(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 …
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 …
(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 …
(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 …
(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 …
(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 …
(a) Top (left) and bottom (right) views, and (b) side views of hFzd5 colored by rainbow with blue on the N-terminus and red on the C-terminus of the structural model. (c) Comparison of the …
Cell-surface hFzd5 variants were fluorescently labeled, and frequency distribution of FACS data are presented for each variant (red) with a negative control (black).
Raw FACS histogram data for Figure 2—figure supplement 1 showing cell-surface expression of hFzd5 variants.
(a, b) Structural comparison of hFzd5, hFzd4 and hSmo (a) at the cytoplasmic end of TM5, and (b) the K/R6.32-W7.55 ionic lock with the aromatic network around H8. (c) Bottom views of hFzd5 and hFzd4 …
Schematic presentation of the ternary complex formation, and hFzd5-mediated signaling upon stimulation by (a) Wnt3a and (b) surrogate Wnt agonist. Non-lipidated, water-soluble surrogate Wnt would …
Raw data points for the signaling experiments shown as circles in Figure 4.
hFzd5ICL3BRIL/anti-BRIL Fab/anti-Fab Nb (EMD-21927) (PDB ID: 6WW2) | |
---|---|
Data collection and processing | |
Magnification | 81,000x |
Voltage (kV) | 300 |
Electron exposure (e/Å2) | 50 |
Defocus range (μm) | −0.8 to −2.0 |
Pixel size (Å) | 1.078 |
Symmetry imposed | C1 |
Initial particle images (no.) | 6,483,398 |
Final particle images (no.) | 369,704 |
Map resolution (Å) FSC threshold | 3.70 0.143 |
Map resolution range (Å) | 3.2 to 5.9 |
Refinement | |
Initial model used (PDB code) | 5L7D, 6BD4, 6ANI, 6CBV |
Model resolution (Å) FSC threshold | 3.7/4.1 0.143/0.5 |
Map sharpening B factor (Å2) | −124.4 |
Model composition Non-hydrogen atoms Protein residues | 7,594 982 |
B factors (Å2) Protein | 89.59 |
R.m.s. deviations Bond lengths (Å) Bond angles (°) | 0.010 1.083 |
Validation MolProbity score Clashscore Poor rotamers (%) | 1.87 5.73 0 |
Ramachandran plot Favored (%) Allowed (%) Disallowed (%) | 89.59 10.31 0.10 |
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Cell line (Spodoptera frugiperda) | Sf9 | ATCC | CTL-1711 (RRID:CVCL_0549) | Insect cells used for baculovirus production |
Cell line (Homo sapiens) | HEK293S GnTI- | Gift from Prof. H Gobind Khorana (PMID:12370423) | Mammalian cells used for baculovirus expression of hFzd5FL and hFzd5ICL3BRIL | |
Cell line (Homo sapiens) | HEK293T with Fzd1/2/4/5/7/8 KO | Gift from Prof. Michael Boutros (PMID:28733458) | For signaling assay | |
Cell line (Mus musculus) | Mouse monoclonal M1 hybridoma | Gift from Prof. Brian K Kobilka (PMID:17962520) | To purify anti-FLAG M1 (mouse IgG2a) antibody to prepare FLAG affinity column (15 mg/ml resin). This was used for purification of hFzd5FL and hFzd5ICL3BRIL. | |
Strain, strain background (Escherichia coli) | BL21(DE3) | New England Biolabs | C2527I | E. coli strain for expression of the nanobody |
Strain, strain background (Escherichia coli) | BL21 (gold) | Agilent Technologies | 230130 | E. coli strain for expression of the Fabs |
Transfected construct (Homo sapiens) | pRK5 hFzd5 wild-type | Yu et al., 2012 (PMID:23095888) | For signaling assay | |
Transfected construct (Homo sapiens) | pRK5 hFzd5 mutants | this study | For signaling assay | |
Recombinant DNA reagent | 7xTCF-Ffluc | Addgene | 24308 | For signaling assay |
Recombinant DNA reagent | BestBac Linearized Baculovirus DNA 2.0 | Expression Systems | 91–002 | For baculovirus production |
Peptide, recombinant protein | Wnt3a | R and D Systems | 5036-WN | For signaling assay |
Peptide, recombinant protein | Surrogate Wnt | Miao et al. Cell Stem Cell 2020 | For signaling assay | |
Peptide, recombinant protein | DRPB_Fz8 | Dang et al., 2019 (PMID:31086346) | For signaling assay | |
Commercial assay or kit | Luciferase assay kit | Promega | E152A | For signaling assay |
Chemical compound, drug | n-Dodecyl-β-D-Maltoside (DDM) | Anatrace | D310S | Membrane protein purification |
Chemical compound, drug | Lauryl Maltose Neopentyl Glycol (LMNG) | Anatrace | NG310 | Membrane protein purification |
Chemical compound, drug | Glyco-Diosgenin (GDN) | Anatrace | GDN101 | Membrane protein purification |
Chemical compound, drug | Cholesterol Hemisuccinate tris Salt (CHS) | Anatrace | CH210 | Membrane protein purification |
Chemical compound, drug | Digitonin | EMD Millipore | 300410 | Cryo-EM specimen freezing additive |
Software, algorithm | DigitalMicrograph | Gatan | Microscope alignment and cryo-EM data collection | |
Software, algorithm | serialEM | https://bio3d.colorado.edu/SerialEM/ (PMID:16182563) | version 3.6 | Cryo-EM data collection |
Software, algorithm | MotionCor2 | https://emcore.ucsf.edu/ucsf-motioncor2/ (PMID:28250466) | Motion correction of cryo-EM movies | |
Software, algorithm | cryoSPARC | https://cryosparc.com/ (PMID:28165473) | version 2.12.14 | Cryo-EM data processing |
Software, algorithm | Coot | https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/ (PMID:20383002) | Structure model building | |
Software, algorithm | Isolde | https://isolde.cimr.cam.ac.uk/ (PMID:29872003) | Structure model building/refinement | |
Software, algorithm | Phenix suite | https://www.phenix-online.org/ (PMID:20124702) | Structure refinement | |
Software, algorithm | UCSF Chimera | https://www.cgl.ucsf.edu/chimera/ (PMID:15264254) | Initial homology model docking | |
Software, algorithm | UCSF ChimeraX | https://www.rbvi.ucsf.edu/chimerax/ (PMID:28710774) | Structural visualization/figure preparation/Isolde execution | |
Software, algorithm | PyMol | Schrödinger | Structural visualization/figure preparation | |
Software, algorithm | GraphPad Prism 7 | GraphPad | analysis of signaling data | |
Other | CNBr-Activated Sepharose 4 Fast Flow | GE Healthcare | 17098101 | For preparation of anti-FLAG sepharose |
Other | Lipofectamine 2000 | Invitrogen | 11668030 | Transfection reagent for signaling assay |