Advances in experimental phasing

An introduction to the 2015 CCP4 Study Weekend on Advances in Experimental Phasing.

A history of experimental phasing in macromolecular crystallography

The development of phasing methods in protein crystallography is presented.

Efficient merging of data from multiple samples for determination of anomalous substructure

The benefits of using local scaling and optimization of anomalous signal (as implemented in PHENIX) for merging data sets from many crystals for determination of the substructure for weak anomalous scatterers are examined.

An overview of heavy-atom derivatization of protein crystals

This review summarizes the reasons why the heavy-atom derivatization of protein crystals can be useful, how to select heavy atoms, how to produce a heavy-atom-modified crystal that still diffracts and how to determine whether the protein has been modified.

Phasing and structure of bestrophin-1: a case study in the use of heavy-atom cluster compounds with multi-subunit transmembrane proteins

The structure of a eukaryotic ion channel solved using tantalum bromide SAD phasing is discussed in the context of basic challenges common to membrane proteins.

Tackling the crystallographic structure determination of the COP9 signalosome

The low-resolution structure determination of the COP9 signalosome, a large multi-protein complex, from crystals affected by rotational pseudo-symmetry and twinning is detailed.

Using selenome­thionyl derivatives to assign sequence in low-resolution structures of the AP2 clathrin adaptor

A selenomethionine marker strategy allowed the identification of a region of disconnected electron density at low resolution and despite poor selenomethionine incorporation, thereby building a structural framework for understanding how the clathrin adaptor AP2 regulates clathrin binding in mammalian cells.

Can I solve my structure by SAD phasing? Anomalous signal in SAD phasing

The useful anomalous correlation and the anomalous signal in a SAD experiment are metrics describing the accuracy of the data and the total information content in a SAD data set and are shown to be related to the probability of solving the anomalous substructure and the quality of the initial phases.

Can I solve my structure by SAD phasing? Planning an experiment, scaling data and evaluating the useful anomalous correlation and anomalous signal

Algorithms for evaluating and optimizing the useful anomalous correlation and the anomalous signal in a SAD data set are described.

A log-likelihood-gain intensity target for crystallographic phasing that accounts for experimental error

A new Rice-function approximation for the effect of intensity-measurement errors improves the treatment of weak intensity data in calculating log-likelihood-gain scores in crystallographic applications including experimental phasing, molecular replacement and refinement.

Radiation damage and derivatization in macromolecular crystallography: a structure factor's perspective

Both site-specific radiation damage and heavy-atom derivatization result in small changes to the intensity of reflections. The size of the change owing to each is calculated and compared for individual reflections.

Radiation-damage-induced phasing: a case study using UV irradiation with light-emitting diodes

A case study of radiation-damage-induced phasing is discussed using ultraviolet light-emitting diodes to induce specific radiation damage.

Data collection with a tailored X-ray beam size at 2.69 Å wavelength (4.6 keV): sulfur SAD phasing of Cdc23^Nterm

Data collection with a tailored 50 µm diameter X-ray beam at 4.6 keV (λ = 2.69 Å) on the newly established EMBL beamline P13 at PETRA III allowed the crystal structure determination of the Cdc23^Nterm homodimer (65.4 kDa; 12 Cys and ten Met residues) by sulfur SAD phasing at 3.1 Å resolution while overcoming crystal twinning.

Facilitating best practices in collecting anomalous scattering data for de novo structure solution at the ESRF Structural Biology Beamlines

Best practices in data collection for experimental phasing at the ESRF Structural Biology beamlines are described.

Data-collection strategy for challenging native SAD phasing

The successful structure solution of the integral membrane diacylglycerol kinase and the CRISPR-associated endonuclease RNA–DNA complex by native SAD phasing is demonstrated. The structures were solved with a combined low-dose multi-orientation, multi-crystal data-collection strategy.

In-vacuum long-wavelength macromolecular crystallography

The motivation for, and the first results from, the novel in-vacuum long-wavelength MX beamline I23 at Diamond Light Source are presented.

Initiating heavy-atom-based phasing by multi-dimensional molecular replacement

A strategy is presented to set up an n-dimensional molecular-replacement parameter matrix (MRPM) search using anomalous difference Fourier maps from related data sets to uncover weak, but correct, molecular-replacement solutions for heavy-atom substructure determination and subsequent experimental phasing.

Progress in low-resolution ab initio phasing with CrowdPhase

New developments in CrowdPhase, a collaborative online game for tackling the low-resolution phase problem, are presented. The new features address several crystallographic issues and extend the reach of CrowdPhase to a broader range of experimental data sets.