biostudies-literatureJernigan RJNIGMS NIH HHS138-151.e5https://www.ebi.ac.uk/biostudies/studies/S-EPMC9830665biostudies-literatureUnknown31(2)NendoU from SARS-CoV-2 is responsible for the virus's ability to evade the innate immune system by cleaving the polyuridine leader sequence of antisense viral RNA. Here we report the room-temperature structure of NendoU, solved by serial femtosecond crystallography at an X-ray free-electron laser to 2.6 Å resolution. The room-temperature structure provides insight into the flexibility, dynamics, and other intrinsic properties of NendoU, with indications that the enzyme functions as an allosteric switch. Functional studies examining cleavage specificity in solution and in crystals support the uridine-purine cleavage preference, and we demonstrate that enzyme activity is fully maintained in crystal form. Optimizing the purification of NendoU and identifying suitable crystallization conditionStructure (London, England : 1993)Room-temperature structural studies of SARS-CoV-2 protein NendoU with an X-ray free-electron laser.PMC9830665R01 GM095583R01 GM094450R01 GM149864R01 GM117342Nagaratnam NBatyuk AKirian RAChen JJTolstikova AKaschner EKShelby MLBotha SSonker MYung MCHayes BLisova SSchmidt MYang JHGrant TDSheikh MZFrank MColeman MASierra RGRos AFromme RMariani VKupitz CZaare SFromme PMartin-Garcia JMHunter MSKetawala GNazari RRabbani MTBoutet SHansen DTDoppler DJernigan RJLogeswaran DZacks MAfalseRoom-temperature structural studies of SARS-CoV-2 protein NendoU with an X-ray free-electron laser.NendoU from SARS-CoV-2 is responsible for the virus's ability to evade the innate immune system by cleaving the polyuridine leader sequence of antisense viral RNA. Here we report the room-temperature structure of NendoU, solved by serial femtosecond crystallography at an X-ray free-electron laser to 2.6 Å resolution. The room-temperature structure provides insight into the flexibility, dynamics, and other intrinsic properties of NendoU, with indications that the enzyme functions as an allosteric switch. Functional studies examining cleavage specificity in solution and in crystals support the uridine-purine cleavage preference, and we demonstrate that enzyme activity is fully maintained in crystal form. Optimizing the purification of NendoU and identifying suitable crystallization condition2023-01-01T00:00:00Z2023 Feb2026-05-10T06:46:57.462Z2025-02-19T04:20:21.919ZS-EPMC98306653663096010.1016/j.str.2022.12.009