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Crystallization of Nucleic Acids and ProteinsA Practical Approach$
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Arnaud Ducruix and Richard Giegé

Print publication date: 1999

Print ISBN-13: 9780199636792

Published to Oxford Scholarship Online: November 2020

DOI: 10.1093/oso/9780199636792.001.0001

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PRINTED FROM OXFORD SCHOLARSHIP ONLINE (oxford.universitypressscholarship.com). (c) Copyright Oxford University Press, 2021. All Rights Reserved. An individual user may print out a PDF of a single chapter of a monograph in OSO for personal use. date: 24 September 2021

Biochemical Aspects and Handling of Macromolecular Solutions and Crystals

Biochemical Aspects and Handling of Macromolecular Solutions and Crystals

Chapter:
2 (p.17) Biochemical Aspects and Handling of Macromolecular Solutions and Crystals
Source:
Crystallization of Nucleic Acids and Proteins
Author(s):

B. Lorber

R. Giegé

Publisher:
Oxford University Press
DOI:10.1093/oso/9780199636792.003.0006

The quality and quantity of the macromolecular samples are important prerequisites for successful crystallizations. Proteins and nucleic acids extracted from living cells or synthesized in vitro differ from small molecules by additional properties intrinsic to their chemical nature and their larger size. They are frequently difficult to prepare at a high degree of purity and homogeneity. Besides traces of impurities, harsh treatments may decrease their stability and activity through different kinds of alterations. Consequently, the quality of biomacromolecules depends on the way they are prepared and handled. As a general rule purity and homogeneity are regarded as conditions sine qua non. Accordingly, purification, stabilization, storage, and handling of macromolecules are essential steps prior to crystallization attempts. Other difficulties in crystal growth may come from the source of the biological material. It is advisable to have at disposal a few milligrams of material when starting first crystallization trials although structures were solved with submilligram quantities of protein (1). Once crystals suitable for X-ray analysis can be produced, additional material is often needed to improve their quality and size and to prepare heavy-atom derivatives. It is thus essential that isolation procedures are able to supply enough fresh material of reproducible quality. Similar situations are encountered with multi-macromolecular assemblies (e.g. viruses, nucleosomes, ribosomal particles, or their subunits). This chapter discusses biochemical methods used to prepare and characterize macromolecules intended for crystallization assays. Practical aspects concerning manipulation and qualitative analyses of soluble proteins will be emphasized. The cases of nucleic acids and membrane proteins are described in more detail in Chapters 8 and 9. Peculiar aspects of molecular biology that are important for crystallogenesis are presented in Chapter 3. They include the design of engineered macromolecules with new physical properties or modified to simplify purification or crystallographic analysis. Finally, methods for identification of macromolecular content of crystals and measurements of their density are presented as well. Many biological functions are sustained by classes of proteins and nucleic acids universally present in living organisms so that the source of macromolecules may seem unimportant. In fact, better crystallization conditions or better diffracting crystals are frequently found by switching from one organism to another.

Keywords:   ageing, cross-linking, expression systems, gel electrophoresis, handling samples, homogeneity of samples, impurities, microheterogeneity of samples, nuclease inhibitors

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