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Analytical Chemistry

NMR Protein Structures vs. X-Ray ones

Here’s a neat paper that’s shown up on arXiv.org on protein structures. The authors, from Yale and Edinburgh, are specifically comparing X-ray crystallographic structures with NMR-determined ones in solution. It’s widely known that when you look at the same (or nearly the same) proteins by both those methods that you see small but real differences: the question is why those exist. Crystal-packing in the solid state is certainly a different situation than floating around in buffer solution, so that’s a first-order answer, but what specifically is going on? And can you be sure that what you’re seeing is a real difference in structure, or is it just an artifact of the two experimental techniques?

The team identified 16 cases where proteins have been solved with high-quality data by both techniques (although, as they note, there isn’t a universally agreed-on framework for quality of NMR protein structures, so they used the ones where all residues had the appropriate number of restraints applied). Edit: originally misconstrued! Separately, they also created lists of high-quality protein structures solved by either method without the other one available, to try to exclude effects caused by ease of crystallization, solubility, and other factors peculiar to one system or the other. The differences that people had seen in the past definitely showed up this time – specifically, root-mean-square deviation (RMSD) of core alpha-carbon positions (higher in the NMR structures), higher “packing fraction” in the cores of NMR structures, changes in backbone and/or side chain dihedral angles, etc.

What’s interesting is that they go on to suggest a common physical basis for all these effects. They find that computational simulations of packing that don’t have an explicit temperature component (athermal) generate protein cores that look like the X-ray data, while adding some thermal annealing generates cores that look much more like the NMR structures (in fact, they can end up even more tightly packed). That second link above is an earlier paper that suggested that the differences seen have to do with the greater dynamics of the solution structures (as opposed to the limited mobility of the crystals), and this work ties in well with that.

So the “thermalized” systems can pack more tightly in their cores than the athermal ones, and that might well be just a function of the greater mobility in the NMR situation. But there might still be room for a “just an artifact of the technique” explanation when you consider that NMR structures are not only in solution, they’re done around room temperature. And X-ray structures are not only from solid crystals, they’re usually done at very low temperature (to slow the atomic motions and get tighter data). I suppose that one way to deal with that would be experimentally, but there’s a limit to how cold you can get buffer solutions (and the decreased motion might well broaden the NMR resonances?) And you could take RT protein crystal X-ray measurements, but the increased motion will almost certainly decrease the quality of the data for those as well. Never the twain shall meet, or not?

12 comments on “NMR Protein Structures vs. X-Ray ones”

  1. sgcox says:

    Derek, I think the following is incorrect, in fact they did opposite.

    “although, as they note, there isn’t a universally agreed-on framework for quality of NMR protein structures, so they used the ones where the least number of restraints has been applied.”

    The paper said – and I agree with authors the number of restrains per residue is extremely important in NMR:

    “Specifically, we determined the number of NMR restraints per residue beyond which structures do not change significantly with the addition of more restraints, and only used structures with at least this number of restraints per residue on average. “

    1. Derek Lowe says:

      My brain was thinking of having to add X-ray restraints! Fixed, and thanks. . .

  2. Kaleberg says:

    That is a really useful piece of work. I’m glad someone took the time to do this.

  3. 10 Fingers says:

    Lots of food for thought in this paper, actually. An interesting and dense read.

    Reminds me of an MD experiment done many years ago with old colleagues (who are fondly remembered, indeed). Our simulations had trouble reproducing a certain, well-defined crystallographic water network seen in our high resolution structures – and one in particular that was most intimate with the inhibitor bound in the active site. Sure enough, slow quenching (lowering the temperature of the simulation) down to 77K (the crystallographic condition) sharpened up the entire network – mostly by eliminating a set of small conformational wagglings about a minima that were “smearing out” the water densities from the simulation. Almost every crystallographic water in the first few solvation shells of the active site was reproduced, and it was striking to see the overlaps.

    It seemed obvious afterwords that this made sense, and that we hadn’t been giving kT its proper due in our thinking about this and other aspects of inhibitor binding and SAR.

    1. MPK says:

      Wow, a real protein structure reproduced by MD?? What magic force field was that?

  4. Scott says:

    Most of the science here is *way* over my head, but even I know it’s very important to compare different methods of getting the ‘same’ data!

  5. Ramesh says:

    Didn’t find this very insightful! Pretty unidimensional to attribute everything to thermal fluctuations. Might as well have named it “Of thermal fluctuations in different structure determination techniques”!!!

    Proteins whose structures are determined by NMR generally have lower MW and that is a limitation of the technique itself. Hence the packing fractions of proteins in that MW range should have been analyzed and compared. At least they should have touched upon this point.

    In spite of all this, it was a good effort to put it together and the community will surely be provoked to think about it.

  6. Gorka says:

    “but there’s a limit to how cold you can get buffer solutions (and the decreased motion might well broaden the NMR resonances?)”
    Derek, I may be wrong because I don’t have experience with protein NMR, but one should expect that, lowering the temperature, the spectra will exhibit narrower resonances. Yes, perhaps more resonances (a broad signal at RT may be resolved into two or more signals at low temperature), but narrow ones.

    1. Pedwards says:

      Not necessarily. If part of the structure is fluctuating between two distinguishable conformers at a rate that is faster than the NMR timescale, lowering the temperature will eventually cause the resonances associated with that part of the structure to broaden (as the exchange slows down to the NMR timescale). Going down further will eventually cause the peak to split into separate peaks, which will then sharpen as the temperature decreases further.

      1. PM says:

        At ultra low temperatures proteins have extremely broad peaks even in solid state NMR, taking away anisotropic interactions by magic angle spinning. The reason is that below the protein glass transition, there is heterogeneity of the protein structure ensemble (due to the dynamic before stopping the motions), with each conformation having individual chemical shift.
        For typical spectra look for the protein DNP NMR.

  7. Bryan says:

    >And X-ray structures are not only from solid crystals, they’re usually done at very low temperature (to slow the atomic motions and get tighter data).

    IIRC, while you do benefit from lowered thermal motions with crystallography at cryogenic temperatures, I think the main benefit is reduced radiation damage to the crystal. In more recent times, with fast, pulsed synchotron x-ray sources, scientists have been able to get good quality room temperature X-ray structrues (e.g. some of the more recent work on time-resolved crystallography). Tom Alber published a paper in 2011 (see link in handle) comparing room temp and cryo X-ray structures of various proteins and see some important differences in regions known to be flexible or dynamic. It would be interesting to take another look at the data to see if some of the differences in core residue packing are also apparent in the cryo vs RT structures.

  8. Bill says:

    The tumbling rates of protein molecules in solution decrease at lower temperatures. This leads to shorter T2 relaxation and. broader lines. The tumbling rate also decreases with increasing protein size and this, in the past, has limited the size of proteins that can be investigated by solution NMR. More recently,, higher magnetic fields, selective labelling and other new techniques now allow investigation of much larger proteins than in the past. However, as Lewis Kay in particular has demonstrated, the real value of NMR of proteins is its ability to investigate protein dynamics. X-ray crystallography will almost always be the easier method for protein structure determination since it doesn’t require 13C and 15N labelling.

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