I am currently working with inhibitors for an enzyme (about 500Da MW). We were able to collect data and processing it, we were able to get a map with a 3.9A resolution. The problem is that in the active site we do,'t see any type of density that corresponds to the inhibitor (previously incubated with the enzyme).
I suppose that it is most likely because not all the particles I selected have the inhibitor. For the processing, I classified them into 200 2D classes and reconstructed 1 class 3D map.
I wanted to ask you, how or what could I do to be able to analyze the active site? How could I differentiate particles that have the inhibitor from those that don’t? What could I use to get the density of the inhibitor?
If there is a correlated change in conformation or dynamics upon binding (and if your ligand is bound with an appreciable occupancy), you may be able to separate the apo and holo states by 3D classification.
If there is minimal conformational change on binding though, then this will be very challenging or impossible to solve due to the small size of the ligand.
3D classification can/will do it, but it’s always challenging for something small. I had excellent results with a ligand approximately 600 Da and identifying bound/unbound, but the map was <2 Ang. At ~4 Ang I’d be very cautious.
A friend has some amazing results showing binding of an ~240 Da ligand, but resolution is <2.5 Ang and structural changes are minimal, it just binds in a really beautiful spot which can’t be missed.
Also, as an aside, just because something is incubated with the relevant inhibitor, doesn’t mean it’s bound, especially if conditions have been adjusted to optimise grids (this was a massive headache with a project over the summer). Do you have biochemical evidence that it’s binding in conditions used to make grids?
If at all possible, see if you can get resolution to <3 Ang, then I’d be pretty hopeful.
@rbs_sci Thank you for your reply.
If we obtained a map at the resolution <3-2A, how were you able to identify the ligand? And how were you able to separate the particles bound to the ligand from those not bound to it?
Best, David
Identifying the ligand was easy; it was either there, or not there. For one dataset (my friends) it’s actual two datasets, but for mine, it was just 3D classification of the complex. Nothing else to really explain.