44, hydrogen bonding between the amino group of Am3760 (A1913 in E. coli ) of H69 and N3 of A1824 replaces the stacking interaction between these two bases observed in bacteria where Am3760 resides inside h44. A1825 is disordered rather than, as observed in bacteria, flipped out close to its ‘monitoring’ position, possibly due to the empty space available to it inside h44 due to the distal position of Am3760 in human compared to bacteria. G626 is in an anti-conformation, rather than in a syn conformation as observed in bacteria, it is stacked with C614 and positioned away from the decoding centre. Ribosomal proteins uS12 and eS30 are disengaged. In the GA and AC complexes the decoding centre is fully structured around the codon–anticodon minihelix and the monitoring bases occupy positions like those observed in bacteria. A1824 and A1825 reside outside h44 forming A-minor interactions with the codon–anticodon pair. Am3760 has moved away from its CR position and is hydrogen bonded to the aa-tRNA base 3′ of the anticodon, as in bacteria. Domain closure has brought G626, C614 and uS12 roughly 3 Å further into the decoding centre. G626 now hydrogen bonds with A1824 and the first and second bases of the anticodon. C614 coordinates a Mg 2+ molecule with the third base of the mRNA codon and uS12 hydroxy-pro62. Additionally, uS12 Gln61 forms hydrogen bonds with the second base of the mRNA codon and A1824. C1331 (C1054 in E. coli ) forms a Pi-stacking interaction with the third base of the anticodon while C1698 (C1397 in E. coli ) intercalates into the mRNA one base 3′ of the codon. The N-terminal tail of eS30 has become structured and Met1 hydrogen bonds with A1825 while His3 hydrogen bonds to the aa-tRNA residue 3′ of the anticodon and forms a salt bridge to C615. View of the same process from the SSU side highlighting intercalation of C1698 into the mRNA ( b ). Close-up view of the same sequence of events focused on the h44 side of the decoding centre shown with cryo-EM density ( c ). Density for A1824 is strong in all four reconstructions, indicating stable localization and a switch-like behaviour while density for A1825 is absent in IC, weak in CR and strong in GA and AC, indicating a stepwise transition from disordered to ordered positioning of this base. Strong density places G626 in an anti-conformation in all four reconstructions ( inset ), unlike the syn to anti flip observed in bacteria in the CR to GA transition, due to the stacking between G626, C614 and G625 these bases appear to move as a rigid body with the rest of the shoulder domain. d , Equivalent cryo-EM structures of decoding intermediates from E. coli from the same view as (c). From left to right, POST complex (PDB-ID: 7N31 ), ‘Structure I’ (PDB-ID: 5UYK ), ‘Structure III’ (PDB-ID: 5UYM ), PRE-C complex (PDB-ID: 7N1P ). All cryo-EM density is filtered by local resolution and is contoured at 5 σ.

Article Snippet: Human polymix buffer was further supplemented with 5 mM MgCl 2 and reducing agent (1 mM DTT for cryo-EM and 1.5 mM 2-mercaptoethanol for smFRET).

Techniques: Sequencing, Cryo-EM Sample Prep

Journal: Nature

Article Title: mRNA decoding in human is kinetically and structurally distinct from bacteria

doi: 10.1038/s41586-023-05908-w

Figure Lengend Snippet: a , Zoomed-out view of Fig. , showing interactions between eEF1A, the SSU and the LSU in the CR complex ( top ) and the GA complex ( bottom ). In the GA complex the eEF1A G domain packs against the SRL and its α2 expansion segment forms a temporary bridge B8 between h14 and uL14. b , View of same process from the head domain of the SSU with aa-tRNA hidden for clarity, showing the interaction between DII of eEF1A with the C-terminus of uS12. c , View of the interaction between the eEF1A G domain and the ribosome as seen from the GAC in the CR complex ( top ) and the GA complex ( bottom ). The GTPγS molecule is tightly coordinated in the G-domain by switch I (SW-I), switch II (SW-II), the P loop and other G-domain elements. In the GA complex, the G domain of eEF1A docks onto the GAC, packing the α2 expansion segment tighter against SW-I. The SRL coordinates Arg69 of the SW-I element and the ‘catalytic’ His95 of the SW-II element, respectively, priming eEF1A for GTP hydrolysis. Cryo-EM density from a focused refinement on eEF1A is shown for the GA complex. d , e , The geometry of the catalytic His95 in the eEF1A G domain in the G interaction between the eEF1A G domain and the ribosome as seen from the GAC in the CR complex ( top ) and the GA complex. Contour levels for cryo-EM density are indicated in σ units.

Article Snippet: Human polymix buffer was further supplemented with 5 mM MgCl 2 and reducing agent (1 mM DTT for cryo-EM and 1.5 mM 2-mercaptoethanol for smFRET).

Techniques: Cryo-EM Sample Prep

Journal: Nature

Article Title: mRNA decoding in human is kinetically and structurally distinct from bacteria

doi: 10.1038/s41586-023-05908-w

Figure Lengend Snippet: a , b , Flowcharts of cryo-EM data processing of the ribosome structures stalled with (a) plitidepsin, anisomycin, lactimidomycin and GTPγS (PLT, ANS, LTM and GTPγS) and (b) SR-A3, homoharringtonine, cycloheximide and GTPγS (SR-A3, HHT, CHX and GTPγS). For 3D classifications, the number of classes (K) and the regularization parameter (T) are indicted. For focused 3D classification and refinements, regions contained within the soft mask are indicated. To generate high-resolution consensus maps, the particles circled in light grey line were merged, pooled, filtered for duplicates and refined with the full pixel sizes. Focused refinements with signal subtraction and 3D classifications with signal subtraction were performed with ‘shiny’ particles reextracted from refined consensus LSU metadata. All processing was conducted in RELION 3.1 , unless otherwise noted. Processing of consensus maps and final refinements/postprocessing of all maps was conducted in RELION 4.0 . c – j , Cryo-EM maps filtered and coloured by local resolution ( left ) and Fourier shell correlation (FSC) curves ( right ) obtained by masking the two half maps and calculating the cross-correlation between the masked volumes in RELION 4.0 for (c) consensus LSU, (d) consensus SSU, (e) initiation (IC), (f) CR, (g) GA and (h) AC complexes stalled with PLT, ANS, LTM and GTPγS and (i) consensus LSU and GA complexes stalled by SR-A3, HHT, CHX and GTPγS. Resolution was estimated using the 0.143 cutoff criterion (black dotted line). Cross-validation was used to optimize the weight on the experimental density in REFMAC to prevent overfitting . Cryo-EM density contour levels are indicated in σ units. Refinement procedures are described in Methods . For more details on cryo-EM processing, see also Extended Data Table .

Article Snippet: Human polymix buffer was further supplemented with 5 mM MgCl 2 and reducing agent (1 mM DTT for cryo-EM and 1.5 mM 2-mercaptoethanol for smFRET).

Techniques: Cryo-EM Sample Prep

Journal: Nature

Article Title: mRNA decoding in human is kinetically and structurally distinct from bacteria

doi: 10.1038/s41586-023-05908-w

Figure Lengend Snippet: a , Cryo-EM density of the SSU of the GA complex coloured by backbone r.m.s.d. compared with the AC complex, contoured at 3 σ . b , Overview of the factor-binding site in the GA (left) and AC (right) complexes, showing closure of the eEF1A-binding site between the SSU and LSU due to SSU rolling. aa-tRNA has been omitted for clarity. The black bar shows the distance between the phosphates of LSU rRNA G4600 and SSU rRNA A464. c , Example smFRET trace (blue) and an HMM idealization (red) from the perspective of FRET between the two tRNAs of an uninhibited decoding reaction showing reversible excursions from the GA to the AC state preceding stable AC-state formation (left). Transition density plot showing the FRET efficiency before and after each transition detected in this population of traces by HMM idealization after the first visit to the AC state (right), showing persistent fluctuations back to the GA state. d , Intersubunit bridges formed and broken by SSU rolling in the GA-to-AC transition (bottom), contoured at 3 σ .

Article Snippet: Human polymix buffer was further supplemented with 5 mM MgCl 2 and reducing agent (1 mM DTT for cryo-EM and 1.5 mM 2-mercaptoethanol for smFRET).

Techniques: Cryo-EM Sample Prep, Binding Assay

Journal: Nature

Article Title: mRNA decoding in human is kinetically and structurally distinct from bacteria

doi: 10.1038/s41586-023-05908-w

Figure Lengend Snippet: a , Overview of all intersubunit bridges on the human ribosome. b , Overviews of bridges formed (cyan) and not formed (yellow) in the four decoding complexes. c , Overview of the changes in bridging interactions during the transitions between the four decoding complexes. All cryo-EM density is filtered by local resolution and is contoured at 3 σ. See for more details.

Article Snippet: Human polymix buffer was further supplemented with 5 mM MgCl 2 and reducing agent (1 mM DTT for cryo-EM and 1.5 mM 2-mercaptoethanol for smFRET).

Techniques: Cryo-EM Sample Prep

Journal: Nature

Article Title: mRNA decoding in human is kinetically and structurally distinct from bacteria

doi: 10.1038/s41586-023-05908-w

Figure Lengend Snippet: a , Overlay of the interaction between eIF5A and P-site tRNA for the rolled AC complex (solid) and the unrolled GA complex (transparent), showing movements of eIF5A towards the P site and the P-site tRNA towards the E site as a response to SSU rolling. LSU rRNA base G4385 is shown in red. b , Overlay of the E site for the rolled AC complex (solid) and the unrolled GA complex (transparent), showing conformational changes in the E site as a consequence of SSU rolling. c , Cryo-EM density for the N-terminal tail of eIF5A in the GA ( left ) and AC ( right ) complexes, showing its restructuring as a response to SSU rolling. Cryo-EM density is filtered by local resolution and contoured at 2 σ with a 1 σ gaussian filter. d , Catalytic efficiency of decoding on fast ribosomes as a function of the ligand bound in the ribosomal E site, slow ribosomes carried out the decoding reaction with 10-20× lower speed. e , Fraction of ribosomes that carried out decoding fast as a function of the ligand bound in the ribosomal E site. Approximately 50% of ribosomes with an empty E site carry out the decoding reaction slowly, whereas with any E site ligand only about 15% do so. This implies that natural as well as small-molecule ligands that bind the E site are able to affect the conformation of the ribosome in a way that accelerates binding of ternary complex to the A site. Each dot represents one experimental replicate, the horizontal bar represents the average.

Article Snippet: Human polymix buffer was further supplemented with 5 mM MgCl 2 and reducing agent (1 mM DTT for cryo-EM and 1.5 mM 2-mercaptoethanol for smFRET).

Techniques: Cryo-EM Sample Prep, Binding Assay

Journal: Nature

Article Title: mRNA decoding in human is kinetically and structurally distinct from bacteria

doi: 10.1038/s41586-023-05908-w

Figure Lengend Snippet: a , b , Overview of the binding sites for PLT (red) ( a ) and SR-A3 (coral) ( b ) on eEF1A between domain III (DIII, cyan) and the G domain (blue) bound to a GA-ribosome complex. Focus refined on eEF1A. The black arrow indicates the hydroxyl moiety that differentiates SR-A3 from ternatin-4. c – e , Structures of ANS (light orange) ( c ) and HHT (orange) ( d ) in the PTC and LTM (dark purple) ( e ) and cycloheximide (CHX, purple) ( f ) in the E site from consensus LSU focused refinements. The cryo-EM density from structures stalled with either PLT, ANS, LTM and GTPγS ( a – c ) or SR-A3, HHT, CHX and GTPγS ( d – f ) is shown. Known resistance mutations (green), waters (red), Mg 2+ (lime green) and K + (steel blue) are indicated. Contour levels for cryo-EM density are indicated in σ units.

Article Snippet: Human polymix buffer was further supplemented with 5 mM MgCl 2 and reducing agent (1 mM DTT for cryo-EM and 1.5 mM 2-mercaptoethanol for smFRET).

Techniques: Binding Assay, Cryo-EM Sample Prep