ABSTRACT
Christian Riekela, Manfred Burghammera, and Dmitry PopovbaEuropean Synchrotron Radiation Facility, B.P.220, F38043 Grenoble Cedex, FrancebHPCAT, Geophysical Laboratory, Carnegie Institution of Washington, 9700 South Cass Ave., Bldg. 434E, Argonne, IL 60439, USA riekel@esrf.fr We review in this article the current status of protein microcrystal-lography and the scope for protein nanocrystallography at third-generation synchrotron radiation sources. Practical issues of sample environments, radiation damage, and sample manipulation will be discussed. 1.1 INTRODUCTIONSynchrotron radiation (SR) centers worldwide are making considerable efforts of developing high-throughput protein crystallography for structural proteomics (Arzt et al., 2005). The production of welldiffracting protein crystals remains, however, the limiting step in protein structure analysis (Derewenda, 2004). Synchrotron Radiation and Structural Proteomics Edited by Eugenia Pechkova and Christian Riekel Copyright © 2012 Pan Stanford Publishing Pte. Ltd. www.panstanford.com
A complementary approach for studying “difficult” protein structures is protein microcrystallography (µPX) (Riekel et al., 2005; Schneider, 2008). “Difficult” implies here proteins which are difficult to crystallize such as membrane proteins. Other reasons for using SR microbeams are the reduction of background scattering from sample environments (Sanishvili et al., 2008) or the study of more perfect domains in a larger crystal. Finally, the mosaic spread can be reduced as the use of microcrystals increases flash-cooling rates and allows using more diluted cryoprotectants (Garman and Mitchel, 1996; Garman and Schneider, 1997; Chinte et al., 2005). An overview on selected µPX beamlines (BLs) which are operational or in the commissioning phase is given in Table 1.1. The present review will focus principally on µPX developments at the ESRF-ID13 BL. Table 1.1 Selected parameters of several µPX beamlines which are operational or in the commissioning phase. µPX capabilities are also planned for SOLEIL, Spring-8, Photon Factory, PETRA III, MAX IV, and NSLS-II. (BL: beamline). SRweb page BLname
1Evans et al., 2007; 2Fischetti et al., 2009. Protein nanocrystallography can be defined as: “using nanotechnology for the production and characterization of protein crystals at the nano-and subnano-scale” (AFM, µPX…) (Pechkova and Nicolini, 2004). The extent to which protein crystallography
with nanometer-sized beams (nanoPX) will find practical applications will depend not only on the scientific interest in studying ultrasmall crystalline domains but also on systematic studies of radiation damage issues, the availability of advanced sample environments including high precision goniometers, sample characterization, and manipulation tools. Pushing the limits to smaller crystals and smaller beam sizes will require the integration of more and more nanotechnology in a µPX BL and annex laboratories, which justifies the term nanocrystallography. Finally, we mention two further complementary approaches to high-throughput PX which do not require single crystals. Small-angle X-ray scattering (SAXS) allows obtaining low-resolution, averaged solution scattering structures (Svergun, 2007). The use of µSAXS (Riekel et al., 2009) is of interest for microfluidic environments and combinatorial approaches (Toft et al., 2008). Lensless imaging by scanning coherent diffraction imaging (CXDI) (Faulkner et al., 2004; Rodenburg and Faulkner, 2004) using coherent beams could provide in the future complimentary real space information in the <30 nm resolution range (Schroer et al., 2008; Huang et al., 2009; Lima et al., 2009; Nishino et al., 2009). 1.2 SR SOURCES AND INSTRUMENTATION
Figure 1.1 Schematic design of mirror focusing system. The horizontal and vertical source size (sh,v) is demagnified at the focus by: sh,v × (F2/F1) and the source divergence (s΄h,v) by: s΄h,v × (F1/F2).Any focusing optical system will reduce the source size and increase the beam divergence as shown schematically in Fig. 1.1 for mirror focusing. A compromise between spot size and divergence at the sample position is therefore required (Riekel et al., 2000).
