Structure of the two-component S-layer of the archaeon Sulfolobus acidocaldarius
- Living Systems Institute, University of Exeter, United Kingdom
- Faculty of Environment, Science and Economy, University of Exeter, United Kingdom
- Faculty of Health and Life Sciences, University of Exeter, United Kingdom
- Department of Theoretical Biophysics, Max Planck Institute for Biophysics, Germany
- Malopolska Centre of Biotechnology, Jagiellonian University, Poland
- Institute of Psychiatry and Neurosciences of Paris, Inserm UMR1266 - Université Paris Cité, France
- GHU Psychiatrie et Neurosciences de Paris, France
- Henry Wellcome Building for Biocatalysis, Biosciences, Faculty of Health and Life Sciences, University of Exeter, United Kingdom
Figures
Figure 1 with 6 supplements
Atomic model of S. acidocaldarius S-layer protein SlaA at pH 4.
(a, b), SlaA30–1069 atomic model obtained by single-particle cryo electron microscopy (cryoEM) in ribbon representation and cyan–grey–maroon colours (N-terminus, cyan; C-terminus, maroon) with …
Figure 1—figure supplement 1
Relion processing workflow for the pH 4 dataset of SlaA.
Figure 1—figure supplement 2
Representative cryoEM micrographs and 2D classes for SlaA.
Representative cryoEM micrographs (from a total of 3687 for (a), 3163 for (b), and 5046 for (c)). (d–f) 2D classification examples of S. acidocaldarius polished SlaA particles at pH 4 (a, d), pH 7 (b…
Figure 1—figure supplement 3
Resolution estimation for the cryoEM maps of SlaA.
(a) Gold-standard Fourier Shell Correlation (FSC) and (b) local resolution estimations for the SlaA map obtained at pH 4, 7, and 10. Red, phase randomised masked; green, unmasked; blue, masked; …
Figure 1—figure supplement 4
SlaA30–1069 cryo electron microscopy (cryoEM) map and atomic model.
(a) CryoEM map of SlaA30–1069 at 3.1 Å global resolution. (b) Atomic model of SlaA30–1069 (ribbon representation, cyan–grey–maroon colours; N-terminus, cyan; C-terminus, maroon). (c) Fitting of the …
Figure 1—figure supplement 5
SlaA flexibility.
(a) 2D classification of negatively stained micrographs of SlaA purified from S. acidocldarius. The white arrowheads point at domains D5 and D6 in different orientations, highlighting the flexibilit …
Figure 1—figure supplement 6
Five Alphafold predictions of SlaA914–1424.
(a–e) SlaA30–1069 is shown in ribbon representation and cornflower blue; glycans are in ball-stick representation and rusty brown. The Alphafold predictions are coloured according to domains, …
Figure 2 with 1 supplement
N-glycosylation of S.acidocaldarius SlaA.
(a) Atomic model of SlaA in ribbon representation. SlaA30–1069 as solved by cryoEM is in cornflower blue; SlaA1070–1424 as predicted by Alphafold is in purple (boxed). 19 Asn-bound N-glycans were …
Figure 2—figure supplement 1
Entropic contribution of glycans to protein conformation.
Position of N-glycosylated sites and globular domains on SlaA primary structure (upper panel) and changes of the number of possible N-glycan conformers at each glycosylated site (bar graphs). Top, …
Figure 3 with 3 supplements
Structural comparison and electrostatic surface potentials of S.acidocaldarius SlaA at different pH conditions.
(a) SlaA30–1069 cryo electron microscopy (cryoEM) maps at pH 4 (light blue, res. 3.1 Å), pH 7 (orange, res. 3.9 Å), and pH 10 (magenta, res. 3.2 Å). (b) r.m.s.d. (root-mean-square deviation) …
Figure 3—figure supplement 1
Relion processing workflow for pH 7 dataset.
The image processing pipeline incuded the collection of two datasets (a and b), which were subsequently merged.
Figure 3—figure supplement 2
Relion processing workflow for pH 10 dataset.
Figure 3—figure supplement 3
Impact of glycosylation on the electrostatic surface charge of SlaA at different pH values.
Comparison of the SlaA electrostatic surface charge with (left) and without (right) glycans at pH 4 (a), 7 (b), and 10 (c). The glycosylation increases the overall surface negative charge of SlaA, …
Figure 4 with 4 supplements
S. acidocaldarius SlaA assembly into exosome-bound S-layers.
(a) Extracellular view of assembled SlaA monomers in rainbow colours and surface representation. (b) Extracellular view of assembled SlaA in ribbon representation with SlaA dimers forming a …
Figure 4—figure supplement 1
Subtomogram averaging of the S-layer on exosomes and fitting of SlaA.
CryoEM map of the S-layer assembled on exosomes in extracellular (a), intracellular (b), and side (c) views at 11.2 Å resolution. The membrane-distal face of the map is shown in magenta, and the …
Figure 4—figure supplement 2
CryoET processing, STA and resolution estimation.
(a) CryoET and STA workflow using Warp–Relion–M. (b) Gold-standard FSC of the STA map.
Figure 4—figure supplement 3
Comparison between current and previously reported (Gambelli et al., 2019) S. acidocaldarius SlaA assembly models.
(a) Side view of the SlaA dimer (ribbon representation; cyan, N-terminus; maroon, C-terminus). Within the dimer, the long axes of two SlaA monomers include an angle of ~160°. The dimer has a height …
Figure 4—figure supplement 4
Isolated SlaA-only S-layer from S.acidocaldarius.
(a) Surface representation of the atomic model of the SlaA dimer, as it occurs in the S-layer. (b) Negative stain electron tomography of isolated SlaA-only S-layer. (c) 1–4 are cut-outs from (b) …
Figure 5 with 6 supplements
S. acidocaldarius S-layer assembly.
(a, b) SlaB trimer (ribbon representation, N-terminus, cyan; C-terminus, maroon) as predicted by Alphafold v2.2.0 (Jumper et al., 2021). (c–e) Ribbon representation of the assembled SlaA and SlaB …
Figure 5—figure supplement 1
Alphafold v2.2.0 predictions of SlaB monomer and trimer.
(a, b) Alphafold v2.2.0 predictions of SlaB monomer and trimer, respectively. The ribbon is coloured by pLDDT (per-residue confidence score) where red indicates low, and blue high confidence. The …
Figure 5—figure supplement 2
Structural prediction of S. acidocaldarius SlaB.
(a) Structure of SlaB as predicted by Alphafold v2.2.0 (ribbon representation, cyan–grey–maroon from N-terminus to C-terminus). Amino acids from 1 to 24 (blue) are predicted as signal peptide by …
Figure 5—figure supplement 3
Subtomogram average of the exosome-bound S-layer and SlaB fitting.
(a–d) SlaA hexamer (cornflower blue) and SlaB trimer (magenta) fitting into the cryoEM map. (d) Cross-section through the boxed region in (c), showing the interaction between SlaA and SlaB. Scale …
Figure 5—figure supplement 4
Structure of archaeal and bacterial S-layer proteins.
(a–c) Atomic models are shown in ribbon representation in cyan–grey–maroon from the N-terminus to the C-terminus. S. acidocaldarius SlaA (domains D5 and D6 as predicted by Alphafold v 2.2.0) and …
Figure 5—figure supplement 5
Stability and charge heatmaps for S. acidocaldarius SlaA30–1069, SlaA, and SlaB.
(a, c, e) Calculated folded state stability heatmaps for SlaA30–1069 (a), SlaA (c), and SlaB (e), respectively. SlaA30–1069, SlaA, and SlaB predicted to be stable across pH 2–8. (b, d, f) calculated …
Figure 5—figure supplement 6
Stability and charge heatmaps for C.crescentus and H. volcanii S-layer proteins.
(a, c, e) calculated folded state stability heatmaps for C. crescentus S-layer protein RsaA N-terminus (a) and C-terminus (c) domains, and H. volcanii S-layer protein csg (e). The RsaA N-terminus …
Videos
Video 1
Atomic structure and glycosylation of SlaA30–1069. The SlaA30–1069 cryo electron microscopy (cryoEM) map is shown in cornflower blue.
The atomic structure is shown in ribbon representation and coloured in cyan–grey–maroon. N-terminus, cyan; C-terminus, maroon. The glycosylated Asn residues are in orange and the glycans are …
Video 2
Flexibility of SlaA.
Sequence of 2D classifications of negatively stained SlaA obtained in Relion 3. D2-4 were aligned, showing the flexibility of D1, D5, and D6.
Video 3
Comparison of SlaA30–1069 structure at pH 4 and 10. Root-mean-square deviation (r.m.s.d.) alignment between SlaA30–1069 atomic models at pH 4 and 10.
Smaller deviations are shown in blue and larger deviations in red, with mean r.m.s.d. = 0.79 Å, as in Figure 3b.
Video 4
Model of the assembled S. acidocaldarius S-layer.
Additional files
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Supplementary file 1
CryoEM statistics.
(a) Statistics of data collection, 3D reconstruction, and validation. (b) Mapping of glycan residues from the structure file to residues of the GLYCAM force field or the newly charge-derived SG0 and SG4 residues, representing the 1-substituted and 1,4-substituted SMA. (c) RESP charges derived for residue SG0 on the HF/6-31G*//HF/6-31G* level of theory (see Methods for details). (d) RESP charges derived for residue SG4 on the HF/6-31G*//HF/6-31G* level of theory (see Methods for details).
- https://cdn.elifesciences.org/articles/84617/elife-84617-supp1-v2.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/84617/elife-84617-mdarchecklist1-v2.docx
