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Crystallographic methods and protocols, methods in molecular biology, volume 56
trends in analytical
chemistry
Crystallographic
XI
vol.1 6, no.6, 1997
methods
Cystallographic Methods and Protocols, Methods in Molecular Biology, Volume 56, C. Jones, B. Mullex M.R. Sanderson, Humana Press, 1996, (xii + 394 pages), ISBN 0-89603-259-O Crystallographic Methods and Protocols, Volume 56 in the series Methods in Molecular Biology, details up-todate procedures of macromolecular X-Ray structure determination. Thanks to the current state of the art of three-dimensional structure determination, a detailed knowledge of structure is more available to the biochemist and molecular biologist than ever before. The result is a subtle change in approach to biochemical problems where three-dimensional structural details are more likely to be considered in the design of experiments. With this evolution of thought, an understanding of the nature of the crystallographic experiment becomes useful as the segregation between sample provider and structure solver becomes less apparent. This volume targets those advanced biochemists and molecular biologists who are interested in at least understanding and possibly participating in the steps involved in a full crystal structure determination. Thus the impetus for the publication is to offer a how-to guide for the non-crystallographer to take part in the solution of the structure of interest. A standard overview of the diffraction experiment is given in Chapter 1 and the chapter is appended by an excellent bibliography of additional texts and other sources. Chapter 2 sets this volume apart from other similar texts by addressing methods of overexpression and isolation of proteins prior to any attempt at crystallization. The success of the crystallographic experiment depends on the ability to grow crystals of sufficient quality. The molecular biologist is in a position to engineer a protein to ultimately optimize crystal and heavy atom derivative quality. Chapter 3 covers mounting of crystals and preliminary crystal characterization and is the launching point
for the practical X-Ray experiment. Chapter 4 describes modern methods of home source X-Ray data collection for which dedicated equipment is necessary. At this stage, the neophyte is likely to require assistance from a local X-Ray specialist. Subsequent to data collection, phase information is obtained from the preparation of heavy atom derivatives and comparison of heavy atom data with that from native crystals. Chapters 4 and 5 deal with the collection of heavy atom derivative data. The MAD multi-anomalous dispersion) technique described in Chapter 5 requires the availability of tunable wavelengths at a synchrotron source. Because of the necessity for specialized synchrotron radiation, the technique is, althoughvery powerful, not routine. Instead, the more conventional multiple isomorphous replacement (MIR) method is more typical and is covered in Chapter 6. A common alternative to the MIR method is possible when coordinates of similar protein structures are available where the crystallographer can use known structural information to solve the phase problem for the unknown structure. This method, called molecular replacement (MR), is described in Chapter 7. The MR techinvolves first finding the nique orientation of a similar (i.e. structurally homologous) molecule or fragment in the cell of the unknown crystal followed by a three-dimensional translation search. The chapter also touches upon the different approaches of the various computer programs in use for MR structure solution. Once the structure is solved, the model must be built into resulting electron density maps. These maps, calculated from experimental phases, may require further improvement in order to make them interpretable, especially in the case of an MIR solution. Chapter density 8 on modification suggests ways to improve the initial electron density map. This is generally an iterative procedure where the molecular envelope is identified and information about crystal solvent and non-crystallographic symmetry is used to improve phase
quality. Refinement of the initial model is carried with respect to observed intensity data. A number of computer programs are available for this purpose with wide-ranging approaches to the refinement procedure. An overview of commonly used programs, from least-squares methods to energy minimization by simulated annealing, is given in Chapter 9. The R factor as a means of judging correctness of the model is described. Also discussed are the available graphics programs used to adjust the model in the improved electron density map after successive rounds of refinement. Chapter 10 concentrates on some advances in refinement using simulated annealing techniques and introduces the concept of validity by monitoring the free R factor. Chapters 11 through 14 describe extensions of standard crystallographic techniques required for the specific structural problems of oligonucleotides, protein-DNA complexes, viruses and membrane proteins consecutively. Advances in hardware and software for macromolecular crystallography are frequent and the editors are to be commended for offering such a timely edition. Written for the biologist interested in embarking on a first structure solution, this volume will serve crystallographers also as a general update to the current status of the field. MARY TURNER Dr M. Turner is at the Division of Research Biochemistry, Hospital for Sick Children, Toronto, Canada.
Notice to Advertisers TrAC offers a unique opportunity to inform all those who use and develop analytical methods in research, industrial, government and clinical laboratories about new products and publications. TrAC has: l worldwide circulation and l special rates for series advertisement.
chemistry
Crystallographic
XI
vol.1 6, no.6, 1997
methods
Cystallographic Methods and Protocols, Methods in Molecular Biology, Volume 56, C. Jones, B. Mullex M.R. Sanderson, Humana Press, 1996, (xii + 394 pages), ISBN 0-89603-259-O Crystallographic Methods and Protocols, Volume 56 in the series Methods in Molecular Biology, details up-todate procedures of macromolecular X-Ray structure determination. Thanks to the current state of the art of three-dimensional structure determination, a detailed knowledge of structure is more available to the biochemist and molecular biologist than ever before. The result is a subtle change in approach to biochemical problems where three-dimensional structural details are more likely to be considered in the design of experiments. With this evolution of thought, an understanding of the nature of the crystallographic experiment becomes useful as the segregation between sample provider and structure solver becomes less apparent. This volume targets those advanced biochemists and molecular biologists who are interested in at least understanding and possibly participating in the steps involved in a full crystal structure determination. Thus the impetus for the publication is to offer a how-to guide for the non-crystallographer to take part in the solution of the structure of interest. A standard overview of the diffraction experiment is given in Chapter 1 and the chapter is appended by an excellent bibliography of additional texts and other sources. Chapter 2 sets this volume apart from other similar texts by addressing methods of overexpression and isolation of proteins prior to any attempt at crystallization. The success of the crystallographic experiment depends on the ability to grow crystals of sufficient quality. The molecular biologist is in a position to engineer a protein to ultimately optimize crystal and heavy atom derivative quality. Chapter 3 covers mounting of crystals and preliminary crystal characterization and is the launching point
for the practical X-Ray experiment. Chapter 4 describes modern methods of home source X-Ray data collection for which dedicated equipment is necessary. At this stage, the neophyte is likely to require assistance from a local X-Ray specialist. Subsequent to data collection, phase information is obtained from the preparation of heavy atom derivatives and comparison of heavy atom data with that from native crystals. Chapters 4 and 5 deal with the collection of heavy atom derivative data. The MAD multi-anomalous dispersion) technique described in Chapter 5 requires the availability of tunable wavelengths at a synchrotron source. Because of the necessity for specialized synchrotron radiation, the technique is, althoughvery powerful, not routine. Instead, the more conventional multiple isomorphous replacement (MIR) method is more typical and is covered in Chapter 6. A common alternative to the MIR method is possible when coordinates of similar protein structures are available where the crystallographer can use known structural information to solve the phase problem for the unknown structure. This method, called molecular replacement (MR), is described in Chapter 7. The MR techinvolves first finding the nique orientation of a similar (i.e. structurally homologous) molecule or fragment in the cell of the unknown crystal followed by a three-dimensional translation search. The chapter also touches upon the different approaches of the various computer programs in use for MR structure solution. Once the structure is solved, the model must be built into resulting electron density maps. These maps, calculated from experimental phases, may require further improvement in order to make them interpretable, especially in the case of an MIR solution. Chapter density 8 on modification suggests ways to improve the initial electron density map. This is generally an iterative procedure where the molecular envelope is identified and information about crystal solvent and non-crystallographic symmetry is used to improve phase
quality. Refinement of the initial model is carried with respect to observed intensity data. A number of computer programs are available for this purpose with wide-ranging approaches to the refinement procedure. An overview of commonly used programs, from least-squares methods to energy minimization by simulated annealing, is given in Chapter 9. The R factor as a means of judging correctness of the model is described. Also discussed are the available graphics programs used to adjust the model in the improved electron density map after successive rounds of refinement. Chapter 10 concentrates on some advances in refinement using simulated annealing techniques and introduces the concept of validity by monitoring the free R factor. Chapters 11 through 14 describe extensions of standard crystallographic techniques required for the specific structural problems of oligonucleotides, protein-DNA complexes, viruses and membrane proteins consecutively. Advances in hardware and software for macromolecular crystallography are frequent and the editors are to be commended for offering such a timely edition. Written for the biologist interested in embarking on a first structure solution, this volume will serve crystallographers also as a general update to the current status of the field. MARY TURNER Dr M. Turner is at the Division of Research Biochemistry, Hospital for Sick Children, Toronto, Canada.
Notice to Advertisers TrAC offers a unique opportunity to inform all those who use and develop analytical methods in research, industrial, government and clinical laboratories about new products and publications. TrAC has: l worldwide circulation and l special rates for series advertisement.