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.
Fig 2.
Final processing pipeline of cryo-EM dataset EMPIAR-10930.
(A) Final processing steps were performed in CryoSPARC [18] v4.5 or RELION [22,23] v5. Post-processing was performed in RELION or using deepEMhancer [24]. (B and C) Conical FSC summary plots corresponding to the final maps of the 2:1 (B) and 2:2 (C) ALK:ALKAL2 complexes, generated via “Orientation Diagnostics” in cryoSPARC v4.5. The resolution at FSC = 0.143 is annotated via dotted blue lines. (D) Azimuth plot showing the distribution of orientations over Azimuth (x-axis) and Elevation (y-axis) angles for the particle set corresponding to the final map of the 2:1 ALK-ALKAL2 complex, obtained after Orientation Rebalancing in cryoSPARC v4.5 and 3D refinement with Blush regularization [23] in RELION v5. (E) Display of the 20 most populated classes after 2D classification of the final particle stack corresponding to the 2:1 ALK-ALKAL2 complex. New side and top views not observed in initial 2D classification runs are shown as blue squares.
Fig 3.
Comparison of map (an)isotropy following data processing strategies with or without Orientation Rebalancing and/or 3D refinement with enabled Blush Regularization.
(A) Processing workflows (1–5) of cryo-EM dataset EMPIAR-10930 starting from 876,806 selected particles as outlined in Methods. Workflow 1 does not include Orientation Rebalancing (CryoSPARC v4.5) nor 3D refinement with Blush regularization (RELION v5). Workflow 2 is the same as 1 but with a final 3D refinement with Blush regularization in RELION v5 instead of NU-refinement in CryoSPARC v4.5. Workflow 3 utilizes Orientation Rebalancing in CryoSPARC v4.5 followed by NU-refinement in CryoSPARC. Workflow 4 includes Orientation Rebalancing in CryoSPARC v4.5 followed by regular 3D refinement in RELION (without Blush regularization), while workflow 5 combines Orientation Rebalancing in CryoSPARC with 3D refinement with enabled Blush regularization in RELION. (B) Conical FSC summary plots for the final 3D refinements obtained in processing workflows 1, 2, 3, 4, and 5, generated via “Orientation Diagnostics” in cryoSPARC v4.5. The resolution at FSC = 0.143 is annotated in blue.