International
Tables for
Crystallography
Volume F
Crystallography of biological macromolecules
Edited by E. Arnold, D. M. Himmel and M. G. Rossmann

International Tables for Crystallography (2012). Vol. F, ch. 8.1, pp. 197-200   | 1 | 2 |

Section 8.1.8. Scientific utilization of SR in protein crystallography

J. R. Helliwella*

aDepartment of Chemistry, University of Manchester, M13 9PL, England
Correspondence e-mail: john.helliwell@manchester.ac.uk

8.1.8. Scientific utilization of SR in protein crystallography

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There are a myriad of applications and results of the use of SR in crystallography. Helliwell (1992)[link] gave an extensive survey and tabulations of SR and macromolecular crystallography applications; Chapter 9 therein concentrates on anomalous scattering and Chapter 10 on high resolution, large unit cells, small crystals, weak scattering efficiency and time-resolved data collection. The field has expanded so dramatically, in fact, that an equivalent survey today would be vast. Table 8.1.4.1[link] lists the web pages of the facilities, where the specifications and details of the beamlines can be found (e.g. all the publications at Daresbury in the protein crystallography area organized by beamline instrument are to be found at http://dlwebres.dl.ac.uk/dl_public/publications/index.jsp ). The examples below cite extreme cases of the large unit cell (virus and multi-macromolecular) cases, weak anomalous-scattering signal in MAD, fast time-resolved Laue studies and the ultra-high-resolution and even valence-density structure determinations to date. Another phasing technique involving multiple (`n-beam') diffraction is also being applied to proteins [Weckert & Hümmer (1997)[link] at the ESRF and the NSLS]. These examples at least indicate the present bounds of capability of the various sub-fields of SR and macromolecular crystallography.

8.1.8.1. Atomic and ultra-high-resolution macromolecular crystallography

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The use of high SR intensity, cryo-freezing of a protein crystal to largely overcome radiation damage and sensitive, automatic area detectors (CCDs and/or image plates) is allowing diffraction data to be recorded at resolutions equivalent to smaller-molecule (chemical) crystallography. In a growing number of protein crystal structure studies, atomic resolution (1.2 Å or better) is achievable (Dauter et al., 1997[link]). The `X-ray data to parameter' ratio can be favourable enough for single and double bonds, e.g. in carboxyl side chains, to be resolved [Fig. 8.1.8.1[link]; Deacon et al. (1997)[link] for concanavalin A at 0.94 Å resolution]. Along with this bond-distance precision, one can see the reactive proton directly. This approach complements H/D exchange neutron diffraction studies. Neutron studies have expanded in scope by employing Laue geometry in a synergistic development with SR Laue diffraction (Helliwell & Wilkinson, 1994[link]; Helliwell, 1997b[link]; Habash et al., 1997[link], 2000[link]); a comprehensive survey of neutron macromolecular crystallography, instruments and results in determining the atomic details of protonation and hydration has been given by Blakeley (2009)[link]. In particularly well ordered protein structure cases, valence-electron-density descriptions are possible for those atoms with B factors ~<3 Å2 [see e.g. Guillot et al. (2008)[link] and Luger (2007)[link]]. The scope and accuracy of protein crystal structures has been transformed. A diffraction-component precision index for characterizing the overall precision of protein structures has been given by Cruickshank (1999)[link] and cast in terms of experimental parameters by Blow (2002)[link].

[Figure 8.1.8.1]

Figure 8.1.8.1 | top | pdf |

Determination of the protonation states of carboxylic acid side chains in proteins via hydrogen atoms and resolved single and double bond lengths. After Deacon et al. (1997)[link] using CHESS. Reproduced by permission of The Royal Society of Chemistry.

8.1.8.2. Small crystals

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Compensating for small crystal sample volume by increasing the intensity at the sample has been of major interest from the outset, and tests showed that the use of ~20 µm-sized samples is feasible (Hedman et al., 1985[link]). Third-generation high spectral brightness sources were optimized for this application via micron-sized focal spot beams, as described in the ESRF Foundation Phase Report (1987)[link]. Applications of the ESRF microfocus beamline diffractometer (Perrakis et al., 1999[link]) include as an example the determination of the structure of the bacterio­rhodopsin crystal at high resolution from micro-crystals (Pebay-Peyroula et al., 1997[link]). Experiments using extremely thin plates involving only 1000 protein molecular layers are described by Mayans & Wilmanns (1999)[link] on the BW7B wiggler beamline at DESY, Hamburg. A variety of small crystals and SR, including tabulated sample scattering efficiencies, can be found in Helliwell (1992)[link], pp. 410–414 and more recently in Riekel et al. (2005)[link]. The ESRF Upgrade (Fig 8.1.2.2[link]) will push this into the sub-micron territory.

8.1.8.3. Time-resolved macromolecular crystallography

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Time-resolved SR Laue diffraction of light-sensitive proteins, such as CO Mb studied with sub-nanosecond time resolution in pump–probe experiments (see Srajer et al., 1996)[link], have shown direct structural changes as a function of time. Enzymes, likewise, are being studied directly by time-resolved methods via a variety of reaction initiation methods, including pH jump, substrate diffusion and light flash of caged compounds pre-equilibrated in the crystal. Flash freezing is increasingly used to trap molecular structures at optimal times in a reaction determined either by microspectrophotometry or repeated Laue `flash photography'. Enzyme reaction rates can be altered through site-directed mutagenesis (e.g. see Niemann et al., 1994[link]; Helliwell et al., 1998[link]) and matched to diffraction-data acquisition times. For overviews, see the books edited by Cruickshank et al. (1992)[link] and Helliwell & Rentzepis (1997)[link], the recent review by Bourgeois & Weik (2009)[link] and the companion chapter by Moffat (Chapter 8.2[link] in this volume).

8.1.8.4. Multi-macromolecular complexes

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There is now a wealth of results in this field and the reader must be referred to books such as Liljas et al. (2009)[link] for a detailed exposition. Multi-macromolecular complexes, such as viruses (Rossmann et al., 1985[link]; Acharya et al., 1989[link]; Liddington et al., 1991[link]) (Fig. 8.1.8.2[link]), the nucleosome (Luger et al., 1997[link]), light-harvesting complex (McDermott et al., 1995[link]) and the 13-subunit membrane-bound protein cytochrome c oxidase (Tsukihara et al., 1996[link]), and large-scale molecular assemblies like muscle (Holmes, 1998[link]) are very firmly recognizable as biological entities whose crystal structure determinations relied on SR. These single-crystal structure determinations involved extremely large unit cells and became tractable despite very weak scattering strength. The crystals often showed extreme sensitivity to radiation (hundreds, even a thousand, crystals have been used to constitute a single data set). Cryocrystallography radiation protection is now used extensively in crystallographic data collection and was critical for work with ribosome crystals (Hope et al., 1989[link]); SR has been essential for these structure determinations (see e.g. Yonath, 1992[link]; Yonath et al., 1998[link]; Ban et al., 1998[link]; Wimberley et al., 2000[link]; Noller, 2005[link]). A very large multi-protein complex solved using data from the Daresbury SRS wiggler is the F1 ATPase structure (Fig. 8.1.8.3[link]), for which a share in the Nobel Prize for Chemistry in 1997 was awarded to John Walker in Cambridge. The structure (Abrahams et al., 1994[link]; Abrahams & Leslie, 1996[link]) and the amino-acid sequence data, along with fluorescence microscopy, show how biochemical energy is harnessed to drive the proton pump across biological membranes, thus corroborating hypotheses about this process made over many years. This study, made tractable by the SRS wiggler high-intensity protein crystallography station (Fig. 8.1.4.1[link]), illustrates the considerable further scope that became possible with third-generation SR sources, such as the 780 Å diameter blue tongue virus (Grimes et al., 1997[link], 1998[link]) and the nucleosome core particle (Luger et al., 1997[link]). Spectacular progress has been made in the structural biology of photosynthesis using SR sources, which is not only yielding answers on this vital natural process but also stimulating much research to help address artificial energy sources based on this natural system. This research could have profound climate-change impacts with `greener energy sources'. One such structure is the Photosystem II (PSII; see Fig. 8.1.8.4[link]). This topic has been reviewed recently by Barber (2009)[link]; the atomic details of the Mn4Ca cluster of PSII have proved to be especially challenging to X-ray study (Yano et al., 2005[link]). These large-scale molecular assemblies often combine electron-microscope and diffraction techniques with SR X-ray crystallography and diffraction for low-to-high resolution detail, respectively.

[Figure 8.1.8.2]

Figure 8.1.8.2 | top | pdf |

A view of SV40 virus (based on Liddington et al., 1991[link]) determined using data recorded at the SRS wiggler station 9.6 (Fig. 8.1.4.1a[link]).

[Figure 8.1.8.3]

Figure 8.1.8.3 | top | pdf |

The protein crystal structure of F1 ATPase, one of the largest non-symmetrical protein structure complexes, solved using SR data recorded on an image plate at the SRS wiggler 9.6, Daresbury. The scale bar is 20 Å long. Reprinted with permission from Nature (Abrahams et al., 1994[link]). Copyright (1994) MacMillan Magazines Limited.

[Figure 8.1.8.4]

Figure 8.1.8.4 | top | pdf |

Side view of the structure of Photosystem II, the water-splitting enzyme of photosynthesis, determined using X-ray crystallography based on data recorded at the SLS and ESRF (Ferreira et al., 2004[link]). Kindly provided by Professor So Iwata, Imperial College, London.

8.1.8.5. Optimized anomalous dispersion (MAD), improved multiple isomorphous replacement (MIR) data and `structural genomics'

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Rapid protein structure determination via the MAD method, notably involving seleno protein variants (Hendrickson et al., 1990[link]) as well as xenon pressure derivatives (see e.g. Schiltz et al., 1997[link]; Cianci et al., 2001[link]), and improved heavy-atom isomorphous replacement data are removing a major bottleneck in protein crystallography: that of phase determination. Databases of successful heavy-atom compounds (see e.g. Sugahara et al., 2009[link]) have been compiled and are increasingly sophisticated. Overall, as the number of protein structures in the Protein Data Bank doubles every few years, the possibility of considering whole genome-level structure determinations arises (Chayen et al., 1996[link]; Chayen & Helliwell, 1998[link]). The human genome comprises some 35 000 genes. Of these, some 40% are coding for membrane-bound proteins, which are more difficult to crystallize. Since a MAD protein crystal structure currently requires less than 1 day of SR BM beamtime, the new `bottlenecks' are protein production and crystallization. Thus, structural genomics projects have established `pipelines' for protein structure determination with a view to creating a complete `protein folds space'. This approach, along with homology modelling and genetic alignment techniques, opens the immense potential for structural genomics to yield huge numbers of experimentally derived protein structures and thereby a much better basis for understanding and controlling disease through structure-based drug design and discovery [for an early description see Bugg et al. (1993)[link], for an example of pharmaceutical company collaboration see the Industrial Macromolecular Crystallography Association (IMCA) Collaborative Access Team at APS in Chicago (http://www.imca.aps.anl.gov/ ) and for a recent European perspective see e.g. Maclean et al. (2006)[link].]

8.1.8.6. Radiation damage

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The successes of these sources in macromolecular crystallography have been spectacular, so much so that provision of such beamlines now accounts for ~40% of the APS and ESRF insertion-device sectors. Nevertheless, X-radiation damage has been a continuing concern not least as the `cutting edge' of capability to study ever-smaller crystals and ever-larger unit cells at synchrotron-radiation sources has been improved and optimized. A notable development has been a series of International Workshops on X-ray Damage to Crystalline Biological Samples. Most of these workshops have resulted in special issues of the Journal of Synchrotron Radiation (Volume 9 part 6, Volume 12 part 3, Volume 14 part 1 and Volume 16 part 2). The latest of these has a mini-review by Garman & Nave (2009)[link].

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