As a result of bacterial infection with viruses, bacteria have developed CRISPR-Cas as an adaptive immune system, which allows them to destroy the viral genetic material introduced via infection. However, viruses have also evolved to develop multiple anti-CRISPR proteins, which are capable of inactivating the CRISPR-Cas adaptive immune system to combat bacteria. In this study, we aimed to elucidate the molecular mechanisms associated with anti-CRISPR proteins by determining a high-resolution crystal structure (1.3 angstrom) of Type I-E anti-CRISPR protein called AcrIE2. Our structural analysis revealed that AcrIE2 was composed of unique folds comprising five antiparallel beta-sheets (beta 1 similar to beta 5) surrounding one alpha-helix (alpha(1)) in the order, beta(2)beta(1)alpha(1)beta(5)beta(4)beta(3). Structural comparison of AcrIE2 with a structural homolog called AcrIF9 showed that AcrIE2 contained a long and flexible beta 4-beta 5 connecting loop and a distinct surface feature. These results indicated that the inhibitory mechanism of AcrIE2 might be different from that of AcrIF9. This unique structure of AcrIE2 indicates its special mode of CRISPR-Cas inhibitory activity. Therefore, this study helps us understand the diversity in the inhibitory mechanisms of Acr family. (C) 2020 Elsevier Inc. All rights reserved.