Overcoming cryo-EM map anisotropy reveals ALK-cytokine assemblies with distinct stoichiometries

Activation of Anaplastic lymphoma kinase (ALK) and leukocyte tyrosine kinase (LTK) by their cognate cytokines ALKAL2 and ALKAL1 play important roles in development, metabolism, and cancer. Recent structural studies revealed ALK/LTK-cytokine assemblies with distinct stoichiometries. Structures of ALK-ALKAL2 and LTK-ALKAL1 complexes with 2:1 stoichiometry determined by X-ray crystallography contrasted 2:2 ALK-ALKAL2 complexes determined by electron cryo-microscopy (cryo-EM) and X-ray crystallography. Here, we show based on a reanalysis of the cryo-EM data deposited in EMPIAR-10930 that over half of the particles in the dataset correspond to ALK-ALKAL2 complexes obeying a 2:1 stoichiometry besides the originally reported structure displaying 2:2 stoichiometry. Unlike particles representing the 2:2 ALK-ALKAL2 complex, particles for the 2:1 ALK-ALKAL2 complex suffer severely from preferred orientations resulting in cryo-EM maps which display strong anisotropy. Here, we show that extensive particle orientation rebalancing in cryoSPARC followed by 3D model refinement with Blush regularization in RELION constitutes an effective strategy for avoiding map artefacts relating to preferred particle orientations and report a 3D reconstruction of the 2:1 ALK-ALKAL2 complex to 3.2 Å resolution. This new cryo-EM structure together with the crystal structures of ALK-ALKAL2 and LTK-ALKAL1 complexes with 2:1 stoichiometry, reconcile a common receptor dimerization mode for ALK and LTK poised for signalling.