[3dem] some problems of crosslinking complex for cryoEM

[赵河豫]

 Hi，everyone
We encountered difficulties in crosslinking while attempting to stabilize our protein for cryoEM samples. Post cross-linking, the particles appeared more homogeneous in micrographs compared to the native state. However, the quality of the 2D classification deteriorated, resulting in blurred details and reduced clarity. This cases happened when using cross-linking using BS3 and glutaraldehyde as well as Grafix. Nevertheless, these observations are primarily evident in proteins with a molecular weight below 300 kDa. Conversely, for proteins exceeding 400 kDa, crosslinking has been shown to enhance their quality, as confirmed through cryoEM analysis.The comparison between raw images and 2D classifications is shown in the attachment.We find ourselves perplexed by this disparity.Is there any way to explain the phenomenon of having good particle results but poor 2D classifications? Additionally, do you have any valuable suggestions to address these issues?
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