Who here works on X-ray crystallography to resolve protein structures?

[Dr. Chiquita]

Hey guys. I want to hear pros and cons of working in a lab doing X-ray crystallography to resolve protein structures, then go after its function in a traditional way (by that I mean doing typical molecular/biochemical work). I am a layperson in terms of X-ray crystallography so go easy on me. No jargons.

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[fyli260]

Hey guys. I want to hear pros and cons of working in a lab doing X-ray crystallography to resolve protein structures, then go after its function in a traditional way (by that I mean doing typical molecular/biochemical work). I am a layperson in terms of X-ray crystallography so go easy on me. No jargons.

I have been working in X-ray crystallography for about 4-5 years now (not full time continuously though). It's a very frustrating field to work in since you don't really get any really meaningful results until the end (i.e. when you solved the structure). Hence, it's not a good field to work in if you're very result oriented. It's quite a bit of gamble because you're project is usually all or nothing. Until you get the structure, people outside of the field probably will not appreciate your progress. You may be able to do some biochemistry along the way, but it's usually very hard to draw any strong conclusions from in vitro studies without the support of a structure. But if you have the determination and work ethic to persist in this field, it can be very rewarding. A single structure can reveal an important point that may take 5-6 experiments to show. And most structure papers publish very well (usually impact factor greater than 5) no matter how unimportant the structure may be. And for the hard projects, you get to excise quite bit of creativity because most problems are not solved by a standard set of rules or methods. Crystallography is not a very well understood technique, so you can use your imagination and learn something new everyday.

 

[microTAS]

any opinions also from crystallography peeps on Fluidigm's Topaz crystallizer chips? There is definitely a lot of hype surrounding the technology.

 

[logos]

What ive been told by a number of people is that its a crapshoot. I guess you just try huge numbers of different conditions in hopes of getting a crystal. Could take you forever to get it, or a day. Have heard that once you have the crystal its pretty easy.

 

[fyli260]

What ive been told by a number of people is that its a crapshoot. I guess you just try huge numbers of different conditions in hopes of getting a crystal. Could take you forever to get it, or a day. Have heard that once you have the crystal its pretty easy.

It's not always a total crapshoot though. The really experienced and good crystallographers may be able to pick out conditions that could yield crystals with some modifications even if they didn't yield crystals in the first place. Thus, if you're good, it shouldn't take you forever to get a structure of some kind. But it definitely takes much more than a day to complete a structural project. If it's a new structure, it will take at the very least several weeks (that's if you're super lucky). So it does depend quite a bit on luck (probably more so than the other fields), but everyone will have luck at some time, and luck will only favor the prepared mind. It's true that getting crystals is one of the biggest bottlenecks, but it can still be quite problematic after you get crystals. For example, your crystals may not diffract well enough, or they may not have the right morphology to give a good data set. Again, if you're a good crystallographer, you should be able to eventually get the structure in some way once you get crystals, although it may not be very easy.

 

[structurelab]

I have been working in X-ray crystallography for about 4-5 years now (not full time continuously though). It's a very frustrating field to work in since you don't really get any really meaningful results until the end (i.e. when you solved the structure). Hence, it's not a good field to work in if you're very result oriented. It's quite a bit of gamble because you're project is usually all or nothing. Until you get the structure, people outside of the field probably will not appreciate your progress. You may be able to do some biochemistry along the way, but it's usually very hard to draw any strong conclusions from in vitro studies without the support of a structure. But if you have the determination and work ethic to persist in this field, it can be very rewarding. A single structure can reveal an important point that may take 5-6 experiments to show. And most structure papers publish very well (usually impact factor greater than 5) no matter how unimportant the structure may be. And for the hard projects, you get to excise quite bit of creativity because most problems are not solved by a standard set of rules or methods. Crystallography is not a very well understood technique, so you can use your imagination and learn something new everyday.

I disagree that you do not get any meaningful results until you solve the structure. I've worked in an x-ray crystallography lab for two years now and can say that although the "big picture" isnt complete until the structure is solved, you can publish good papers based on the biochemistry of your protein. In fact, that's what most non-structure and structure based labs do. Pick up JBC and Biochemistry and you'll see my point. I do not personally know a single crystallography lab that focuses solely on structure determination. They focus on solving structures as well as the biochemistry or cell biology of their protein/s. You can either use this information to supplement your structure paper or publish it on its own. For example, you can look at allosteric regulation of the protein. The process of getting suitable crystals is a paper all in its own. You also learn a great deal about the proteins solubility based on the crystal screens, which help you develop new protocols for protein purification. Acta crystallographica loves this sort of thing. And, if you think this journal is not very good, just look at its impact factor. This sort of research is exactly what I've done. I have a first author paper based on the protein purification and crystal preparation of my protein. I'm currently writing another paper based on the biochemistry of my protein before I knew its structure. And, I have a joint first author paper based on its structure and cell biology, which is in a top-notch journal. So, results are based on you, not the technique of crystallography. Protein structure determination is a technique that is very powerful and can gain you a lot of recognition. It is difficult, but what isn't. It seems to me that people are very vision oriented. We can look at a large object and decipher how it works because we can visually see how it works. As items get smaller, we can only do 'blind' experiments to determine how it works. Crystallography helps 'regain your sight' of how proteins and DNA interact. This is a very poweful tool. So, I'll shut up and give a big

 

[fyli260]

I disagree that you do not get any meaningful results until you solve the structure. I've worked in an x-ray crystallography lab for two years now and can say that although the "big picture" isnt complete until the structure is solved, you can publish good papers based on the biochemistry of your protein. In fact, that's what most non-structure and structure based labs do. Pick up JBC and Biochemistry and you'll see my point. I do not personally know a single crystallography lab that focuses solely on structure determination. They focus on solving structures as well as the biochemistry or cell biology of their protein/s. You can either use this information to supplement your structure paper or publish it on its own. For example, you can look at allosteric regulation of the protein. The process of getting suitable crystals is a paper all in its own. You also learn a great deal about the proteins solubility based on the crystal screens, which help you develop new protocols for protein purification. Acta crystallographica loves this sort of thing. And, if you think this journal is not very good, just look at its impact factor. This sort of research is exactly what I've done. I have a first author paper based on the protein purification and crystal preparation of my protein. I'm currently writing another paper based on the biochemistry of my protein before I knew its structure. And, I have a joint first author paper based on its structure and cell biology, which is in a top-notch journal. So, results are based on you, not the technique of crystallography. Protein structure determination is a technique that is very powerful and can gain you a lot of recognition. It is difficult, but what isn't. It seems to me that people are very vision oriented. We can look at a large object and decipher how it works because we can visually see how it works. As items get smaller, we can only do 'blind' experiments to determine how it works. Crystallography helps 'regain your sight' of how proteins and DNA interact. This is a very poweful tool. So, I'll shut up and give a big

I agree with all these points. I didn't mean to say that purely biochemistry papers are not meaningful. It's just that it is better that they're supported by a structure. That's why biochemistry papers are usually published in JBC or Biochemistry (which definitely are not bad journals), while adding a structure may get the paper into Cell or Molecular Cell. Hence, I think it's usually easier and better to try to get a structure first and then verify the findings with biochemistry experiments. But certainly if you can't get the structure, then you would have no choice but to do biochemistry experiments.

 

[brotherbu]

I have been working in a crystallography lab for 2 years. I spent the first year determining the proper conditions for crystallization for an enzyme and spent teh past year refining the structure of the enzyme. I personally feel that though at times, it can be frustrating, the ultimate rewards of solving a structure is well worth it. Refining a structure is like playing a video game all day, making residues fit electron density maps. Maybe its just me, but such a visual science is just cool to do.

Most crystallography labs do a lot of other biochemical experiments and projects as well. Your crystallography work also applies to such projects as transmemebrane mutations, and protein folding, and you will constantly be reading papers and collaborating with other researchers on these topics as well as almost anything else about proteomics.

If you have any specific questions you want to ask me about working in crystallography, just let me know

Brotherbu

 

[structurelab]

I have been working in a crystallography lab for 2 years. I spent the first year determining the proper conditions for crystallization for an enzyme and spent the past year refining the structure of the enzyme.

Brotherbu

Wow, the past year refining the structure. Must be a large structure and/or the electron density is hard to fit. Kudos to you. Good luck, I'm sure it'll be an excellent paper.

 

[Dr. Chiquita]

Thanks everybody for all the helpful comments!

Can somebody go over a very basic step-by-step protocol with me? I imagine you first purify your protein of interest, right? That's as far as I know.

Thanks.

 

[fyli260]

Thanks everybody for all the helpful comments!

Can somebody go over a very basic step-by-step protocol with me? I imagine you first purify your protein of interest, right? That's as far as I know.

Thanks.

It really depends on what kind of project you're going to work on. If you want to get the structure of a novel protein, then your first steps will be cloning different constructs of the protein into expression vectors to test for solubility. If you manage to get soluble protein, then you can try to purify it, although you may encounter some problems with your protein here as well (e.g. your protein may aggregate, get degraded, or precipitate out during concentration). But if you manage to purify reasonably well behaved protein at a high concentration (concentration necessary for crystallization may depend on the size of your protein) and purity, then you can set up crystallization trials to find a condition that can yield crystals of your protein. If you manage to get crystals of some form from these initial broad screening, usually you have to experiment around these conditions to optimize the crystals. If the optimized crystals can diffract, then you can collect data using these native crystals. However, novel proteins will require a second data set to solve the phase problem. This usually is solved by purifying the same protein containing sellenium-Methionine (SeMet) and growing crystals of the SeMet protein. If this does not work, then you may want to try soaking heavy metals into your native crystals and collecting the data again. Although this may sound quite straight forward, it may take months to years to overcome some of the obtacles that you may face along the way.