Subtraction of liposome signals in cryo-EM structural determination of protein-liposome complexes

Reconstituting membrane proteins in liposomes and determining their structure is a common method for determining membrane protein structures using single-particle cryo-electron microscopy(cryo-EM).However,the strong signal of liposomes under cryo-EM imaging conditions often interferes with the structural determination of the embedded membrane proteins.Here,we propose a liposome signal subtraction method based on single-particle two-dimensional(2D)classification average images,aimed at enhancing the reconstruction resolution of membrane proteins.We analyzed the signal distribution characteristics of liposomes and proteins within the 2D classification average images of protein–liposome complexes in the frequency domain.Based on this analysis,we designed a method to subtract the liposome signals from the original particle images.After the subtraction,the accuracy of single-particle three-dimensional(3D)alignment was improved,enhancing the resolution of the final 3D reconstruction.We demonstrated this method using a PIEZO1-proteoliposome dataset by improving the resolution of the PIEZO1 protein.

CAMSAP2 and CAMSAP3 localize at microtubule intersections to regulate the spatial distribution of microtubules

Microtubule networks support many cellular processes and exhibit a highly ordered architecture.However,due to the limited axial resolution of conventional light microscopy,the structural features of these networks cannot be resolved in three-dimensional(3D)space.Here,we used customized ultra-high-resolution interferometric single-molecule localization microscopy to characterize the microtubule networks in Caco2 cells.We found that the calmodulin-regulated spectrin-associated proteins(CAMSAPs)localize at a portion of microtubule intersections.Further investigation showed that depletion of CAMSAP2 and CAMSAP3 leads to the narrowing of the inter-microtubule distance.Mechanistically,CAMSAPs recognize microtubule defects,which often occur near microtubule intersections,and then recruit katanin to remove the damaged microtubules.Therefore,the CAMSAP–katanin complex is a regulatory module for the distance between microtubules.Taken together,our results characterize the architecture of cellular microtubule networks in high resolution and provide molecular insights into how the 3D structure of microtubule networks is controlled.