The phase problem

An overview of macromolecular phasing methods for those new to structural biology.

New ways of looking at experimental phasing

The application of likelihood has had a great impact on experimental phasing. New approaches based on multivariate statistics give new insights into current methods and suggest areas for future improvement.

Heavy-atom derivatization

The practical procedures involved in derivatizing protein crystals with heavy-atom compounds are discussed.

A new class of lanthanide complexes to obtain high-phasing-power heavy-atom derivatives for macromolecular crystallography

Seven gadolinium complexes used as highly concentrated solutions for co-crystallization or soaking experiments are shown to be a very powerful tool for obtaining heavy-atom derivatives for macromolecular structure determination. SAD phasing experiments have successfully been carried out with several proteins of known structure, as well as with a 450 kDa protein assembly of previously unknown structure.

Triiodide derivatization in protein crystallography

The procedures for preparing triiodide derivatives are described and examples of SAD and SIRAS phasing for six test proteins using conventional and synchrotron sources are presented.

Selling candles in a post-Edison world: phasing with noble gases bound within engineered sites

The lack of noble-gas binding sites in native proteins has been addressed by introducing new sites using protein engineering. In addition to facilitating phase determination, these sites may also prove invaluable during model building and molecular replacement.

Optimizing data collection for structure determination

Methods used to obtain experimental phases and ways of optimizing these experiments are described.

Away from the edge: SAD phasing from the sulfur anomalous signal measured in-house with chromium radiation

The use of Cr Kα radiation combined with other advances in hardware and software makes sulfur SAD phasing of protein diffraction data rioutinely achievable in the home laboratory.

Is it jolly SAD?

Numerous examples show that with accurate diffraction data, SAD phasing leads to a rapid solution of macromolecular crystal structures. The experiment is demanding and several factors can result in failure.

Substructure search procedures for macromolecular structures

An overview of Patterson methods and direct methods is presented. This is followed by an outline of a new hybrid substructure search.

On symmetries of substructures

An overview of substructure symmetries and the implications for experimental phasing procedures are presented.

'MAD'ly phasing the extracellular domain of the LDL receptor: a medium-sized protein, large tungsten clusters and multiple non-isomorphous crystals

The extracellular domain of the LDL receptor, a medium-sized protein with 700 residues, was determined from multiple non-isomorphous crystals at low resolution in a MAD experiment by exploiting the anomalous signal from two and a half 12-tungstophosphate clusters present in the asymmetric unit. A case study is presented.

Optimization of selenium substructures as obtained from SHELXD

The accuracy of Se substructures obtained by different routes using SHELXD is assessed by comparing substructure solutions with the selenium positions in the refined models.

Introduction to twinning

A twinned crystal is an aggregate in which different domains are joined together according to a specific symmetry operation: the twin law. Reflections from different domains may overlap and twinned crystals fall broadly into two categories in which either all reflections or only certain zones of reflections are affected by overlap. The former occurs when a crystal lattice belongs to a higher point group than the crystal structure itself; the latter frequently occurs when the twin law is a symmetry operation belonging to a higher symmetry supercell.

Twinned crystals and anomalous phasing

The effect of merohedral or pseudomerohedral twinning of macromolecular crystals on the process of structure solution is discussed and illustrated by application in the single-wavelength anomalous diffraction (SAD) mode.

MIR phasing using merohedrally twinned crystals

To date, only six structures have been solved by isomorphous replacement from merohedrally twinned crystals. The main obstacle appears to be a large variation in twin fractions between data sets.

Generation, representation and flow of phase information in structure determination: recent developments in and around SHARP 2.0

Successive formulations of experimental phasing methodology are reviewed from a historical and conceptual perspective, including the latest developments in SHARP. An improved method for encoding and exploiting phase information in the complex plane is presented. Directions for further research are indicated.

Measurement errors and their consequences in protein crystallography

A discussion of the types of measurement errors, their estimates and their consequences in further stages of the crystallographic process is presented.

Phasing the AP2 core complex with Xe, Hg and Se

Retrospective analysis of the relative phasing effectiveness of different derivatives shows that Xe provides the best phasing in this case. Inclusion of bad data degrades the phases.

Phasing the 30S ribosomal subunit structure

High-resolution structures of both subunits of the bacterial ribosome have recently been determined at high resolution by X-ray crystallography. The methods involved in phasing the 30S ribosomal subunit will be reviewed and analysed.