Hello, I have never done EM, sorry, so it would help me to know how grids are stored on that sort of timescale. Presumably under liquid nitrogen. Is there the possibility of warming to temperatures where a protease or microbial impurity could become active e.g. if topping up the liquid nitrogen relied on human endeavour.
Best wishes, Jon Cooper. jon.b.coo...@protonmail.com Sent from Proton Mail Android -------- Original Message -------- On 23/05/2025 11:47, Firdous Tarique wrote: > Hi everyone > > I am encountering an issue during data processing of a membrane protein > construct fused to BRIL (apocytochrome b562RIL) at the N-terminus. Cryo-EM > grids were prepared from the same batch of purified protein, but data > collection was performed at two different time points: one immediately after > grid preparation, and the other after three years of storage. > > In the initial dataset, BRIL is clearly visible in the 2D class averages and > contributes well to the 3D reconstruction. However, in the dataset collected > three years later from the same grid batch, no BRIL density is observed in > either the 2D or 3D reconstructions. > > Has anyone observed similar behavior with BRIL fusion constructs, > particularly the loss of BRIL density over time or during extended sample > handling? This is my first experience working with BRIL, and I am curious > whether this could be due to structural degradation, autocleavage, or > conformational flexibility of the fusion over time. > > If anyone is aware of published literature or reports discussing the > instability or potential autocleavage of BRIL in similar contexts, I would > greatly appreciate references or insights. > > Best wishes > > Firdous > > --------------------------------------------------------------- > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
