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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: 02 December 2021

Soaking Techniques

Soaking Techniques

Chapter:
13 (p.365) Soaking Techniques
Source:
Crystallization of Nucleic Acids and Proteins
Author(s):

E. A. Stum

T. Gleichmann

Publisher:
Oxford University Press
DOI:10.1093/oso/9780199636792.003.0017

Once crystals of a macromolecule are obtained there are many circumstances where it is necessary to change the environment in which the macromolecule is bathed. Such changes include the addition of inhibitors, activators, substrates, products, cryo-protectants, and heavy atoms to the bathing solution to achieve their binding to the macromolecule, which may have sufficient freedom to undergo some conformational changes in response to these effectors. In fact, macromolecular crystals have typically a high solvent content which ranges from 27-95% (1, 2). Although, part of this solvent, ‘bound solvent’ (typically 10%) is tightly associated with the protein matrix consisting of both water molecules and other ions that occupy well defined positions in refined crystal structure it can be replaced in soaking experiments, at a slower rate compared to the ‘free solvent’. In this chapter we will consider the relative merits of various methods for modifying crystals, the restraints that the lattice may impose on the macromolecule, and the relative merits of soaking compared to co-crystallization. The size and configuration of the channels within the lattice of macromolecular crystals will determine the maximum size of the solute molecules that may diffuse in. The solvent channels are sufficiently large to allow for the diffusion of most small molecules to any part of the surface of the macromolecule accessible in solution except for the regions involved in crystal contacts, although in some cases lattice forces may hinder conformational changes or rearrangements of the macromolecule in crystal. In other cases, the forces that drive the conformational changes can be sufficient to overcome the constraints imposed by the crystalline lattice leading to the disruption of intermolecular and crystal contacts resulting in the cracking and dissolution of the crystals. Some lattices may be more flexible and capable of accommodating conformational changes, and while crystals may crack initially, they may subsequently anneal into a new rearrangement and occasionally improve their crystallinity. In general small changes are easily accommodated and many macromolecules maintain their activity in the crystalline state. This is exploited in time-resolved crystallography to obtain structural information of transition states of enzymes.

Keywords:   additives, cross-linking, gold compounds, iridium compounds, isomorphous replacement, krypton, lead compounds, niobium compounds, osmium compounds, platinum compounds

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