Hello,

well, if the grids had warmed up for some time before the next liquid nitrogen 
refill then there would be no good amorphous ice anymore, hence loss of image 
contrast and no data collection possible.
We have had grids for > 10 years in liquid nitrogen, and recently collected 
excellent data on them.

Considering that it’s a cytochrome, could the protein maybe be light-sensitive? 
Were the grids looked at by fluorescence microscopy (at the time, or now to 
cross-check)?

Best,

Bruno



From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> On Behalf Of Jon Cooper
Sent: vendredi 23 mai 2025 16:59
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] BRIL as a Chaperone


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<mailto: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



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