Introduction to phasing

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.

Radiation damage in macromolecular crystallography: what is it and why should we care?

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.

Selenium incorporation using recombinant techniques

An overview of techniques for recombinant incorporation of selenium and subsequent purification and crystallization of the resulting labelled protein.

A rational approach to heavy-atom derivative screening

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.

Progress in rational methods of cryoprotection in macromolecular crystallography

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.

The magic triangle goes MAD: experimental phasing with a bromine derivative

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.

Know your dose: RADDOSE

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.

Carrying out an optimal experiment

Diffraction data collection parameters leading to optimal data quality are discussed in the context of different applications of these data.

The minimum crystal size needed for a complete diffraction data set

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.

Optimization of data collection taking radiation damage into account

Software implementing a new method for the optimal choice of data-collection parameters, accounting for the effects of radiation damage, is presented.

With phases: how two wrongs can sometimes make a right

Combining experimental phases and those from refinement of very incomplete models significantly improves electron-density maps.

Diffraction data analysis in the presence of radiation damage

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.

Temperature-dependent macromolecular X-ray crystallography

The dynamical behaviour of crystalline macromolecules and their surrounding solvent as a function of cryo-temperature is reviewed.

`Broken symmetries' in macromolecular crystallography: phasing from unmerged data

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.

Experimental phasing: best practice and pitfalls

The pitfalls of experimental phasing are described.

Recent developments in classical density modification

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: combining chain tracing with density modification

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.

Features and development of Coot

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.