Yeast Tel1 and its highly conserved human orthologue ATM are large protein kinases central
to the maintenance of genome integrity. Mutations in ATM are found in ataxia-telangiectasia
(A-T) patients and ATM is one of the most frequently mutated genes in many cancers. Using
cryo electron microscopy, we present the structure of Tel1 in a nucleotide-bound state. Our
structure reveals molecular details of key residues surrounding the nucleotide binding site and
provides a structural and molecular basis for its intrinsically low basal activity. We show that
the catalytic residues are in a productive conformation for catalysis, but the PIKK-regulatory
domain-Insert (PRD-I) restricts peptide-substrate access and the N-lobe is in an open
conformation, thus explaining the requirement for Tel1 activation. Structural comparisons with
other PIKKs suggest a conserved and common allosteric activation mechanism. Our work also
provides a structural rationale for many mutations found in A-T and cancer.