Wednesday, March 21, 2012

Actin part two: Formin

Like everything in the cell, the nucleation and elongation of actin filaments is highly regulated. Here to perform part of this regulation is the Formin family of proteins. Formins are cool because they stay associated with the growing end of an actin filament, stepping along the elongating filament as each monomer is added. In this way they protect the growing filament from being capped (and its growth terminated) by the abundant capping protein. Formins are usually big long proteins with many different domains, so the domain I've chosen to represent is the Formin Homology 2 (FH2) domain, because it's the one responsible for keeping the Formin protein stepping along at the growing end of the actin filament.
Here's a single FH2 domain from the formin bni1p (pdb code 1y64). The N-terminus is on the top right and the C terminus at the bottom. Check out the lasso domain! That is how the FH2 domain dimerizes; that lasso loops around the post (knob at bottom left) of another monomer. Actin binds in the middle of the resulting doughnut shape. The thin linker part was printed along with the main portion of the protein, and the lasso was made separately. Then I glued the lasso on, but the linker part broke so that's why I'm holding it like that.

This is how the two FH2 monomers would be oriented in the dimer. These are printed with the lasso wrapped around the post, but I didn't print the linkers because I wanted to glue the monomers together with something flexible to emulate how they would move in real life. I tried gluing a nylon rope but the glue I used didn't seem to take.. I'll update when I come up with something better. For now, imagine the linkers there.
And here's a dimer bound to two actin monomers (almost like in the pdb structure!) Imagine this sitting at the top of a filament, the FH2 domains walking up along the filament when new actin monomers get added.

So that's it for now; I'm building a new extruder with a smaller nozzle, so hopefully soon I'll get that working and I will be getting even better print resolution. Or it will explode/melt. Time will tell.

Tuesday, March 13, 2012

Actin Crazy

So I work in a lab that studies actin so why not print out the actin structure so I can learn more about it?

Here is a nice overview of actin and what it does. Briefly, actin is polymerized into filaments for lots of different purposes inside the cell.
Here are two structures that show actin bound to Dnase I (ATN1 on pdb; left) and actin bound in a filament (2Y83 on pdb; right). Notice how the pointed-end (that's the bottom in the picture) is more compressed in the one on the right, that is supposed to fit in a filament. Whereas the one on the left has the pointed end sort of splayed out, because there's supposed to be a DNase I bound to the DNase I loop (biologists are creative aren't they). Haven't had a chance to print out more of the right structure so I can make a filament, but I will do that as soon as possible.

Also I need to work out a concrete set of scale. I sort of eyeballed these and it shows, the one on the right is noticeably larger.

Woo! First protein structure print. You can see I was messing with options so the rightmost one came out stringy and hole-y.

Check it out! Half an actin filament! I need a better way to hold the monomers together.. double sided tape doesn't look great. Eventually I'll put pegs in them but it would also be great to be able to tack them on wherever, just for play purposes.

Monday, March 12, 2012

Mission Statement

As a biochemist I love looking at protein structures. But what I love even more than looking at something is having it, physically, in my hands to play with. Sadly you can't put together a physical protein out of a bunch of elementary plastic parts like those chemical model kits (darn chemists get to have all the fun), and if you tried to make a protein out of chemical model parts you would be broke pretty quick (and you'd need to move into a hangar).

So the next best thing is 3d printing. There are websites that will 3d print protein models for you, and other websites that will print any old thing for you, but they tend to be ridiculously expensive and if I was going to do this for real (with the intent of learning something about proteins from the process) I would want to 3d print lots of different things, for example proteins in different conformations, maybe at different scales with different ligands, etc. I want to be able to play with the concept, not feel constrained by the price tag every time (and thus afraid to make a mistake).

So, simply, what I've done is built my own 3d printer for the purpose of printing these models.
This is Heffy. He can move his axes up to 4 mm/s, and has a nozzle size of 0.8 mm. Print quality is steadily increasing, but as of now I can achieve 0.4 mm layers with a minimum feature size of 1 mm. The cartesian robot part is based off this, with these electronics and a stepper-driven extruder similar to this.

So I'll post pictures of what I've made along with a short description and a link to the stl I used to make them.