Structural basis for distinct operational modes and protease activation in AAA+ protease Lon.

Initial rigid body docking was done using UCSF Chimera

Solutions were made fresh from concentrated and filtered using a 0.1 um syringe filter to avoid microbial contamination. Buffers were stored on ice and used within 15 minutes of mixing in order to avoid excess ATP hydrolysis.

Complexes consisting of homohexameric Lon protease from Yersinia pestis were isolated using size-exclusion chromatography

Images were collected in counting mode at 5 frames per second

Coma-free alignment procedure from Herzik & Wu, Nature Methods (2017). Preliminary grid screening was performed manually prior to data collection.

Particles were selected from a subset of micrographs using BoxNet automated particle picker

Leginon software was used for automated data collection

Coot was used for de novo atomic model building

cryoSPARC was used to assign initial angles

cryoSPARC was used to assign final euler angles

cryoSPARC was used to perform final classification

cryoSPARC was used to perform final reconstruction

Phenix 1.11.1 was used to perform real space refinement using the atomic model and experimentally-derived EM map

4 uL of sample was applied per grid and manually blotted for 4 seconds followed by immediately plunge-freezing in liquid ethane cooled by liquid nitrogen.

ALKRKIEAAKMPKDAREKTEAELQKLKMMSPMSAEATVVRGYIDWMLQVPWNSRSKVKKDLVKAQEVLDTDHYGLERVKD
RILEYLAVQSRVSKIKGPILCLVGPPGVGKTSLGQSIAKATGRQYVRMALGGVRDEAEIRGHRRTYIGSMPGKLIQKMAK
VGVKNPLFLLDEIDKMASDMRGDPASALLEVLDPEQNVAFNDHYLEVDYDLSDVMFVATSNSMNIPAPLLDRMEVIRLSG
YTEDEKLNIAKQHLLPKQFERNAIKKGELTIDDSAIMSIIRYYTREAGVRSLEREISKLCRKAVKNLLMDKTVKHIEING
DNLKDFLGVQKVDYGRADTENRVGQVTGLAWTEVGGDLLTIETACVPGKGKLTYTGSLGEVMQESIQAALTVVRARADKL
GINPDFYEKRDIHVHVPEGATPKDGPSAGIAMCTALVSCLTGNPVRADVAMTGEITLRGLVLPIGGLKEKLLAAHRGGIK
VVLIPDDNKRDLEEIPDNVIADLEIHPVKRIDDVLAIALEHPA

National Institutes of Health/National Institute of Biomedical Imaging and Bioengineering (NIH/NIBIB)

National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)

National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)

National Institutes of Health/National Institute on Aging (NIH/NIA)

National Institutes of Health/Office of the Director

6ON2 contains the same protein with a E424Q mutation in complex with a substrate, Y2853

AAA+ ATPase, Quality Control, Protease, HYDROLASE