Macromolecular Structure Determination by X‐ray Crystallography


For more than 50 years, single‐crystal X‐ray diffraction has remained at the forefront of structural studies of biological macromolecules and complex molecular machines. Diffraction occurs when X‐rays interact with the electrons in the crystallised macromolecule or a complex. Importantly, only amplitudes, not phases, are recorded during an X‐ray diffraction experiment. This ‘phase’ problem is resolved by incorporating electron‐dense, anomalous scatterers into the protein or nucleic acid molecules. Major technical advances in crystal handling, synchrotron beamlines, free‐electron lasers, detector technology, software development and computer hardware have allowed X‐ray crystallography to become essentially a routine method for obtaining high‐resolution images of biomolecules. Much of our insights into macromolecular structure/function relationships are, in fact, based on single‐crystal X‐ray crystallography.

Key Concepts:

  • X‐ray crystallography is one of the fundamental methods of structural biology and remains the most widely used technique to study the structure/function relationships of macromolecules.

  • X‐ray diffraction is not an imaging technique as the scattered X‐ray intensities need to be associated with phases to assemble an image.

  • The phase problem in X‐ray crystallography can be overcome by determining the anomalous scattering generated by suitable heavy atoms.

  • Crystallisation of biological macromolecules remains a bottleneck in X‐ray crystallography.

Keywords: structural biology; structure/function relationships; X‐ray crystallography; diffraction; phase problem

Figure 1.

X‐ray diffraction patterns. (a) The first diffraction pattern of rocksalt (NaCl) recorded in 1911 by Laue and coworkers. (b) A high‐resolution diffraction image of a lysozyme crystal recorded with a prototype CCD detector at beamline A1 at MacCHESS, Cornell, USA.

Figure 2.

The interaction of X‐ray photons with a biological matter. Principally, the electric field of the X‐ray photons induces in‐phase dipole oscillations in the electrons of the sample, which in turn give rise to coherently diffracted radiation. (a) The two electrons in the sample are separated by the distance r, the vectors s0 and s are unit vectors describing the direction of the incident primary beam and the scattered rays, respectively. The path difference gives rise to interference between the scattered beams. (b) Scattering from a crystalline lattice. The incident beam approaching the lattice plane at an angle θ is being reflected from that plane at an equal angle (Glanzwinkel).

Figure 3.

This figure shows a primitive, a face centred and a body centred orthorhombic unit cell. The system is characterised by orthogonal unit cell vectors of different length (a≠b≠c and α=β=γ=90).

Figure 4.

Graphical evaluation of a phase angle. (a) Phase circle or Argand diagrams showing observed amplitudes from the native (FP) and a derivatised form of a protein (FPH′) in the complex plane. The phase angle can assume two different values (A or B) and, thus, the phase ambiguity cannot be resolved by a single derivative. (b) With information from two isomorphous derivatives (FPH′ and FPH″) it is possible to unabmiguously assign a phase value by three intersecting phase circles (B).

Figure 5.

Myoglobin (left panel) and haemoglobin (right panel) were the first proteins for which complete crystallographic structures were obtained at near atomic resolution.

Figure 6.

ABC transporters typically consist of two distinct domains, a transmembrane domain (shown in teal and light orange), and a nucleotide‐binding domain (shown in blue and dark orange). The latter domain is responsible for the binding and the hydrolysis of ATP (shown in red; sodium ions, green spheres).

Figure 7.

The recently determined crystal structure of the 80S yeast ribosome shows a deep channel large enough to accomodate the aminoacylated tRNAs (sites A, P and E), indicates binding sites for elongation factors and reveals a long tunnel which the emerging polypetide chain is using to exit the assembly.



Afonine PV, Grosse‐Kunstleve RW, Echols N et al. (2012) Towards automated crystallographic structure refinement with phenix. refine. Acta Crystallographica Section D 68: 352–367.

Astbury WT and Bell FO (1941) Nature of the intramolecular fold in alpha-keratin and alpha-myosin. Nature 147: 696.

Astbury WT and Street A (1931) X-ray studies of the structures of hair, wool and related fibres. I. General. Philosophical Transactions of the Royal Society of London A230: 75–101.

Ben‐Shem A, Garreau de Loubresse N, Melnikov S et al. (2012) The structure of the eukaryotic ribosome at 3.0 Å resolution. Science 334(6062): 1524–1529.

Bernal JD and Crowfoot D (1934) X-ray photographs of crystalline pepsin. Nature 133: 794–795.

Bernal JD, Fankuchen I and Perutz M (1938) X-ray study of chymotrypsin and haemoglobin. Nature 141: 523–524.

Blaha GM, Polikanov YS and Steitz TA (2012) Elements of ribosomal drug resistance and specificity. Current Opinion in Structural Biology 22(6): 750–758.

Blake CCF, Koenig DF, Mair GA et al. (1965) Structure of hen egg-white lysozyme: a three-dimensional Fourier synthesis at 2 Å resolution. Nature 206: 757–761.

Boutet S, Lomb L, Williams GJ et al. (2012) High-resolution protein structure determination by serial femtosecond crystallography. Science 337: 362–364.

Blanchet CE and Svergun DI (2013) Small-angle X-ray scattering on biological macromolecules and nanocomposites in solution. Annual Review of Physical Chemistry 64: 37–54.

Bragg WH and Bragg WL (1913) The Reflection of X‐rays by Crystals. Proceedings of the Royal Society of London A88: 428–438.

Dawson RJP and Locher KP (2007) Structure of the multidrug ABC transporter Sav1866 from Staphylococcus aureus in complex with AMP-PNP. FEBS Letters 581: 935–938.

DeLano W (2010) The PyMOL Molecular Graphics System, Version 1.0r.

Drenth J, Jansonius JN, Koekoek R, Swen HH and Wolthus BG (1968) Structure of papain. Nature 218: 929–932.

Emsley P and Cowtan K (2004) Coot: model-building tools for molecular graphics. Acta Crystallographica Section D D60: 2126–2132.

Friedrich W, Knipping P and von Laue M (1912) Interferenz-Erscheinungen bei Röntgenstrahlen. Sitzungsberichte der Kgl Bayerischen Akademie der Wissenschaften 1912: 303–322.

Hope H (1988) Cryocrystallography of biological macromolecules: a generally applicable method. Acta Crystallographica Section B 44: 22–26.

Kendrew JC, Dickerson RE, Strandberg BE et al. (1960) Structure of myoglobin: a three-dimensional Fourier synthesis at 2 Å resolution. Nature 185: 422–427.

Ladner RC, Heidner EG and Perutz MF (1977) The structure of horse methaemoglobin at 2.0 Å resolution. Journal of Molecular Biology 114: 385–414.

Langer G, Cohen SX, Lamzin VS and Perrakis A (2008) Automated macromolecular model building for X-ray crystallography using ARP/wARP version 7. Nature Protocols 3(7): 1171–1179.

Lipscomb WF, Coppola JC, Hartsuck JA et al. (1966) Structure of carboxypeptidase A: molecular structure at 6 Å resolution. Journal of Molecular Biology 19: 423–441.

Liu W, Wacker D, Gati C et al. (2013) Serial femtosecond crystallography of G protein-coupled receptors. Science 342: 1521–1524.

Maddox B (2003) The double helix and the ‘wronged heroine’. Nature 421: 407–408.

Pauling L, Corey RB and Branson HR (1951) The structure of proteins: two hydrogen-bonded helical configurations of the polypeptide chain. Proceedings of the National Academy of Sciences of the USA 37: 205–211.

Perutz MF (1951) New X-ray evidence on the configuration of polypeptide chains: polypeptide chains in poly-γ-benzyl-L-glutamate, keratin and haemoglobin. Nature 167: 1053–1054.

Perutz MF, Rossman MG, Cullis AF et al. (1960) Structure of haemoglobin: a three-dimensional Fourier synthesis at 5.5-Å. resolution, obtained by X-Ray analysis. Nature 185: 416–418.

Pettersen EF, Goddard TD, Huang CC et al. (2004) UCSF Chimera–a visualization system for exploratory research and analysis. Journal of Computational Chemistry 25(13): 1605–1612.

Read RJ and McCoy AJ (2011) Using SAD data in phaser. Acta Crystallographica Section D 67(Pt 4): 338–344.

Rodgers DW (1994) Cryocrystallography. Structure 2: 1135–1140.

Schlichting I and Miao J (2012) Emerging opportunities in structural biology with X-ray free-electron lasers. Current Opinion in Structural Biology 22(5): 613–626.

Sheldrick GM and Schneider TR (1997) SHELXL: high resolution refinement. Methods in Enzymology 277: 319–343.

Sigler PB, Blow DM, Matthews BW and Henderson R (1968) Structure of crystalline-chymotrypsin. II. A preliminary report including a hypothesis for the activation mechanism. Journal of Molecular Biology 35: 143–164.

Stuart DI and Abrescia NG (2013) From lows to highs: using low-resolution models to phase X-ray data. Acta Crystallographica Section D 69(Pt 11): 2257–2265.

Terwilliger TC (2003) SOLVE and RESOLVE: automated structure solution and density modification. Methods in Enzymology 374: 22–36.

Vonrhein C, Blanc E, Roversi P and Bricogne G (2007) Automated structure solution with autoSHARP. Methods in Molecular Biology 364: 215–230.

Watson HC (1969) The stereochemistry of the protein myoglobin. Program Stereochemistry 4: 299–305.

Watson JD and Crick FHC (1953a) Molecular structure of nucleic acids; a structure for deoxyribose nucleic acid. Nature 171(4356): 737–738.

Watson JD and Crick FHC (1953b) Genetical implications of the structure of deoxyribonucleic acid. Nature 171(4361): 964–967.

Wider G and Wuethrich K (1999) NMR spectroscopy of large molecules and multimolecular assemblies in solution. Current Opinion in Structural Biology 9: 594–601.

Winn MD, Isupov MN and Murshudov GN (2001) Use of TLS parameters to model anisotropic displacements in macromolecular refinement. Acta Crystallographica Section D 57(Pt 1): 122–133.

Wright CS, Alden RA and Kraut J (1969) Structure of subtilisin BPN' at 2.5 Å resolution. Nature 221(5177): 233–242.

Wuethrich K, Shulman RG and Peisach J (1968) High-resolution proton magnetic resonance spectra of sperm whale cyanometmyoglobin. Proceedings of the National Academy of Sciences of the USA 60: 373–380.

Wyckoff HW, Doscher M, Tsernoglu D et al. (1967) Design of a diffractometer and flow cell system for X-ray analysis of crystalline proteins with applications to the crystal chemistry of ribonuclease-S. Journal of Molecular Biology 27: 563–578.

Further Reading

McPherson A (2009) Introduction to Macromolecular Crystallography [Paperback]. Hoboken, NJ: Wiley & Sons Inc. ISBN 9780470185902.

Rupp B (2010) Biomolecular Crystallography: Principles, Practice, and Application to Structural Biology [Hardcover]. New York, NY: Garland Science. ISBN 9780815340812.

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Jaeger, Joachim(Jul 2014) Macromolecular Structure Determination by X‐ray Crystallography. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1002/9780470015902.a0002723.pub2]