Edited by Wolfgang Baumeister, Max Planck Institute of Biochemistry, Martinsried, Germany, and approved July 20, 2016 (received for review May 16, 2016)
Cryo-EM and 3D reconstruction of Iho670 filaments. (A) Images of frozen hydrated Iho670 filaments. The arrow points to a bulbous structure sometimes seen at one end of the filaments, likely the basal body (60). The yellow line is an arc with a radius of curvature of 1,500 Å. (Scale bar: 500 Å.) (B) A cutaway view of the 3D reconstruction, with a ribbon representation of the atomic model shown in a different color for each subunit. The core of the filament is entirely α-helical, with each subunit contributing a single highly hydrophobic helix. (Scale bar: 50 Å.) (C) A single subunit from the model. The gray density shown is the region where we were unable to build a unique model, accounting for residues 135–195.
Secondary structure is clearly resolved. (A) A β-sheet with two bulky aromatic residues. The presence of such large side chains allows for an unambiguous threading of the sequence through the density map. (B) A portion of the N-terminal α-helix, again showing two clearly resolved bulky aromatics.
The resolution of the reconstruction has been estimated using FSC comparisons (A) between the map and the refined atomic model and (B) between two half-maps generated from independent nonoverlapping datasets. (A) The blue curve is from the comparison between the model and the full map (including the region containing 61 residues where no model has been built), whereas the red curve is from masking off both the model and the map, so that only the α-helical core is being compared. The vertical line suggests that the overall reconstruction is somewhat worse than 4.0 Å, whereas the inner core is somewhat better than 4.0 Å. In B, no masking of the poorly ordered outer region has been done, and the overall resolution using an FSC = 0.143 criterion is 4.3 Å.
A comparison between the globular domains of Iho670 (magenta) and FlaF (cyan; PDB ID code 4P94). The hydrophobic N-terminal domain of FlaF was removed before crystallization and is, thus, not available for this comparison, whereas the Iho670 model was truncated at residue 40. The two views are related by a rotation of 80° about the vertical axis.
The predicted α-helices (light blue) and β-strands (light red) for a number of archaeal flagellin-like proteins as well as the observed α-helices (dark red) and β-strands (dark blue) for Iho670 (in the text) and Sulfolobus acidocaldarius FlaF (PDB ID code 4P94). The secondary structure predictions were made using the Phyre2 server (61). The N-terminal domain of the FlaF protein was removed for crystallization (33), so that only the predicted secondary structure is available in that region. The sequences shown, from top to bottom, are Iho670 from Ignicoccus hospitalis (iho670; Uniprot A8AAA0), FlaF (flaf_sa; Uniprot Q4J9K8) and FlaB (flab_sa; Uniprot Q4J9K5) from Sulfolobus acidocaldarius; FlaB from Sulfolobus solfataricus (flab_ss; Uniprot Q97WB2); FlaB from Thermoplasma acidophilum (flab_ta; Uniprot Q9HKP4); flgB2 from Haloferax mediterranei (fglb2_hm; Uniprot I3R3X2); flgB2 from Halobacterium salinarum (flgb2_hs; Uniprot B0R4J0); and Mhun_3140 from Methanospirillum hungatei (mhun; Uniprot Q2FUM4). The alignment numbering at the top is arbitrary and does not correspond to any particular sequence.
A tube of extra density projects out from Asn227 into a pocket formed by three subunits. This extra density is consistent with glycosylation, and such glycosylation has been confirmed by MS (Fig. S5).
MS of Iho670. (A) MALDI-TOF mass analysis showing that the actual mass is from 1 to 4 kDa higher than that predicted from the protein sequence. The single largest species (35,794 Da) has a mass that is ∼3.3 kDa greater than expected. (B) Digestion of the protein (yellow) shows the unmodified peptides that were identified. Cys104 (green) was determined to be modified with a carbamidomethyl group (57 Da). The region that we were unable to trace (residues 135–195) in building an atomic model is shown within a box. (C) Fragmentation analysis of several ions showed that mass loss involved the removal of hexose, establishing that glycosylation was present.
The helical net of Iho670 using the conventions that the surface is unrolled and that we are looking from the outside. There is a right-handed one-start helix that passes through every subunit, and subunits are labeled along this helix (e.g., n − 1, n, n + 1, etc.). The outer globular domains only make significant contacts along the right-handed seven-start helices and the left-handed three-start helices, whereas the inner N-terminal helices also make contacts along the one-start helix and a four-start helix.
Details of the subunit–subunit interfaces, with residues highlighted that were identified as hotspots by the alanine scanning. A close-up view of the interface with the n + 3 subunit (blue and green) is shown in Left. (Upper) The n/n + 7 interface (blue and orange) involves the C terminus. Arg236 interacts with two subunits (n + 7 and n + 3). The major interactions of the helical core with subunits n + 3 and n + 7 involve hydrophobic contacts (Val37 and Ile13) as well as Ser–Ser hydrogen bonds.
Potential deformations of the filament from the CONCOORD analysis. (A) The first eigenvector shows an expansion of a subunit along the right-handed seven-start helix along with a compression along the left-handed three-start helix (and vice versa). This motion induces a change of the length of the filament (Movie S1). (B) The second eigenvector describes an opening and closing of the wedge-shaped subunits, which affect the bending of the filament (Movie S2).
| Category | Result |
| Data collection | |
| No. of overlapping segments | 146,696 |
| No. of unique molecules | ∼300,000 |
| Pixel size (Å) | 1.08 |
| Defocus range (µm) | 0.5–3.5 |
| Voltage (kV) | 300 |
| Electron dose (e− Å−2) | 20 |
| Refinement | |
| Helical symmetry | 106.65°/5.45 Å |
| Resolution (Å) | 4.0 |
| Free shell (Å) | 4.3–4.0 |
| FSCavg (work)/FSCavg (free) | 0.81/0.45 |
| Cwork/Cfree | 0.81/0.43 |
| Rwork/Rfree | 31.8/48.5 |
| Deviations from idealized geometry | |
| Bond lengths (Å) | 0.0018 |
| Bond angles (°) | 0.47 |
| Model quality | |
| Molprobity score | 2.15 (100th percentile) |
| Clash score, all atoms | 7.38 (100th percentile) |
| Good rotamers (%) | 91.7 |
| EMRinger score | 3.42 |
| Ramachandran statistics | |
| Favored (%) | 96.2 |
| Outlier (%) | 1.7 |
| Right-handed seven start | Left-handed three start | |
| No. of residues in interface | 20 | 44 |
| Buried interface area (Å2) | 1,044 | 1,623 |
| Hydrophobic interface area (Å2) | 514 | 754 |
| Polar interface area (Å2) | 550 | 931 |
| Interface energy (Rosetta units) | −5.92 | −7.50 |
| Alanine scanning hotspot residues | Ser26 | Ile13 |
| Val37 | Gln84 | |
| Ser41 | His105 | |
| Asp235 | Asp107 | |
| Arg236 | Tyr215 | |
| Gln280 | Arg226 | |
| Ser301 | Arg236 | |
| Ile302 | Gln262 |
| Interface of helical core n with | ||||||||
| n − 7 | n − 4 | n − 3 | n − 1 | n + 1 | n + 3 | n + 4 | n + 7 | |
| No. of residues in interface | 39 | 15 | 29 | 10 | 10 | 21 | 14 | 11 |
| Buried interface area (Å2) | 1,187 | 553 | 1,054 | 352 | 373 | 692 | 568 | 399 |
| Hydrophobic interface area (Å2) | 946 | 508 | 837 | 271 | 294 | 611 | 559 | 392 |
| Polar interface area (Å2) | 241 | 44 | 217 | 81 | 78 | 81 | 9 | 7 |
| Interface energy (Rosetta units) | −4.91 | −5.91 | −2.79 | −2.38 | −1.91 | −4.04 | −5.35 | −1.24 |
The N-terminal helix was cut from one subunit, and the interface with all neighboring (complete) subunits was calculated.
