Sampling the conformational space of the catalytic subunit of human γ-secretase

Abstract

17Human γ--secretase is an intra--membrane protease that cleaves many different 18 substrates. Aberrant cleavage of Notch is implicated in cancer, while abnormalities in 19 cutting amyloid precursor protein lead to Alzheimer's disease. Our previous cryo--EM 20 structure of γ--secretase revealed considerable disorder in its catalytic subunit 21 presenilin. Here, we introduce an image classification procedure that characterizes 22 molecular plasticity at the secondary structure level, and apply this method to identify 23 three distinct conformations in our previous sample. In one of these conformations, an 24 additional transmembrane helix is visible that cannot be attributed to the known 25 components of γ--secretase. In addition, we present a γ--secretase structure in complex 26 with the dipeptidic inhibitor N--[N--(3,5--difluorophenacetyl)--L--alanyl]--S--phenylglycine t--27 butyl ester (DAPT). Our results reveal how conformational mobility in the second and 28 sixth transmembrane helices of presenilin is greatly reduced upon binding of DAPT or 29 the additional helix, and form the basis for a new model of how substrate enters the 30 transmembrane domain.

Structures of the calcium-activated, non-selective cation channel TRPM4

“…The C-terminal part of the coiled coil domain showed poor density, indicating a local structural heterogeneity. Therefore, we performed a focused 3D classification with density subtraction in order to improve the density of the coiled coil domain 50 . In this approach, only the density corresponding to the coiled coil domain was kept in each particle image by subtracting the density for all other parts including the nanodisc from the original particles.…”

Structure of the Full-Length TRPV2 Channel by Cryo-EM

“…Masked 3D classification procedure 56 was performed using RELION 1.4 to improve the densities in the more flexible regions of our full-length TRPV2 structure. A map of the TM region with the MNG detergent belt was generated using the TRPV2 refined model of the TRPV2 cytoplasmic domains and the split map feature in UCSF Chimera.…”

Subunit conformational variation within individual GroEL oligomers resolved by Cryo-EM

“…The focused classification and refinement approaches (7,8) have been used for two primary purposes: sorting out localized structural heterogeneity among particles (9,10) and improving feature resolvability of flexible domains through local refinement (11,12). These molecular machines frequently are composed of multiple protein subunits that can generate correlated and/or uncorrelated motions (13), and no experimental technique has yet captured the atomic structures of individual subunits within a single molecular machine in solution.…”

“…However, these studies have not revealed any heterogeneity among the protomers in each oligomer. Here we used a 3D classification strategy for cryo-EM data (10) to investigate structural heterogeneity among chemically identical subunits and their conformational variation and correlation in a single GroEL.…”