How high throughput fragment screening is reviving old school crystallography tricks

Takeaway: Why: we need lots of well diffracting crystals. How: crystallisation growth optimisation. What: tweaking conditions, phase space understanding, seeding, MMS. At this day and age (2018) many people are thinking that the future of structural molecular biology being about serial XFEL or synchrotron crystallography and electron diffraction, which are great tools to harness the Read More …

Characterisation of HAO1 fragment hits in solution

In my previous post, I described four promising fragments bound to HAO1 structures. Specifically, I described two key areas of HAO1 – the gating loop and active site Trp110 – where fragments 1, 2 and 5 bind. I also described fragment 6 at the subunit interface of HAO1, away from the active site. After analysis 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 …

Screening Compounds of Interest against USP5 Zf-UBD by SPR #2

I used a surface plasmon resonance assay to determine the binding affinities of hit compounds that were identified in 19F NMR spectroscopy screens against the zinc finger ubiquitin binding domain of USP5. To see how these assays work, check out some of my previous posts! You can see details of the SPR experiment on Zenodo. 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 …

Structure Solution for BMPR1B/FKBP12 complex bound to M4K2009.

Structure Solution for BMPR1B/FKBP12 complex bound to M4K2009. Good news everybody! From the crystals I sent to the Diamond Light Source (a synchrotron, or particle accelerator, that gives very high quality x-rays) last week, I got a data set for BMPR1B/FKBP12 with M4K2009 bound to it! This is one of the compounds that the M4K Read More …