Update on crystal hits, XChem and more purification

Things have been really busy in the lab the last few weeks. I’ve done a couple of purifications of ACVR1 (ALK2), which I’ll post in Zenodo (getting loads of protein now), but probably won’t write up the details of every purification every single time anymore, as it’s the same process every time for this construct. Read More …

Some structure pictures for your viewing pleasure

So I reported here a couple of weeks ago that I’d obtained datasets for ACVR1 bound to M4K2117 and M4K2121, at 1.25 and 2 Å resolution, respectively, and I put up a link to the data. I’m now refining the data and got some pretty good structures so far. These are what the current stats look Read More …

Some good news! I have data! Not just any data – 1.25 Å data!

So, the week has been super busy, but all for good reasons this time!! I’m absolutely thrilled to say that amongst all the salt crystals I found in my crystal plates, I also found the crystals below, one of which diffracted to 1.25 Å. This is definitely the highest resolution I’ve ever worked with. It’s amazing! Read More …

More protein purification and some fine screens

As mentioned in my last post, I got 26 mg of ACVR1 on my last purification (as shown in my Zenodo log here). This is fabulous as it has allowed me to follow up a number of conditions for crystallisation. In particular, one of these hits was in G10 of my composite screen. I’ve therefore Read More …

Lots of protein now! A massive composite screen and a few crystal hits

Now that our workhorse ACVR1 construct is expressing again, I’m getting loads of protein – 26 mg on the last 3 L scale up! Because of this, I’ve been really busy in the lab trying to get things back up to speed again. While waiting for the ACVR1 to work, I purified some TGFBR1 (ALK5) Read More …

Virus titration and test expression of ACVR1 – some good news!

As I’ve mentioned in recent posts, our workhorse ACVR1 constructs have, for mysterious reasons, stopped expressing. This is clearly a massive problem, as with no protein we can not progress the project, and in the meantime, another DIPG patient has died, Aubreigh Nicholas, the 11-year-old girl who started the lemon face challenge. This is tragic, Read More …

Short form TGFBR1 (ALK5) purification and co-crystallisation with M4K2006 and M4K2089

In the absence of our “workhorse” protein, ACVR1 residues 208-499 (constitutively active Q207D mutant) (as we are still trying to work out why it won’t express at the moment), and the complete lack of crystals/diffracting crystals for the TGFBR1/FKBP12 complex, I have shifted to some other constructs we have to keep things moving. I had Read More …

Still no protein, and no diffraction from TGFBR1/FKBP12 crystals, but outreach is fun!

I’m sad to report that a couple of test expressions later, we’re still no wiser as to why ACVR1 isn’t expressing. It’s worked on a couple of small scale expression test plates, but just doesn’t want to scale up anymore. We may be looking at having to remake the virus. In the meantime, I had Read More …

Things still not going according to plan …

I ended last time saying Shubhash had saved the day by finding a better virus stock and growing up some more cells for me. Sadly, this did not work – still no expression. So we tried halving the amount of virus used, as using too much can infect too many cells leaving not enough making Read More …

A bad day in the lab – science never goes as expected!

I promised to post again today with an update on in-house screening of my hit for fragment screening, but sadly, there’s nothing to update, as yesterday turned into a litany of disappointment (not to be too much of a drama queen!). First, I started a fresh purification of ACVR1 to prepare for setting up new Read More …