But we looked and we see no effect. Combined with cool subcelluar bedaquilne imaging (pmc.ncbi.nlm.nih.gov/articles/PMC...) we conclude that there is insufficient bedaquiline in mitochondria to inhibit significantly. Either it doesn't get in or gets in and is pumped out

There a lot here but by comparing to know uncouplers and K+/H+ ionophores, we can see they cause very different redistributions of electrons in the chains compared to bedaquiline- allowing us to exclude uncoupling and ionophoric activity

We used decomposition to unmix the muddled mixture of signals into the main three types of cytochrome, allowing for insight into more subtle changes and straight-forward statistical analysis. Most of the signals come from the supercomplex, with an addition ‘b557’ signal from other complexes

Exposed to bedaquiline, we can clearly see electrons immediately accumulate in the electron transport chain in the model Mycobacterium smegmatis. This effect is greatly diminished in a resistant mutant. We also see it happen in Mycobacterium tuberculosis

Using a special spectroscopic set up that collects light backscattered (‘re-emitted’) from dense bacterial suspensions, we can measure these changes in living cells, allowing us to measure the status of the electron transport chain in intact, unperturbed, wild-type cells and in real-time

To answer this we applied a novel technique: remission spectroscopy. Heam groups absorb light strongly (think haemoglobin) and that absorbance changes with oxidation state. See the isolated supercomplex spectrum below

We explore 3 hypotheses for what might be happening based on the literature and bioenergetic principles. Bedaquiline either acts as one of two flavours of ion-carrier (H+ or K+) or inhibits like oligomycin but TB has adaptive systems to relieve backpressue/redirect flux, increasing O2 consumption

In a respiring organism, you have an electron transport chain that couples transfer of electrons from cytoplasmic metabolism to O2 with proton pumping out of the cell, building a proton-motive force (PMF). This PMF then powers ATP synthase and through that, the cell

Our Glacios has an energy filter on!

As ever, @cellarchlab.com and Manon provided stunning cutting edge Cryo-ET data to see what happens when shell4 is deleted

Our small contribution here were to just explore the in vitro behaviour of the Shell1 and 4 components

In another paper Sabina Musial helped Onyou Nam explore the recently discovered pyrenoid shell he discovered through extensive proteomics, working with out Adam the York facility. This includes spatial information

Excitingly, it works on (some) higher plants as well! In the future this might lead to it being used as a engineering tool

It binds weakly but forms many interactions so it was challenging to get it to (reasonably) high-res but we managed with symmetry expansion

Firstly, PhD student James Barrett found a new cross linker that phase-separates Rubisco into pyrenoids using a homebrew bioinformatics pipeline, which was a covid project

It’s great to finally share the cryo-EM work I’ve been carrying out with Luke Mackinder over the last few years- 2 papers on how Rubisco is condensed to form CO2 concentrating pyrenoids and how a shell is formed around them
www.nature.com/articles/s41...
www.sciencedirect.com/science/arti...

I was skeptical at first but I gave it a go and really liked the community and tone here. And then the other place had an active role is promoting race riots and that cemented by move over here