Thank you Paula!

Thanks Jamie!

Thanks Kate!

And of course the biggest amount of thanks to Douglas who did the majority of this work. Moving lab, COVID, and shifting focus from a CRISPR based project was not easy but Douglas overcame these challenges to successfully do something we have wanted to do for a long time.

Big thanks to the University of York, with their Glacios which we used for screening and another big thanks to eBIC which was used to do the larger data collections that produced our reported structures. Without these facilities our cryo-EM studies would have been far more difficult.

There is still much to do in understanding human MTR in terms of its flexibility, and ability form complexes to support its function. Understanding these would give us a deeper understanding on how things go wrong in associated inherited disorders and guide better treatments.

Coupled with recently reported thermophilic structures of the MTR homologue MeTH from the Ando and Koutmos labs we have attempted to contextualise these conformations within cobalamin loading, reactivation, and catalytic cycles.

Using recombinant material, we could confirm that MTR does form a complex with MTRR and that complex formation can still occur (albeit weakly) without the FMN domain present.

We next turned to AlphaFold to understand how MTR interacts with its binding partners. AlphaFold of MTR with its rescue partner MTRR threw a surprise our way: AF3 suggested two regions of interaction with one newly suggested that deviated from the previously known interaction with MTRR’s FMN domain.

Douglas also collected cryo-EM data of MTR with hydroxycobalamin which does not support activity and methylcobalamin which is the active form of the cofactor. Though MTR again behaves as two separate bodies, we were able to observe conformational changes due to cobalamin binding.

These structures demonstrate that MTR without cofactor acts as two separate modules with the C-half organised as what is called the reactivation state. A state productive for the binding of Cbl and its reactivation aided by MTRR.

Due to its flexibility MTR is a tricky protein to study by classical structural techniques however cryo-EM is well suited for this type of sample. Douglas could successfully purify full-length MTR which allowed him to determine two separate classes of MTR without cbl.

MTR also operates with other assisting proteins such as MMADHC which loads cobalamin into MTR and methionine synthase reductase (MTRR) also rescues MTR from oxidative inactivation of the cbl cofactor by supporting reductive methylation.

MTR is the link between the folate and methionine cycles. It produces methionine while recycling folate using methylcobalamin/vitamin B12 (cbl) as a cofactor. As substrates bind to different active sites MTR requires a high degree of flexibility to function.

It's nice! If you haven't already seen this the CryoSPARC people have it as a case study too: guide.cryosparc.com/processing-d...

Using Leishmania as a model, we determined the cryo-EM structure of the doublet microtubule to pinpoint the position of each dynein. This gave us a detailed map of where every “rower” sits on the ciliary “boat.”

I wonder if there is a global refinement available of the enolase in a membrane and why that wasn't deposited. It is actually that recent Sept 2025 paper and the authors have said it is an oversight that the EMDB code wasn't included: pubpeer.com/publications...

... a sharp image on a piece of film.

What it revealed was this: fuzzy edges with four distinct "spots" in an X pattern in the interior.

This was compared to the chemical structures worked out for the nucleotides themselves, suggesting a stacked pattern of interacting bases.

None of the associated local refinements and the global refinement are available in the EMDB. Where did the micelle come from? How was EMD-47001 reconstructed? #CryoEM