Hi BRIL is attached to the C-terminal not in the N-terminal.
Best On Fri, May 23, 2025 at 11:46 AM Firdous Tarique <[email protected]> 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 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/
