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. 2022 Nov 8;11:e81977. doi: 10.7554/eLife.81977

Figure 1. AlphaFold2 structures of the IFT-A subunits are supported by experimentally-determined intramolecular cross-links.

(A) Sample preparation protocol for obtaining an enriched sample of endogenous IFT-A from T. thermophila. The sample preparation was followed by chemical cross-linking using DSSO and tandem (MS2/MS3) mass spectrometry to identify cross-linked peptides. Image created with BioRender.com. (B) Bar diagrams highlight the extensive intramolecular DSSO cross-links (purple arcs connecting cross-linked amino acid pairs) within each of the IFT-A subunits (bars, numbers denote amino acid positions). (C) Violin plots of the distance between Cɑ atoms of chemically cross-linked residues. Surrounding images show locations of intramolecular crosslinks (black bars) on aligned AlphaFold2 predicted models of IFT-A subunits from Chlamydomonas reinhardtii (green) and Tetrahymena thermophila (pink). A maximum distance of 35 Å between Cɑ atoms is expected for DSSO cross-links. 97% of intramolecular cross-links are satisfied for T. thermophila and 94% for C. reinhardtii.

Figure 1.

Figure 1—figure supplement 1. AlphaFold2 structural predictions for T. thermophila proteins of the IFT-A complex.

Figure 1—figure supplement 1.

Structures are colored based on pLDDT (Tunyasuvunakool et al., 2021) where red represents higher confidence predictions and blue lower confidence predictions.
Figure 1—figure supplement 2. Enrichment for monomeric IFT-A from Tetrahymena cilia.

Figure 1—figure supplement 2.

(A) FPLC size exclusion chromatography elution traces for molecular weight calibrants (lines, left axis), overlaid with mass-spectrometry determined IFT-A protein abundances (in protein spectral matches, PSMs; bars, right axis) measured from Tetrahymena whole cell extract. Using these data, we identified IFT-A fractions to be subsequently collected from ciliary M+M preparations for cross-linking. (B) Linear regression between chromatography elution volumes (Ve, peak elution volume in mL; V0, void volume from Blue Dextran) and molecular weight calibrants (known size calibrants, filled circles; protein complexes of known composition, open circles) provided a molecular weight estimate for the selected IFT-A fractions ranging between 720 kDa and 1.1 MDa, consistent with an IFT-A monomer, whose expected molecular weight is 772 kDa.
Figure 1—figure supplement 3. Tetrahymena IFT-A cross-links mapped onto AlphaFold-predicted structures of human IFT-A proteins.

Figure 1—figure supplement 3.

(A) Locations of intramolecular crosslinks (black bars) are visualized on structural alignments of AlphaFold2 predicted models of IFT-A subunits from Tetrahymena thermophila (pink), Chlamydomonas reinhardtii (green), and humans (blue). (B) Violin plots show the distance between Cɑ atoms of chemically cross-linked residues by DSSO. A maximum distance of 35 Å between Cɑ atoms is expected for DSSO cross-links. 86% of intramolecular cross-links are satisfied for the human structures.