SHELIXIR: automation of experimental phasing procedures using SHELXC/D/E

The program SHELIXIR represents a simple and efficient tool for routine phase-problem solution using data for experimental phasing by the single-wavelength anomalous dispersion, multiwavelength anomalous dispersion, single isomorphous replacement with anomalous scattering and radiation-damage-induced phasing methods. As indicated in its name, all calculation procedures are performed with the SHELXC/D/E program package. SHELIXIR provides screening for alternative space groups, optimal solvent content, and high- and low-resolution limits. The procedures of SHELXE are parallelized to minimize the computational time. The automation and parallelization of such procedures are suitable for phasing at synchrotron beamlines directly or for finding the optimal parameters for further data processing. A simple graphical interface is designed to make use easier and to increase efficiency during beam time.

19F-Tagged metal binding pharmacophores for NMR screening of metalloenzymes

This study demonstrates the screening of a collection of twelve 19F-tagged metal-binding pharmacophores (MBPs) against the Zn(II)-dependent metalloenzyme human carbonic anhydrase II (hCAII) by 19F NMR. The isomorphous replacement of...

IPCAS: a direct-method-based pipeline from phasing to model building and refinement for macromolecular structure determination

A new version (2.0) of the pipeline IPCAS (Iterative Protein Crystal structure Automatic Solution) has been released, in which the program OASIS performs direct-method single-wavelength anomalous diffraction/single isomorphous replacement phasing and direct-method-aided partial-structure extension. IPCAS incorporates the widely used packages CCP4 and PHENIX for locating heavy atoms, density modification, molecular replacement, model building and refinement. Important extensions to the previous version of IPCAS include a resolution screening method for non-crystallographic symmetry searching, an alternate model-building protocol for avoiding premature convergence and direct-method image processing for electron microscopy maps, including single-particle cryo-EM maps. Moreover, a new graphical user interface is provided for controlling and real-time monitoring of the whole dual-space iterative process, which works as a plugin to CCP4i. Applications of the new IPCAS to difficult cases have yielded promising results, including `direct-method phasing and fragment extension' from weak anomalous diffraction signal data and `direct-method-aided partial-structure extension' from low-homology models.

Review on Cement Stabilization/Solidification of Municipal Solid Waste Incineration Fly Ash

Municipal solid waste incineration (MSWI) fly ash must be treated properly prior to being disposed in the security landfill due to its serious pollution toxicity. Nowadays, lots of studies have demonstrated that cement-based stabilization/solidification could reduce the toxicity pollution effectively by encapsulating the heavy metals into cement matrix, which leads to greater capacity and weight. This paper compares and discusses the MSWI fly ash treatment with the mostly used matrix materials such as Portland cement, phosphate cement, aluminate cement, and alkaline activated cement. Moreover, immobilization mechanism introduced by the interaction between the MSWI fly ash and hydrated cement matrix materials, such as the physical cementing effect, adsorption, isomorphous replacement, and complex precipitation, was explored in depth. The paper also pointed out some reasonable development directions for cement-based stabilization/solidification technology to improve the effectiveness and application of cement-based stabilization/solidification technology.

Introducing site-specific cysteines into nanobodies for mercury labelling allowsde novophasing of their crystal structures

The generation of high-quality protein crystals and the loss of phase information during an X-ray crystallography diffraction experiment represent the major bottlenecks in the determination of novel protein structures. A generic method for introducing Hg atoms into any crystal independent of the presence of free cysteines in the target protein could considerably facilitate the process of obtaining unbiased experimental phases. Nanobodies (single-domain antibodies) have recently been shown to promote the crystallization and structure determination of flexible proteins and complexes. To extend the usability of nanobodies for crystallographic work, variants of the Nb36 nanobody with a single free cysteine at one of four framework-residue positions were developed. These cysteines could be labelled with fluorophores or Hg. For one cysteine variant (Nb36-C85) two nanobody structures were experimentally phased using single-wavelength anomalous dispersion (SAD) and single isomorphous replacement with anomalous signal (SIRAS), taking advantage of radiation-induced changes in Cys–Hg bonding. Importantly, Hg labelling influenced neither the interaction of Nb36 with its antigen complement C5 nor its structure. The results suggest that Cys–Hg-labelled nanobodies may become efficient tools for obtainingde novophase information during the structure determination of nanobody–protein complexes.

Membrane protein structure determination by SAD, SIR, or SIRAS phasing in serial femtosecond crystallography using an iododetergent

The 3D structure determination of biological macromolecules by X-ray crystallography suffers from a phase problem: to perform Fourier transformation to calculate real space density maps, both intensities and phases of structure factors are necessary; however, measured diffraction patterns give only intensities. Although serial femtosecond crystallography (SFX) using X-ray free electron lasers (XFELs) has been steadily developed since 2009, experimental phasing still remains challenging. Here, using 7.0-keV (1.771 Å) X-ray pulses from the SPring-8 Angstrom Compact Free Electron Laser (SACLA), iodine single-wavelength anomalous diffraction (SAD), single isomorphous replacement (SIR), and single isomorphous replacement with anomalous scattering (SIRAS) phasing were performed in an SFX regime for a model membrane protein bacteriorhodopsin (bR). The crystals grown in bicelles were derivatized with an iodine-labeled detergent heavy-atom additive 13a (HAD13a), which contains the magic triangle, I3C head group with three iodine atoms. The alkyl tail was essential for binding of the detergent to the surface of bR. Strong anomalous and isomorphous difference signals from HAD13a enabled successful phasing using reflections up to 2.1-Å resolution from only 3,000 and 4,000 indexed images from native and derivative crystals, respectively. When more images were merged, structure solution was possible with data truncated at 3.3-Å resolution, which is the lowest resolution among the reported cases of SFX phasing. Moreover, preliminary SFX experiment showed that HAD13a successfully derivatized the G protein-coupled A2a adenosine receptor crystallized in lipidic cubic phases. These results pave the way for de novo structure determination of membrane proteins, which often diffract poorly, even with the brightest XFEL beams.

Ab initiophasing by molecular averaging in real space with new criteria: application to structure determination of a betanodavirus

Molecular averaging, including noncrystallographic symmetry (NCS) averaging, is a powerful method forab initiophase determination and phase improvement. Applications of the cross-crystal averaging (CCA) method have been shown to be effective for phase improvement after initial phasing by molecular replacement, isomorphous replacement, anomalous dispersion or combinations of these methods. Here, a two-step process for phase determination in the X-ray structural analysis of a new coat protein from a betanodavirus,Grouper nervous necrosis virus, is described in detail. The first step isab initiostructure determination of theT= 3 icosahedral virus-like particle using NCS averaging (NCSA). The second step involves structure determination of the protrusion domain of the viral molecule using cross-crystal averaging. In this method, molecular averaging and solvent flattening constrain the electron density in real space. To quantify these constraints, a new, simple and general indicator, free fraction (ff), is introduced, where ff is defined as the ratio of the volume of the electron density that is freely changed to the total volume of the crystal unit cell. This indicator is useful and effective to evaluate the strengths of both NCSA and CCA. Under the condition that a mask (envelope) covers the target molecule well, an ff value of less than 0.1, as a new rule of thumb, gives sufficient phasing power for the successful construction of new structures.

A pipeline for structure determination ofin vivo-grown crystals usingin cellulodiffraction

While structure determination from micrometre-sized crystals used to represent a challenge, serial X-ray crystallography on microfocus beamlines at synchrotron and free-electron laser facilities greatly facilitates this process today for microcrystals and nanocrystals. In addition to typical microcrystals of purified recombinant protein, these advances have enabled the analysis of microcrystals produced inside living cells. Here, a pipeline where crystals are grown in insect cells, sorted by flow cytometry and directly analysed by X-ray diffraction is presented and applied toin vivo-grown crystals of the recombinant CPV1 polyhedrin. When compared with the analysis of purified crystals,in cellulodiffraction produces data of better quality and a gain of ∼0.35 Å in resolution for comparable beamtime usage. Importantly, crystals within cells are readily derivatized with gold and iodine compounds through the cellular membrane. Using the multiple isomorphous replacement method, a near-complete model was autobuilt from 2.7 Å resolution data. Thus, in favourable cases, anin cellulopipeline can replace the complete workflow of structure determination without compromising the quality of the resulting model. In addition to its efficiency, this approach maintains the protein in a cellular context throughout the analysis, which reduces the risk of disrupting transient or labile interactions in protein–protein or protein–ligand complexes.

5. Seeing atoms

It is clear that knowledge of the relative phases is essential if we wish to find the atoms in a crystal. So what do we do if we do not have phase information? ‘Seeing atoms’ describes the phase problem and the different methods of phase determination used by crystallographers: a difference Fourier map; the Patterson method; electron density maps; multiple isomorphous replacement; multiple-wavelength anomalous dispersion using synchrotron radiation, which is often used in macromolecular crystallography; molecular replacement, commonly used in protein crystallography; the Sayre equation, a mathematical relationship that enables probable values for the phases of some diffracted beams to be found; and a new technique called charge flipping.