Finally, we also performed a chemical cross-linking analysis of the archaeal RtcB protein with its RNA substrate. We observed specific protein-RNA X-links, confirming that the mode of substrate binding is very similar to the AlphaFold3 predictions and observed for the DNA mimic.

We also observed that the tRNA ligase complex with truncated DDX1 helicase showed slightly higher activity. These results suggest that the mobile DDX1 subunit has a negative effect on the ligase activity.

AlphaFold 3 predictions suggest that the tRNA interacts only with a small region of the RtcB protein, which would make it quite mobile. Interestingly, the same region is also predicted to be involved in DDX1 binding.

Despite numerous attempts, we have not been able to obtain a structure with a tRNA substrate. We also did not observe any additional density for the – presumably highly mobile – DDX1 catalytic domains.

To improve the stability and overall behavior of the sample, we reconstituted the complex with truncated variants of the ASHWIN and DDX1 subunits. We verified the functionality of such truncated complex by testing its stability, substrate binding and enzymatic activity in vitro.

I’m happy to share our recent manuscript in which we are structurally and biochemically investigating 3'-5' tRNA splicing ligases, including a #cryoEM structure of the five-subunit D. rerio tRNA ligase complex.
www.jbc.org/article/S002...

Fascinating things happening at the IIMCB nowadays! This is one of the first patterns milled with our new TFS Hydra Bio Plasma-FIB, and - most likely - the smallest IIMCB logo ever!

Come work with us!Let me post this on behalf of IIMCB Warsaw:
📢 Curious about viral RdRPs and their potential inhibitors? Let’s revolutionize virus research together!
🔬Apply for a Postdoc Researcher position in the Laboratory of Protein Structure
📅Deadline: Feb 15th, 2025
🔗 tinyurl.com/PostdocOffer

And what kind of gift did you get for Santa this year? :) #cryoEM #IIMCB

So unboxing a modern cryo-electron microscope is actually packing it into a box...

I did this and it helped...