Reanalysis of cryo-EM data reveals ALK-cytokine assemblies with both 2:1 and 2:2 stoichiometries
Fig 1
Reanalysis of cryo-EM data from EMPIAR entry EMPIAR-10930 deposited by Reshetnyak and colleagues, Nature (2021) [
7]. (A) Ten most populated 2D class averages after performing 2D classification on 11.8 million selected particles. Classes corresponding to 2:1 ALK–ALKAL2 particles are highlighted in blue, while classes corresponding to 2:2 ALK–ALKAL2 particles are highlighted in orange. (B) Ab initio model generation in cryoSPARC [18] results in classes corresponding to both a 2:1 ALK–ALKAL2 and 2:2 ALK–ALKAL2 assembly. Hetero-refinement of the 2:1 and 2:2 ALK–ALKAL2 classes results in the two models displaying 2:1 and 2:2 ALK–ALKAL2 assemblies, respectively. The ALK–ALKAL2 complexes with 2:1 (PDB 7nwz) and 2:2 (PDB 7n00) stoichiometries are shown fitted inside the maps, with ALK colored pink/gray and ALKAL2 colored blue/purple. (C) 3D refinement with Blush regularization [19] in RELION v5 of a trimmed particle set after extensive automatic rebalancing of particle orientations as implemented in cryoSPARC 4.5 (see also Materials and methods and S1 Table). A front view is shown of a map sharpened in RELION using a B-factor of −100 Å2, with ALK colored pink/gray and ALKAL2 colored blue. Front, side, and top views are shown of a transparent map, sharpened using deepEMhancer, with fitted structural model. (D and E) Zoomed-in insets illustrating the quality of the cryo-EM map corresponding to the two major interaction interfaces (site 1 and 2) in the ALK:ALKAL2 complex. Key interacting residues are annotated and shown as sticks. (F) Local resolution estimation performed in RELION v5 of the final map for the 2:1 ALK-ALKAL 2 complex. (G) Fourier shell correlation (FSC) plot corresponding to the final map of the 2:1 ALK:ALKAL2 complex. The corrected FSC (purple lines) is calculated after performing correction by noise substitution [20], and the resolution at FSC = 0.143 is annotated via a dotted purple line. A map-to model FSC curve calculated using the fitted model and an unsharpened, unfiltered full map is shown (gray line) with the resolution at FSC = 0.5 annotated via a dotted gray line.