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I seeing tens of thousands of PDB files on the internet. I really want to determine a 3D structure of my protein of interest. I've heard that 3D structure determination is a complex, expensive, and specialized procedure that can take months or years and is hard to perform routinely or even purchase commercially.

Could you please explain why and how hard it is? What are the requirements and how big of a budget is typically needed to perform this kind of experiment?

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    $\begingroup$ It really depends on the protein. Which protein are you interested in? Or if you want to keep that secret at least tell us what type of protein it is... is it an integral membrane protein, or is it membrane associated? If yes then your experiment will be hard, but not necesarily impossible. If you want to discuss it of thread, send me a mail at (jan@mbg.au.dk) and I will try to help you. I am a trained structural biologist with significant experience in protein crystallography. $\endgroup$ – Jeppe Nielsen Jun 22 '17 at 9:26
  • $\begingroup$ From personal experience, if your protein is not membrane associated and is generally stable in solution, it will usually take ~5-10mg to find possible crystallization conditions. I recommend starting with commercial screens and optimizing from them. Be aware that you may observe crystals that are not protein crystals in certain conditions. If you want help send me an email at the email in my first comment. $\endgroup$ – Jeppe Nielsen Jun 22 '17 at 10:58
  • $\begingroup$ @JeppeNielsen Hi Jeppe ! Thank you for your enthusiastic and kind response. In near future, I'm planning to design a protein (currently non-membrane protein) and just want to see the final 3d structure result of it, I will contact you later if i need help from you. Thank you very much! $\endgroup$ – joe Jun 22 '17 at 13:34
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    $\begingroup$ One warning before going heads in is that crystallography is of course only possible if the protein is capable of crystalizing. If you want to know more about biomolecular crystallography, read the book with the title biomolecular crystallography by Bernhard Rupp, it is more or less the goto source for all protein crystallographers. $\endgroup$ – Jeppe Nielsen Jun 23 '17 at 0:31
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    $\begingroup$ I can attest from experience, the answer is "very"! $\endgroup$ – Joe Healey Jun 27 '17 at 14:10
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Experimental protein structure determination is hard: the most common method is X-ray crystallography, which can be done in a few months if you are lucky and can take years if you're not. The problem with X-ray crystallography is that you need good protein crystals, and in most cases, proteins don't crystallize very well, so it takes a lot of time (and a lot of purified protein) to get the right crystal. (In the comments section Cody added some good links with more in-depth abut X-ray crystallography for structure determination relating to ATP synthase).

The cost is mainly based on the materials & manpower you need for this, and you need to test a lot of different solutions to crystallize your protein in, which aren't very cheap. If you don't have access to specialised equipment for making the crystals (mostly pipetting robots) or the measurement (X-ray beamline), this is going to be super expensive.

Crystallography requires protein crystals, which are sometimes too troublesome to obtain. Other methods that do not require crystals, such as NMR and cryoEM, are even more difficult to perform though less luck dependent. These techniques rely on very expensive equipment and often cannot resolve the structure as precisely as protein crystallography. So unless you can find one of the few people who have experience with these methods, you'll run into a lot of additional problems.

There is also computational structure prediction, which only needs a powerful computer. However, unless your protein is very similar to one with a known structure it will probably not work reliably. As mentioned in the comments there are web server's for the established methods and progress is constantly made on new(er) algorithms, so depending on your needs it's definitely worth a shot to try computational structure prediction.

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    $\begingroup$ For what it's worth, there are many web servers for homology modelling, so all you need for that is a protein sequence and a browser. $\endgroup$ – canadianer Jun 22 '17 at 2:20
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    $\begingroup$ Note that computational predictability recently made a giant leap forward and seems to work quite well (requiring primarily DNA sequences of many species rather than similarity to known protein stucture or computational folding simulations) science.sciencemag.org/content/355/6322/294 $\endgroup$ – tsttst Jun 22 '17 at 4:31
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    $\begingroup$ Two interesting & relevant papers showing how X-ray crystallography is used to determine protein structure are: Structure of ATP synthase from Paracoccus denitrificans determined by X-ray crystallography at 4.0 Å resolution, and a review, The ATP synthase: the understood, the uncertain and the unknown. What's particularly nice about these is they deal with a protein complex virtually everyone is familiar with, yet has been very challenging to characterize using our standard techniques. $\endgroup$ – Cody Gray Jun 22 '17 at 7:19
  • $\begingroup$ In fact, computer approach is so hard (this is a combinatorial problem where exploring all the possibilities is not feasible), that some scientists have designed Fold It!, a game where players must find the best protein folding. There is a field of work where scientists look at best player's strategies to design their own heuristics. $\endgroup$ – aluriak Jun 22 '17 at 10:11
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I'll address NMR for structure determination. It is the less common method, only ~10% of protein structures are determined this way, though it has e.g. advantages for nucleic acids and more than a third of those was solved by NMR. Take any numbers here as very rough estimates, there are a lot of factors that influence the difficulty and cost.

For NMR, the single most important factor is size. A stable, well-behaved protein of 10-20 kD is pretty much routine. Large proteins are either very difficult to measure or outright impossible by NMR.

You need large quantities of protein for X-ray and NMR, but in the case of NMR you also need it isotope-labeled. Uniformly labelling proteins 15N/13C isn't all that expensive if you can express your protein in E. coli in minimal medium (around $100 per liter medium or so, mostly in 13C glucose), but it can get really expensive very fast if you need full medium or anything fancy.

Your protein needs to be stable in solution for a few days at least, a single 3D experiment can take multiple days to measure. If your protein is barely stable enough, you also have to produce much more protein because you need to use many samples to perform all necessary experiments. A complication here is that you can't add a lot of salt to an NMR sample without drastically reducing the sensitivity of the NMR experiments. But some proteins are hard to get stable in a low-salt environment.

You need something like a few weeks of measurement time on a high-field NMR spectrometer, though this depends a lot on the size of the protein and the concentration you can achieve in your sample and the general difficulty of assigning the protein resonances. This kind of spectrometer can cost a few millions, and you need to supply liquid nitrogen and liquid helium to keep them working. Something like a $1000 per day measurement time is a number I've heard about the cost, but there are a lot of factors that go into this and I can't say how accurate this number is.

Then you need to assign the resonances in your protein, which takes a single person several months. This again varies a lot depending on the difficulty.

Then you need to calculate the structure, and typically this requires several rounds of refining the assignment and analysis and redoing the calculations. It's not really expensive in terms of computer power, but it takes quite some work by the person running the calculations and checking them.

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  • $\begingroup$ a 2007 article says "The largest monomeric protein structure solved by NMR to date is that of malate synthase G (MSG), an 81.4 kDa enzyme " ncbi.nlm.nih.gov/pmc/articles/PMC2596980 $\endgroup$ – DavePhD Jun 22 '17 at 15:40
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    $\begingroup$ @DavePhD The fine print is important here, they solved the global fold of the protein, that's not a full structure. And even for that they had to pull out quite a few tricks with specific labeling and deuteration. If you're not Lewis Kay, trying to solve the structure of such large proteins by NMR is probably not a good idea. $\endgroup$ – Mad Scientist Jun 22 '17 at 15:47

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