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 …

Various purifications, and a few crystal hits

Gosh, it’s been a while since I posted – not because I have nothing to report, but because I’ve been super busy in the lab trying to get things done, as well as having loads of meetings and a symposium. So, in the meantime, I’ve done two purifications of ACVR1 and set up crystal plates, Read More …

The power of hope and community action: FOP family and friends conference – 19 May 2018

After the disappointment of looking through ~13000 crystal drop images over the past couple of weeks and finding pretty much nothing in most of them and nothing that actually diffracted nicely in the rest, it was incredibly heartwarming and hugely inspiring to attend the FOP family and friends conference in Manchester last weekend. FOP Friends is Read More …

Purification and co-crystallisation of TGFBR1 (ALK5) and FKBP12 with five ATP-competitive ALK2 inhibitors – so much disappointment

As you are probably aware by now, if you’ve read a few of my blogs, we are testing ATP competitive inhibitors to inhibit ALK2 but not any of the other 6 ALK family members. Thus, most of our activity and cellular assays test ALK2 as well as ALK5 activity in order to check the specificity Read More …

More purification, co-crystallisation and fragment screening data processing

It’s been a little while since I posted. I’ve been really busy, but haven’t had that much to report. I’ve been doing some processing of the fragment screening data we have so far. We found one possible hit in the pocket we are looking at for allosteric ligands, which initially looked very exciting, but on Read More …

Purification of ACVR1 for co-crystallisation with new compounds and for fragment screening crystals

I was away on leave last week, but happily, before I left, I set up 25 crystal plates of ACVR1 with various compounds. The details of the protein purification, the compounds used and some of the crystals obtained are here, on Zenodo. So I was able to at least come back to something after my Read More …

Update on fragment screening of ACVR1 co-crystallised with LDN-193189

I’ve put the Molprobity stats for the model I have refined to run my fragment screening datasets against in Zenodo. It looks pretty good, and I was happy when the parameters finally all went into the green. I have about 90 datasets with resolution lower than 2.6 Å, so hopefully I’ll get at least one Read More …