Reanalysis of cryo-EM data reveals ALK-cytokine assemblies with distinct stoichiometries

Anaplastic lymphoma kinase (ALK) and leukocyte tyrosine kinase (LTK) are receptor tyrosine kinases activated by their cognate ligands ALKAL2 and ALKAL1 resulting in pleiotropic signalling outputs in development, metabolism and cancer. Structural studies by X-ray crystallography and electron cryo-microscopy (cryo-EM) recently revealed distinct assemblies corresponding to ALK-ALKAL2 and LTK-ALKAL1 assemblies with either 2:1 stoichiometry versus ALK-ALKAL2 complexes with 2:2 stoichiometry. Here, we report the reanalysis of cryo-EM data deposited by Reshetnyak et al. (EMPIAR-10930) and show 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. Our workflow critically relied on extensive particle orientation rebalancing in cryoSPARC followed by 3D refinement with Blush regularization in RELION leading to a 3D reconstruction of the 2:1 ALK-ALKAL2 complex to 3.2 Å resolution. This cryo-EM structure together with the reported 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 the facile juxtapositioning of their intracellular kinase domains for signalling.