Macromolecular Structure Determination by X‐ray Crystallography

Abstract

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.

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