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Very happy to share our study on the type III CRISPR CalpLTS cascade, which has been published today in @nature.…

What a great team effort between @Christophe. Rouillon, @Niels Schneberger, @Malcolm White and his student Haotian Chi, @Svergun Group, @Jonathan Schmid-Burgk lab, @Bela Bode lab and @isb_bonn. Here is the story behind it...
Met @Christophe. Rouillon in 2008 when we were both postdocs at @University of St Andrews with @Malcolm White and @James (Jim) Naismith.
Years later in 2019 we both ended up in Bonn and decided to work on the mysterious LonCARF (now CalpL) protein that @Christophe. Rouillon had spotted in this great paper by Shmakov et al.:…
Puzzled by the fact that the predicted transmembrane Lon protease was contained in the soluble fraction, we set up some xtal plates and quickly got some (ugly) xtals.
Enter my very good student @Niels Schneberger: He managed to optimize these xtals and solved the structure via SeMet (these were pre-alphafold times...)
@Christophe. Rouillon discovered the amazing webflags server, which helped us to figure out that the a small protein encoded upstream of CalpL might be the target of the protease.
@Niels Schneberger purified the protein and found that once activated by cA4, CalpL indeed cleaves the small protein that we named CalpT. He also found that the two proteins form a 1:1 complex.
Based on sequence homologies, we suspected CalpT to be a MazF-like RNase. This misled us for a long time, until a collaboration with @Jonathan Schmid-Burgk and his student Katja Blumenstock pulled us out of this rabbit hole. @Bela Bode and @Katrin_Ackermann helped us to escape another one.
Meanwhile, @NielsSchneberg together with @Jonas Möcking and Stefano Da Vela from @Svergun Group figured out, how the protease is activated by cA4 induced oligomerization.
Together with @Malcolm White and his fantastic student Haotian Chi, we finally figured out that the CalpT protein acts as an anti-sigma factor for the sigma-factor CalpS, that is encoded by the third conserved gene in this operon.
Very thankful to everyone involved, also to our three referees who gave us a lot of constructive feedback! Take-home message: #NITO: Never ignore the operon! (coined by @Malcolm White)
Gregor Hagelueken
Structural biologist. Likes proteins - most of the time.
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