The CryoEM structure of the Saccharomyces cerevisiae ribosome maturation factor Rea1
- Institut de Génétique et de Biologie Moléculaire et Cellulaire, France
- Centre National de la Recherche Scientifique, UMR7104, France
- Institut National de la Santé et de la Recherche Médicale, U964, France
- Université de Strasbourg, France
- MRC Laboratory of Molecular Biology, United Kingdom
Figures
Figure 1 with 7 supplements
Function and structure of Rea1.
(A) Schematic representation of the reactions catalysed by Rea1. (B) Cartoon representation of the Rea1 structure showing the N-terminal domain, the AAA+ ring as well as the stem, middle and top …
Figure 1—figure supplement 1
Quality of NTD-AAA + ring and linker cryoEM maps.
(A) Fourier shell correlation (FSC) plot for half-maps of the NTD-AAA+ ring reconstruction. The 0.143 FSC criteria is indicated as horizontal blue line. The final overall resolution is 4.4 Å. (B) …
Figure 1—figure supplement 2
Quality of composite NTD-AAA+ ring linker cryoEM map.
The insets show the map quality in the linker top (purple), the linker middle domain (pink), the linker stem (violet), AAA6L (red) and AAA3L (green).
Figure 1—figure supplement 3
Rea1 mass spectrometry analysis after tryptic digest.
The identified peptides of S. cerevisiae Rea1 are shown in yellow. Methionine and cysteine residues highlighted in green indicate mass shifts due to sulphur oxidation. The sequence coverage level is …
Figure 1—figure supplement 4
Core architecture of the Rea1 AAA +subunits.
(A) Core architecture of AAAL domains. Secondary structure elements are labelled for AAA1L. All Rea1 AAAL’s have β-sheet inserts in H2 as well as between H3 and S4 (red, grey for AAA6L) In AAA2L, …
Figure 1—figure supplement 5
AAA+ ring architecture and nucleotide binding.
(A) In the Rea1 AAA+ ring each AAAL domain is tightly associated with the AAAS domain of the previous AAA+ module to form the following modules: AAA2: AAA2L-AAA1S, AAA3: AAA3L-AAA2S, AAA4: …
Figure 1—figure supplement 6
Nucleotide binding at the Rea1 AAA1 and AAA6 sites.
(A) Nucleotide density of the AAA1 site. (B) Nucleotide density at the AAA6 site. The densities are consistent with an ADP molecule (shown in balls and stick representation).
Figure 1—figure supplement 7
Comparison of the Rea1 AAA1 and AAA6 nucleotide-binding sites with the dynein AAA1 and AAA3 nucleotide-binding sites.
To analyse the degree of site closure, we compared the distance between the terminal phosphate or vanadate group of the nucleotide and the H4 α-helix, which harbours the arginine finger motif. In …
Figure 2 with 2 supplements
Rea1 AAA+ ring architecture and regulation of its ATPase activity.
(A) Architecture of the Rea1 AAA+ ring. For clarity, only AAAL domains are shown. Gaps exits between AAA2L/AAA3L, AAA3L/AAA4L and AAA5L/AAA6L (black arrows) indicating that nucleotide binding sites …
Figure 2—figure supplement 1
Interactions of AAA2L-H2α insert with H2 β-sheets of AAA1L, AAA3L, AAA4L, and AAA5L.
(A) The AAA2L H2 β-sheet interacts with the AAA1L H2 β-sheet. AAA2L-H2α contacts the tip of the AAA3L H2 β-sheet to keep the AAA2 site open. (B) AAA2L-H2α also contacts the tip of the AAA5L H2 …
Figure 2—figure supplement 2
Rea1 AAA+ ring conformation and its comparison with dynein.
(A) In the Rea1 AAA+ ring, the H3 α-helices of AAA1L, AAA2L, AAA3L, AAA5L and AAA6L (red) point towards the pore of the AAA+ ring. The orientation of AAA4L (yellow) deviates from the orientation of …
Figure 3 with 1 supplement
Architecture of the Rea1 linker.
(A) The AAA+ ring linker interface. Left panel: The N-terminal domain (NTD) acts as a scaffold between AAA6L and the linker stem via interactions with AAA6L-H2α and the N-terminal linker stem …
Figure 3—figure supplement 1
Remodelling of the Rea1 linker with respect to the AAA+ ring.
(A) The Rea1 ADP cryoEM structure. (B) 2D projection of the structure shown in A, and low pass filtered to 18 Å. (C) Top to bottom: Negative stain electron microscopy 2D classes of Rea1 published by …
Figure 4 with 6 supplements
CryoEM structures of the Rea1 and Rea1_ΔAAA2L-H2α AMPPNP states.
(A) Front (left panel) and side (middle panel) view of the Rea1 AMPPNP state. The relative orientation of the linker with respect to the AAA+ ring has not changed. The AAA2L-H2α insert sits in the …
Figure 4—figure supplement 1
Quality of the Rea1 AMPPNP map and comparison of the Rea1 ADP and AMPPNP structures.
(A) Fourier shell correlation (FSC) plot for half-maps of the Rea1 AMPPNP reconstruction. The 0.143 FSC criteria is indicated as horizontal blue line. The final overall resolution is 4.3 Å. (B) …
Figure 4—figure supplement 2
Negative stain electron microscopy reconstructions of Rea1 APO and ATP states.
(A) Front (left panel) and side view (right panel) of the Rea1 APO state. The angle between the linker and the AAA+ ring (red) is marked on right panel. It remains around 120° like in the Rea1 ADP …
Figure 4—figure supplement 3
Negative stain electron microscopy reconstructions of Rea1_ΔAAA2L-H2α APO, AMPPNP, ATP and ADP states.
The left panels represent the front views and the right panels the side views of the (A) APO, (B) AMPPNP, (C) ATP and (D) ADP states of the Rea1_ΔAAA2L-H2α deletion mutant. The angle between the …
Figure 4—figure supplement 4
The additional density at the AAA+ ring of the Rea1_ΔAAA2L-H2α APO and AMPPNP states is the MIDAS domain.
(A) A recently published cryoEM structure of a complex between Rea1 and an Rsa4 containing pre60S particle revealed the location of the Rea1 AAA+ ring on the pre60S particle. The Rea1 linker was …
Figure 4—figure supplement 5
CryoEM structure of the Rea1_ΔAAA2L-H2α AMPPNP state.
(A) Fourier shell correlation (FSC) plot for half-maps of the Rea1_ΔAAA2L-H2α AMPPNP reconstruction. The 0.143 FSC criteria is indicated as horizontal blue line. The final overall resolution is 7.8 …
Figure 4—figure supplement 6
Comparison of Rea1 and Rea1_ΔAAA2L-H2α AMPPNP states.
(A) The Rea1_ΔAAA2L-H2α AMPPNP structure (colour coded) superimposed on the Rea1 AMPPNP structure (grey) Left panel: front view, right panel: side view. The superimposition was done by aligning the …
Figure 5
Model for Rea1 mediated Rsa4 assembly factor removal.
In the absence of pre60S particles the Rea1 ATP-hydrolysis activity is inhibited by AAA2L-H2α. When Rea1 binds to pre60S particles, AAA2L-H2α relocates towards Rix1, and the Rea1 ATPase activity is …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference |
|---|---|---|
| Strain, strain background (S. cerevisae) | JD1370 | DOI: 10.1126/science.1212642 |
| Chemical compound | 1,4-dithiotreitol (DTT) | Thermo Fisher SCIENTIFIC |
| Chemical compound | ATP | ACROS Organics |
| Chemical compound | AMPPNP | Jena biosciences |
| Chemical compound | ADP | SIGMA-ALDRICH |
| Chemical compound | Roche cOmplete, EDTA-free Protease Inhibitor | SIGMA-ALDRICH |
| Chemical compound | Dimethylsulfoxide (DMSO) | SIGMA-ALDRICH |
| Chemical compound | Phenylmethylsulfonyl fluoride (PMSF) | SIGMA-ALDRICH |
| Chemical compound | Yeast Nitrogen Base without Amino acids | Formedium |
| Chemical compound | D(+) - Glucose | Formedium |
| Chemical compound | D(+) - Galactose | Formedium |
| Chemical compound | CSM, -Ura | Formedium |
| Chemical compound | Triton X-100 | SIGMA-ALDRICH |
| Commercial assay or kit | EnzChek Phosphate Assay Kit | Thermo Fisher SCIENTIFIC |
| Software, algorithm | Adobe Photoshop version 16.0.3 (for figure preparation) | Adobe Systems, Inc. N/A |
| Software, algorithm | PyMOL(TM) 2.0.6 Schrodinger LLC | https://pymol.org/edu/?q=educational/ |
| Software, algorithm | Chimera Pettersen et al., 2004 | https://www.cgl.ucsf.edu/chimera/download.html |
| Software, algorithm | Gautomatch | https://www.mrc-lmb.cam.ac.uk/kzhang/Gautomatch/ |
| Software, algorithm | Serial EM Mastronarde, 2005 | http://bio3d.colorado.edu/SerialEM |
| Software, algorithm | MotionCor2Zheng et al., 2017 | http://msg.ucsf.edu/em/software/motioncor2.html |
| Software, algorithm | RELION 2.0 Kimanius et al., 2016 | http://www2.mrc-lmb.cam.ac.uk/relion |
| Software, algorithm | COOT Emsley and Cowtan, 2004 | http://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot |
| Software, algorithm | PHENIX Adams et al., 2010 | https://www.phenix-online.org |
Additional files
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Supplementary file 1
Data collection and refinement statistics.
Data collection and refinement statistics for the presented cryoEM maps and structural models. VPP = volta phase plate.
- https://doi.org/10.7554/eLife.39163.023
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Transparent reporting form
- https://doi.org/10.7554/eLife.39163.024
