Crystal screening

SGX-CAT maintains a direct T1 network connection from the Advanced Photon Source in Illinois to SGX San Diego. Database inquiries are handled over this link. Interactions with the database occur in three ways. An extensive web-based system is used for data entry and retrieval. For crystals generated by external users of the beamline, upload of an electronic spreadsheet transfers the required crystal data attributes to the SGX LIMS. For automated operations, such as crystal screening and data collection, custom scripts place the computed results directly into the database. 

Cohen, A. E., McPhUlips, S. E., Song, J. and MiUer, M. D. (2005). Automation of high-throughput protein crystal screening at SSRL. Synchrotron Radiation News 18, 28-35. 

Zhang, Z., Sauter, N. K., van den Bedem, H., SneU, G. and Deacon, A. M. (2006). Automated diffraction image analysis and spot searching for high-throughput crystal screening. /. Appl. Crystallogr. 39,112-119. 

The main technique employed to set up crystal screens is the vapour diffusion method, either in the hanging drop or sitting drop set up. This method is based on slowly concentrating the droplet solution against a reservoir solution of infinite volmne (ml scale) compared to the volume of the droplet ( xl scale, see Fig. 14.2). Other techniques based on diffusion or counter-diffusion in agarose gels (Biertmnpfel et al., 2002) can also be useful. The... 

Figure 14.3 The Genesis workstation from Tecan. (a) A general viewof the workstation. The two mobile arms move above the deck supporting the microplates, (b) A close-up view of the deck supporting the microplates, (c) The Miniprep 75 workstation is very handy when one needs to prepare crystal screening solutions from mother solutions.

Here are provided non-exhaustive guidelines to interpret the droplet content of crystallization screenings (Fig. 14.9) and possible ways to optimize positive hits. See also Ducruix and Giege (1992) for more details. 

The precession camera (Fig. 4.23), although the more complicated in its motion, produces the simplest diffraction pattern. X rays enter through the black tube at left to strike the crystal, mounted in a goniometer head. Beyond the crystal are an annular-screen holder (smaller black square) and a film holder (larger black square). The remaining machinery moves crystal, screen, and film in a precessing motion about the X-ray beam. 

G.G.Z. Zhang, R.F. Henry, T.B. Borchardt, X. Lou, Efficient co-crystal screening using solution-mediated phase transformation, J. Pharm. Sci. 96 (2007) 990-995. 

During these trials, the chip also produced crystals of two targets that had not been seen by conventional screening. A previously unidentified crystal form of the bacterial 70S ribosome was obtained in three conditions of a sparse matrix of precipitants (Hampton Crystal Screen I), demonstrating that large protein-nudeic acid complexes may be crystallized in chip (C. Hansen, A. Vila-Sanjurjo and J. Cate, personal communication). Crystals of a previously uncrystallized mycobacterial RNase were also obtained from a single experimental condition on chip, whereas no crystals had been observed for this sample despite prior extensive... 

Higginson, P. (2000) Automated crystal screening technology. Presented at 2nd International Symposium on Polymorphism and Crystallisation—Chemical Development Issues, Chester, UK. (Organized by Scientific Update, UK) [92]... 

Wiener MC. The development of membrane protein crystallization screens based upon detergent solution properties. Biophys. J. 2002 82 29a. 

Before crystallization trials, the protein was subjected to gel filtration on Superdex-75 (Pharmacia) in 50 mM sodium/potassium phosphate buffer, pH 7.4, containing 1 mM EDTA, 50 mM 2-mercaptoethanol, 150 mM sodium chloride, 5% glycerol and 5% 2-propanol, as described previously (12). The statine-based inhibitor, LP-149 (Ac-Nal-Val-Sta-Glu-Nal-NH2 e Nal is naphtylalanine and Sta is statine) (Fig. 1), was prepared at Lilly Research Laboratories (K. Hui, unpublished results). Crystallization was carried out at 4 °C using the hanging-drop vapor diffusion method as follows 2.5 //I of the FIV PR(D30N) at 7 mg/ml complexed with LP-149 (1 4 molar ratio) in 50 mM imidazole-HCl pH 7.0 containing ImM EDTA and 1 mM dithiothreitol were mixed with an equal volume of 2 M ammonium sulfate, 0.1 M sodium acetate, pH 4.6 (Hampton Crystal Screen, solution 47). Crystals appeared within a few days and reached the size of 0.2 x 0.2 X 0.4 mm in one week. 

Soak complexes of heavy atoms of various kinds into a crystal. Screen diffraction data from heavy-atom derivatives of the protein for differences in intensities from those in the native data set. Calculate difference Patterson maps and locate the positions of the heavy atoms in the unit cell. 

Obtaiinng X-ray quality crystals is usually the most difficult and time-consuming step of a new structure determination project, notably in the case of a novel, poorly characterized gene product. Recent advances in crystallization robotics and the provision of numerous commercial crystallization screens have considerably simplified the process and shortened the time needed to set up extensive crystallization screeiung experiments, while, at the same time, dramatically reducing the amount of material needed. Nowadays, 5-lOmg of a homogeneous protein sample are... 

Several crystallization-screening protocols have been developed over the past decade to help identify initial crystallization conditions. These include footprint screening (Sutra et al., 1992), sparse matrix sampling (Jancarik and Kim, 1991), statistical methods (Carter and Carter, 1979), and MON-48 (Shieh et al., 1991). Reagent kits for some of these screens are now commercially available (Hampton Research [www.hamptonresearch.com], Emerald BioStructures [www.emeraldbio-structures.com]) and are commonly used in the laboratory. 

Automated systems for vapor diffusion and micro-batch crystallization screening are now commercially available from Cyberlab, Inc. [www.gilson.com/cyberlab.htm] and Douglas Instruments Ltd. [www.douglas.co.uk]. These systems are particularly useful for rapid screening of new samples. 

It is very difficult to make definitive conclusions in a review article on polymorphism because the subject is still evolving. Even as our understanding of this phenomenon improves with more structural data pouring in the complexity of systems being smdied is also increasing. Experimental techniques, procedures and automated protocols are being optimized to carrying out crystallization screens for... 

The main book dealing with the precession method is that of Buerger (1964). The precession method is used to record an undistorted representation of a single plane of RLPs and their associated intensities. In order to achieve this the crystal is carefully set so that the plane of the RLPs is perpendicular to the X-ray beam. The normal to this plane, the zone axis, is then precessed about the X-ray beam axis. A layer-line screen with a transparent annulus allows RLPs of the plane of interest to pass through to the film. The screen intercepts all other diffracted rays. The motion of the crystal, screen and film are coupled together to maintain the coplanarity of the film, the screen and the zone. 

Modern industrial crystallization theory dates essentially from the work of Randolph and Larson (1962), who developed the concept of population density insofar as it applies to mixed suspension, mixed product removal crystallization equipment. A detailed treatment of the development of these concepts is given in Chapter 4 of this volume. The population density concept is useful because it allows the user to take the data developed from a crystal screen analysis along with knowledge of the operating parameters of the crystallizer, such as the retention (drawdown) time, slurry density. 

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