Macromolecular Structure Determination by X-ray Crystallography

Joachim Jaeger, Wadsworth Center, Albany, New York, USA

Published online: May 2005

DOI: 10.1038/npg.els.0002723

Full Article on Wiley Online Library

X-ray diffraction is a well-established method to elucidate the atomic structure of single-crystal macromolecules. An image of the macromolecule forming the crystal cannot be directly recorded as the X-ray phase information is lost during the diffraction experiment. Through systematic variation of the chemical content in the crystal and/or through small changes in the wavelength of the incident X-ray beam, however, a sharp image can be reconstituted computationally. Within the Protein Data Bank, the vast majority of three-dimensional structures available have been determined using X-ray diffraction. These structures are used to correlate macromolecular structure with function, to study molecular mechanisms and serve as templates for structure-based drug design of novel therapeutic agents for the treatment of many diseases.

Keywords: diffraction; atomic structure; phase problem; crystallization

	Figure 1. X-ray diffraction patterns. (a) The first diffraction pattern of rocksalt (NACL) recorded in 1911 by Laue and co-workers. (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 matter. Principally, the electric field of the X-ray photons induces in-phase dipole oscillations in the electrons of the two electrons in 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 crystalline lattice. The incident beam approaching the lattice plane at an angle is reflected from that plane at an equal angle (Glanzwinkel).
	Figure 3. This figure shows a primitive, a C-centred, a face-centred and a body-centred orthorhombic unit cell. The orthorhombic system is characterized by orthogonal unit cell vectors of different lengths (a ¹ b ¹ c and = = = 90°).
	Figure 4. Graphical evaluation of a phase angle. (a) Phase circle or Argand diagrams showing observed amplitudes from the native (F_P) and a derivatized form of a protein (F_PH’) 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 (F_PH’ and F_PH’’) it is possible to unambiguously assign a phase value by three intersecting phase circles (B).
	Figure 5. Myoglobin (a) and haemoglobin (b), the first proteins for which full three-dimensional structures were determined at high resolution. (a) Note the electron density corresponding to the haem cofactor in the V-shaped binding pocket. The map is contoured 2.5 Å above the mean electron density. (b) The heterodimer of haemoglobin. Like other members of this family of proteins, it consists entirely of helices (secondary structure). Both proteins play a crucial role in oxygen storage and transport in the muscle.
	Figure 6. Photosynthetic reaction centres (RC) are crucial catalysts in the photosynthetic process, perhaps the most important chemical reaction in the biosphere. The conversion of light to chemical energy is a prerequisite for all higher life on earth. RCs are large multiprotein complexes located in the outer membranes of plants and bacteria. The X-ray structure of the reaction centre is the first structure of an integral membrane protein determined at high resolution. There are four protein chains: H (yellow), L (blue) and M (green) subunits, and cytochrome (red).
	Figure 7. The structure of the Bluetongue virus ( BTV) core has a diameter of 700 Å and represents the largest particle to date solved by X-ray crystallography. The structure illustrates in atomic detail how nearly 1000 protein subunits self-assemble and interact to form a transcriptionally active compartment.
	Figure 8. The structure of the 50S ribosomal subunit has been determined and refined at 2.4 Å resolution, while the 30S particle has been determined to 2.8 Å. The high-resolution structures of the particle show in detail the binding sites for the amino-acylated tRNAs and for elongation factors, and a long tunnel that is used as an exit by the emerging polypeptide chain. The structure of the 50S subunit also reveals that large portions of the subunit are built up from RNA. In fact, the active site entirely consists of RNA, implying that the ribosome is a ribozyme.

Further Reading
	Ban N, Nissen PB, Hansen J, Moore PB and Steitz TA (2000) The complete atomic structure of the large ribosomal subunit at 2.4 Å resolution. Science 289: 905–920.
	Deisenhofer J, Epp O, Miki K, Huber R and Michel H (1984) X-ray structure analysis of a membrane protein complex. Electron density map at 3 Å resolution and a model of the chromophores of the photosynthetic reaction center from Rhodopseudomonas viridis. Journal of Molecular Biology 180(2): 385–398.
	book Drenth JD (1994) Principles of Protein Crystallography. Berlin: Springer-Verlag.
	Grimes JM, Burroughs JN and Gouet P et al. (1998) The atomic structure of the bluetongue virus core. Nature 395(6701): 470–478.
	Hendrickson WA, Horton JR and LeMaster DM (1990) Selenomethionyl proteins produced for analysis by multiwavelength anomalous diffraction (MAD): a vehicle for direct determination of three-dimensional structure. EMBO Journal 9(5): 1665–1672.
	book Perutz MF (1992) Protein Structure – New Approaches to Disease and Therapy. New York: WH Freeman.
	Wimberly BT, Brodersen DE and Clemons WM Jr et al. (2000) Structure of the 30S ribosomal subunit. Nature 407: 327–339.

Article Title:	Macromolecular Structure Determination by X-ray Crystallography
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Macromolecular Structure Determination by X-ray Crystallography

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