The blue whales of crystallography

Fun tour through the big protein complexes crystallographers are stalking these days, such as ribosome, spliceosome, nuclear-pore, HIV trimer, G-protein coupled receptor…

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Wow: a VDJ crystal structure!

Recently my heart was filled with joy to see a paper published by the laboratory where I did my PhD.

David Schatz’s lab at Yale has obtained a crystal structure of part of the protein RAG-1 bound to DNA. My congratulations to first author Fang Fang Yin.

RAG-1 and its sister RAG-2 form the scissors that cut the DNA in immature white blood cells during VDJ recombination, a process where the developing immune system slices and reassembles antibody genes in a unique, partly random way.

Because VDJ recombination can happen in a different way in each cell, the immune system has a library of millions of antibodies that can react with many different antigens out in the environment.

The signals on the DNA that mark off the gene segments that get cut and pasted together again come in two flavors. They look like this:

Antibody gene segment — CACAGTG – 12 letters – ACAAAAACC


Antibody gene segment — CACAGTG – 23 letters – ACAAAAACC

The puzzle that attracted me to this area of research was this: the RAGs need one of each kind, 12 and 23, to cut the DNA. That’s weird.

Another weird aspect is that the part of the DNA opposite the CACAGTG is turned into a hairpin, with one strand of the DNA double-helix joined to the other.

We can imagine a process like this: the RAG proteins grab one of each signal and pull them together, forming a loop. What do the proteins look like when this loop forms? What are the geometric requirements for assembling this complex?

These are the questions I was trying to answer, or at least move closer to answering, during my PhD research.

Besides being a fascinating puzzle and the centerpiece of the adaptive immune system, VDJ recombination has connections to:

  • DNA repair, because all sorts of monkey wrenches can interfere with the immune system’s ability to smush the DNA back together again
  • Evolution, because the transposon-like arrangement suggests how antibodies suddenly appeared in vertebrate biology millions of years ago

When I started it hadn’t rigorously been proven that the RAGs actually cut DNA directly. With the help of Thomas Leu, a postdoc from Switzerland, I put together a system for examining how they cut DNA in the test tube. I also did some experiments where I crosslinked the RAGs to the DNA with UV light.

But actually getting a crystal structure, that’s really impressive and difficult. Now David’s group (and other scientists) can start to build a model of the whole complex.

X-ray crystallography might not allow visualization of an entire ready-to-cut structure with both 12 and 23 signals because the whole shaky bridge might be too flexible for crystals to form.

The structure published in Nature SMB doesn’t include the CACAGTG part, which is where the DNA gets cut, or RAG2, which is necessary for cleavage to occur.

But it does show that two RAG proteins intertwine and that unit binds to two separate ACAAAACC elements at once. That’s a great step forward.

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Impressive visitor

Organ donation advocate Bobby Howard gave an impressive talk to the Emory Transplant Center today. He said that when he started his work with Lifelink Georgia 14 years ago, just 8 percent of donated kidneys in GA came from African Americans, but that figure has increased to more than 30 percent now.

African Americans receive the majority of kidney transplants in GA, because of the prevalence of diabetes and hypertension leading to renal failure, he said.

Howard (more about him) is originally from Pittsburgh (where I grew up) and a former pro football player. He was a very effective speaker.

What I found interesting was: he said when he started out, the main objection to organ donation he experienced was from religious people who wanted to have all their organs when they got to heaven.

That was relatively easy to address compared with the remaining issue: mistrust of the healthcare system. People he talks with are worried that declared organ donors will receive inadequate care because doctors only want their organs! Given the experience of various minority communities with medicine, such mistrust makes sense even if it does impair organ donation and saving lives.

Makes me wonder if everyone was a declared organ donor, how would that affect transplant waiting lists? That deserves another post.

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A simple dinner

I just started reading “A Free Life” by Ha Jin and was struck by this paragraph. The main characters Nan and Pingping have Nan’s friend Danning, who is a link to Nan’s literary aspirations, over for dinner.

Dinner was simple: eggplant stuffed with minced pork, a salad of assorted vegetables, preserved eggs, braised shrimp, and dumplings filled with beef and napa cabbage.

My wife and I were quite pleased with our first few successful preparations of pork pot stickers, (a rough equivalent of the last dish only) but it took work. Making all of those other dishes wouldn’t be simple to me!

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Georgia stem cell/embryo legislation

My interpretation of the modified bill that passed the State Senate Thursday is that it would impact future stem cell research in Georgia less than the original.

From the AP:

The bill’s sponsor said it permits research on existing embryonic stem cell lines, which had been allowed under the Bush administration. It also allows new embryonic stem lines to be brought into the state. The bill does not say whether unused embryos created for fertility treatments could be donated to stem cell research.

That last point will need to be resolved.

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Chili verde recipe

Last week I participated in a chili cook-off at work. My chili verde recipe got third place, behind a more traditional beef chili with cubed steak and black beans and a chicken stew with flavorful broth.

Here is a recreation of my improvisation, which was adapted from a recipe by Rick Bayless:

3 pounds pork shoulder, cut into 1-inch cubes
1 large or 2 small onions, roughly chopped
4-6 cloves garlic, chopped
12 tomatillos, quartered
2 cups pickled jalapenos
1 tablespoon vegetable oil
1 teaspoon ground cumin
1 teaspoon salt
Saute onions and garlic in oil in a wide saucepan or Dutch oven with enough heat so that you get some golden color.
Add tomatillos and distribute so that mostly the tomatillos are facing the floor of the pan. Sprinkle salt and cumin all around. Now add the jalapenos, and then the pork on top.
Turn down the heat (how low depends on how your stove achieves barely simmering). The tomatillos will gradually release their water. Cook for six hours on low heat. If arranged properly, some of the pork pieces should brown as they cook.
Remove pieces of pork with tongs or slotted spoon and set aside. Take sauce and put in a blender along with:
1 cup chopped cilantro
1 cup chopped Italian parsley
Add back pork and two 16-ounce cans cannellini beans, drained.
Store before reheating and serving, or reheat and serve immediately.
Before serving, add:
 juice of two limes
1 tablespoon Worcestershire sauce
salt, pepper and chopped pickled jalapenos to taste.

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I didn’t know the young woman who was fatally burned at UCLA working with t-butyl lithium. It appears that several errors intersected after the reagent spilled and caught fire: she was wearing a flammable sweater, the fume hood wasn’t down far enough, she didn’t run toward the emergency shower.

My first job working in a chemistry lab was at UCLA. It was just for the summer and I didn’t distinguish myself in any big way. I dropped a jug of organic solvent (heptane, perhaps) on my foot and the glass broke. Embarassing, but at least there was no spark!

I also remember a big liquid nitrogen tank having its valve stuck open and the lab folk wheeled it out onto a balcony so it could vent.

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