April 2010 issue
Experimental phasing and radiation damage
Proceedings of the CCP4 study weekend
Cover illustration: Speakers at the CCP4 Study Weekend 2009. Front row, left to right: James Holton, Arwen Pearson, Zbigniew Dauter, Elspeth Garman, Tobias Beck, Zbyszek Otwinowski. Middle row, left to right: Alexander Popov, Sean McSweeney, Garry Taylor, Helen Walden, Karthik Paithankar, Marc Schiltz, Gerard Bricogne. Back row, left to right: Kevin Cowtan, Martin Weik, Clemens Vonrhein, Peter Sun, Douglas Juers, George Sheldrick, Susan Lea, Airlie McCoy.
This introductory paper to the CCP4 weekend on experimental phasing introduces the concept of the `phase problem' for non-experts. Modern methods of phasing are explored, including some recent examples that can be downloaded as tutorials.
The basic causes of the radiation damage inflicted on macromolecular crystals during diffraction experiments are summarized, as well as the current state of research which attempts to understand and to mitigate it.
An overview of techniques for recombinant incorporation of selenium and subsequent purification and crystallization of the resulting labelled protein.
In order to overcome the difficulties associated with the `classical' heavy-atom derivatization procedure, an attempt has been made to develop a rational crystal-free heavy-atom-derivative screening method and a quick-soak derivatization procedure which allows heavy-atom compound identification.
Measurements of the average thermal contractions (294→72 K) of 26 different cryosolutions are presented and discussed in conjunction with other recent advances in the rational design of protocols for cryogenic cooling in macromolecular crystallography.
5-Amino-2,4,6-tribromoisophthalic acid is used as a phasing tool for protein structure determination by MAD phasing. It is the second representative of a novel class of compounds for heavy-atom derivatization that combine heavy atoms with amino and carboxyl groups for binding to proteins.
The program RADDOSE computes the dose absorbed by a macromolecular crystal and here a guide is provided to help to ensure the proper use of the program. In the new version (v.3) described here, modifications to include the energy deposited owing to Compton scattering have been made.
Diffraction data collection parameters leading to optimal data quality are discussed in the context of different applications of these data.
A formula for absolute scattering power is derived to include spot fading arising from radiation damage and the crystal volume needed to collect diffraction data to a given resolution is calculated.
Software implementing a new method for the optimal choice of data-collection parameters, accounting for the effects of radiation damage, is presented.
Combining experimental phases and those from refinement of very incomplete models significantly improves electron-density maps.
Radiation-induced decay of crystal diffraction and additional specific chemical changes of macromolecules forming the crystal lattice are currently two of the main limiting factors in the acquisition of macromolecular diffraction data and macromolecular structure determination. Data-processing and phasing protocols are discussed in the context of radiation-induced changes.
The dynamical behaviour of crystalline macromolecules and their surrounding solvent as a function of cryo-temperature is reviewed.
Site-specific radiation damage and anisotropy of anomalous scattering can induce intensity differences in symmetry-related reflections. If the data are kept unmerged, these symmetry-breaking effects can become a source of phase information.
The pitfalls of experimental phasing are described.
Several new methods are evaluated for use in the improvement of experimental phases in the framework of a classical density-modification calculation. These methods have been implemented in a new computer program, Parrot.
Experimental phasing with SHELXC/D/E has been enhanced by the incorporation of main-chain tracing into the iterative density modification; this also provides a simple and effective way of exploiting noncrystallographic symmetry.
Coot is a molecular-graphics program designed to assist in the building of protein and other macromolecular models. The current state of development and available features are presented.