Macromolecular Structure Determination: Comparison of X‐ray Crystallography and NMR Spectroscopy

Abstract

X‐ray crystallography and nuclear magnetic resonance (NMR) spectroscopy are the most powerful and predominant techniques used to experimentally determine the three‐dimensional structures of biological macromolecules at near atomic resolution. X‐ray diffraction (XRD) studies require a crystalisable protein, whereas NMR is suitable for macromolecules in solution. XRD has no size limitations and provides the most precise atomic detail, whereas information about the dynamics of the molecule may be limited. NMR excels in cases where no protein crystals can be obtained and it provides solution state dynamics, but in turn delivers lower resolution structures and is in general limited to molecular weights below approximately 50 kDa. The two techniques can deliver complementary information. Approximately 90% of the experimentally determined macromolecular structures deposited in Protein Data Bank are crystal structures, with NMR dominating the <10 kDa molecular weight range.

Key Concepts:

  • XRD and NMR are complimentary structure determination methods.

  • XRD has no size limitations and provides the most precise atomic detail, whereas information about the dynamics of the molecule may be limited.

  • NMR excels in cases where no protein crystals can be obtained and it provides solution state dynamics, but in turn delivers less detail and in general practice is limited to molecular weights below approximately 50 kDa.

Keywords: crystallography; NMR; X‐ray; macromolecular structure; diffraction

Figure 1.

The principle of X‐ray structure determination. A crystal mounted on a goniostat with at least one rotatable axis is exposed to a finely collimated, intense X‐ray beam in the 5–20 keV energy range (approximately 2.3–0.6 Å wavelength). Individual diffraction images are recorded during small rotation increments of the crystal and combined into a diffraction data set. Unfortunately, the diffraction images are not direct images of the molecule. The basic mathematical tool of back‐transformation from reciprocal diffraction space into direct space is the Fourier transform (FT), which together with separately acquired phases for each diffraction spot allows synthesising or reconstructing the electron density (blue grid) of the molecules self‐assembled into the diffracting crystal. An atomic model of the structure is then built into the three‐dimensional electron density. From Biomolecular Crystallography: Principles, Practice, and Application to Structural Biology by Bernhard Rupp. Reproduced by permission of Garland Science/Taylor & Francis LLC © 2011.

Figure 2.

Experimental basis of NMR structure determination. Biomolecules in solution at close to physiological conditions are inserted into a magnet. The radio frequency circuit detects the time domain signal corresponding response of the nuclear spins to resonance. This analogue time domain signal detected by the circuit is amplified and digitised prior to Fourier transformation into the spectral domain. A combination of 2D and 3D experiments are generally collected, processed and analysed to obtain NMR restraint parameter that are sensitive to determine both local structural relations and events (through chemical shifts and coupling constants) as well as the global fold (via NOEs) of a protein. Three‐dimensional structural models generated by NMR methods also carry additional information on residue‐specific dynamic motion.

Figure 3.

Flowchart describing the major steps in NMR based structure determination. Four principal elements are combined in the NMR method for protein structure determination: (1) multidimensional NMR experiments that provide the data for all the following steps; (2) sequence‐specific resonance assignment – matching each proton in the protein to respective peaks in the spectra; (3) the Nuclear Overhauser Effect (NOE) data – providing inter‐proton distances to identify the global fold of the protein; (4) computational tools such as distance geometry (restraint) based approach for the structural interpretation of the NMR data and the evaluation of the resulting molecular structures and Each of these elements is critically important in obtaining a good quality NMR structure.

Figure 4.

Sequential assignment strategy. The process starts from first identifying a Cα/Cβ resonance in the CBCANH spectrum of a residue i, as these resonance frequencies vary depending on the protein fold and amino acid type. Comparing the chemical shifts side‐by‐side on strip plots (right) a new NH group is identified in the CBCA(CO)NH spectrum corresponding to the (i+1)th residue. The CBCANH spectrum is again used to identify the Cα/Cβ resonances of this (i+1)th residue and the process continues until all the resonances are identified. In a CBCANNH spectrum the NH group correlates strongly with its own Cα and Cβ, but the correlation is weak with the preceding residue, whereas the CBCA(CO)NH only correlates the NH group to the preceding Cα and Cβ chemical shifts. This combination of information is used to differentiate the intra‐ or inter‐residue correlations. In the strip plots on the right, the Cα atoms are shown by blue, Cβ atoms in green with the intra‐residue peaks repeated in red.

Figure 5.

Data quality determines structural detail and accuracy. The qualitative relation between the extent of X‐ray diffraction, the resulting amount of available diffraction data, and the quality and detail of the electron density reconstruction and protein structure model are evident from this figure: The crystals are labelled with the nominal resolution in Å as determined by the highest diffraction angle at which X‐ray reflections are observed. Above the crystals is a sketch of the corresponding diffraction pattern, which contains significantly more data at higher resolution. As a consequence, both the reconstruction of the electron density (blue grid) and the resulting structure model (stick model) are much more detailed and accurate. The non‐SI unit Å (10–8 cm or 0.1 nm=10–10 m) is generally used in the crystallographic literature, simply because it is of the same order of magnitude as atomic radii (approximately 0.77 Å for carbon) or bond lengths (approximately 1.54 Å for the C–C single bond). From Biomolecular Crystallography: Principles, Practice, and Application to Structural Biology by Bernhard Rupp. Reproduced by permission of Garland Science/Taylor & Francis LLC © 2011.

Figure 6.

HSQC spectrum of ligand‐free and ligand‐bound protein. The 2‐d 1H–15N heteronuclear single‐quantum coherence (HSQC) NMR spectrum of bacterial methionine aminopeptidase (bMAP) with (right) and without (left) a tightly bound novel inhibitor (Evdokimov et al., ). Note the drastic improvement in the discrimination of the spectrum for the bMAP‐ligand complex compared to the apo‐protein. The crystals of the bMAP‐ligand complex diffracted to 0.9 Å resolution. Image courtesy of Artem Evdokimov, Procter & Gamble Pharmaceuticals, Mason, OH. From Biomolecular Crystallography: Principles, Practice, and Application to Structural Biology by Bernhard Rupp. Reproduced by permission of Garland Science/Taylor & Francis LLC © 2011.

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Further Reading

Cavanagh J, Fairbrother WJ, Palmer AG et al. (2007) Protein NMR Spectroscopy, Second Edition: Principles and Practice. New York: Academic Press.

Doucleff M, Hatcher‐Skeers M and Crane NJ (2011) Pocket Guide to Biomolecular NMR. New York: Springer.

Rhodes G (2006) Crystallography Made Crystal Clear. London, UK: Academic Press.

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Krishnan, VV, and Rupp, B(Jun 2012) Macromolecular Structure Determination: Comparison of X‐ray Crystallography and NMR Spectroscopy. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0002716.pub2]