by Peter Nollert
May 6, 2011 09:39
This week I'll prepare my first Iodide soak ever. While we've determined many protein structures at Emerald BioStructures using anomalous data from iodide soaked crystals (see a previous blog post on this topic), I've never prepared iodide-soaked crystals myself. Being a novice I asked Tom Edwards who is an expert in this methodology. Turns out that he's preparing a webinar on this topic for next week: SAD phasing at rotating anode wavelengths using iodide ions. The goal of course is to obtain phases from anomalous X-ray diffraction data, the key to de-novo crystallographic structure determination.
Tom Edwards on "SAD phasing at rotating anode wavelengths using iodide ions"
I asked Tom for his 'standard iodide crystal soaking recipe'. Here is it:
1. prepare a 5 M Sodium Iodide stock and a formulation at 2 x of the crystallization cocktail.
2. Mix iodide to a final concentration of 1 M with the 2 x crystallization cocktail and include the cryo reagent.
3. Transfer a single crystal into 1 uL of 1 M soak and check for crystal damage. If there's no visible damage, test X-ray diffraction. Back down with the iodide concentration (0.75 M, 0.5 M etc.) if the quality of X-ray diffraction pattern suffers (mosaicity, resolution, split spots etc.).
4. Harvest, cool, mount, diffract, collect...
The fun part - such as data treatment - will be covered in Tom's webinar
I'm off to the lab.
Peter