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. 2020 Aug 7;9:e58464. doi: 10.7554/eLife.58464

Figure 1. Design scheme and cryo-EM analysis of hFzd5.

(a) Cartoon representation of the strategy for hFzd5 particle decoration by anti-BRIL Fab and anti-Fab Nb. These chaperones double hFzd5 molecular weight and render it asymmetric in detergent micelle, thus working as a fiducial maker for image alignment. (b) A selected 2D class average showing the side view of monomeric hFzd5ICL3BRIL/Fab/Nb. (c) Overall EM volume around the structural model of hFzd5 (cyan), BRIL and the linker between BRIL and Fzd5 (light blue), Fab heavy chain (pink), Fab light chain (light pink) and Nb (wine). (d) The size-exclusion chromatography profile, SDS-PAGE of peak fractions, and (e) the selected 2D class average of XWnt8/hFzd5FL. The full SDS-PAGE and 2D classes are shown in Figure 1—figure supplement 5. Models of XWnt8/mouse Fzd8CRD (mFzd8CRD) (PDB ID: 4F0A) and hFzd5 are overlaid on the blobs to show the relative size of densities.

Figure 1.

Figure 1—figure supplement 1. Multiple sequence alignment of human class F GPCRs.

Figure 1—figure supplement 1.

Sequence alignment of CRD and transmembrane regions of human class F GPCRs. The CRD region and individual transmembrane helix is labelled above the amino acid sequence. The alignments for CRD-hinge domain was generated using MAFFT (https://www.ebi.ac.uk/Tools/msa/mafft), and for TM1-H8 was taken from GPCR data base (www.gpcrdb.org). The alignment result was displayed using MView (www.ebi.ac.uk/Tools/msa/mview).
Figure 1—figure supplement 2. Purification, data collection and 2D classification of hFzd5ICL3BRIL/Fab/Nb.

Figure 1—figure supplement 2.

(a) Representative size-exclusion chromatography profile of the hFzd5ICL3BRIL/Fab/Nb complex with the Coomassie stained SDS-PAGE around the peak fractions. SDS samples were prepared in reducing condition. The fractions used for EM are indicated by the red bar. (b) Representative raw micrograph of the hFzd5ICL3BRIL/Fab/Nb complex in detergent. (c) 2D class averages of selected particles. The dimer classes are marked by red boxes.
Figure 1—figure supplement 3. Cryo-EM data analysis of hFzd5ICL3BRIL/Fab/Nb.

Figure 1—figure supplement 3.

(a) Cryo-EM data processing scheme. The resulting maps from the final heterogeneous refinement are superimposed for comparison (cyan and brick). (b) Orientation distribution of particles for the final 3D reconstruction. (c) The gold standard FSC curve for the final map. (d) Local resolution estimation of the final map colored in rainbow.
Figure 1—figure supplement 4. Cryo-EM map of representative built in model.

Figure 1—figure supplement 4.

(a) Cryo-EM map (gray) and structural model of hFzd5ICL3BRIL in detergent complexed with anti-BRIL Fab and anti-Fab Nb. The model is colored as follow: the sequences from Fzd5 (cyan), BRIL and the linker between BRIL and Fzd5 (light blue), Fab heavy chain (pink), Fab light chain (light pink) and Nb (brown). (b) The map-model FCS curve generated by Phenix refinement (masked). Cryo-EM volumes (pale cyan) around the (c) extracellular regions and (d) individual transmembrane helices of hFzd5. Except for the whole extracellular region (c right) which only shows disulfide cysteine resides with red colored secondary structures assigned by DSSP, all sidechains are displayed regardless of their quality or model placement.
Figure 1—figure supplement 5. Purification, data collection and 2D class averages of the XWnt8/hFzd5FL complex.

Figure 1—figure supplement 5.

(a) A size-exclusion chromatography profile of the XWnt8/hFzd5FL complex with the Coomassie stained SDS-PAGE. SDS samples were prepared in reducing condition. The peak fractions for the EM analysis are indicated by the red bar. (b) Representative raw micrograph of the XWnt8/hFzd5 complex. (c) 2D class averages of selected particles.