James Foadi

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High-throughput in situ experimental phasing

Acta Crystallographica Section D Structural Biology, 2020

In this article, a new approach to experimental phasing for macromolecular crystallography (MX) a... more In this article, a new approach to experimental phasing for macromolecular crystallography (MX) at synchrotrons is introduced and described for the first time. It makes use of automated robotics applied to a multi-crystal framework in which human intervention is reduced to a minimum. Hundreds of samples are automatically soaked in heavy-atom solutions, using a Labcyte Inc. Echo 550 Liquid Handler, in a highly controlled and optimized fashion in order to generate derivatized and isomorphous crystals. Partial data sets obtained on MX beamlines using an in situ setup for data collection are processed with the aim of producing good-quality anomalous signal leading to successful experimental phasing.

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An effective introduction to structural crystallography using 1D Gaussian atoms

European Journal of Physics, 2017

The most important quantitative aspects of computational structural crystallography can be introd... more The most important quantitative aspects of computational structural crystallography can be introduced in a satisfactory way using 1D truncated and periodic Gaussian functions to represent the atoms in a crystal lattice. This paper describes in detail and demonstrates 1D structural crystallography starting with the definition of such truncated Gaussians. The availability of the computer programme CRONE makes possible the repetition of the examples provided in the paper as well as the creation of new ones.

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The fine art of integral membrane protein crystallisation

Methods, 2018

Integral membrane proteins are among the most fascinating and important biomolecules as they play... more Integral membrane proteins are among the most fascinating and important biomolecules as they play a vital role in many biological functions. Knowledge of their atomic structures is fundamental to the understanding of their biochemical function and key in many drug discovery programs. However, over the years, structure determination of integral membrane proteins has proven to be far from trivial, hence they are underrepresented in the protein data bank. Low expression levels, insolubility and instability are just a few of the many hurdles one faces when studying these proteins. X-ray crystallography has been the most used method to determine atomic structures of membrane proteins. However, the production of high quality membrane protein crystals is always very challenging, often seen more as art than a rational experiment. Here we review valuable approaches, methods and techniques to successful membrane protein crystallisation.

Conformational Flexibility of Proteins Involved in Ribosome Biogenesis: Investigations via Small Angle X-ray Scattering (SAXS)

Crystals, 2018

The dynamism of proteins is central to their function, and several proteins have been described a... more The dynamism of proteins is central to their function, and several proteins have been described as flexible, as consisting of multiple domains joined by flexible linkers, and even as intrinsically disordered. Several techniques exist to study protein structures, but small angle X-ray scattering (SAXS) has proven to be particularly powerful for the quantitative analysis of such flexible systems. In the present report, we have used SAXS in combination with X-ray crystallography to highlight their usefulness at characterizing flexible proteins, using as examples two proteins involved in different steps of ribosome biogenesis. The yeast BRCA2 and CDKN1A-interactig protein, Bcp1, is a chaperone for Rpl23 of unknown structure. We showed that it consists of a rigid, slightly elongated protein, with a secondary structure comprising a mixture of alpha helices and beta sheets. As an example of a flexible molecule, we studied the SBDS (Shwachman-Bodian-Diamond Syndrome) protein that is involved in the cytoplasmic maturation of the 60S subunit and constitutes the mutated target in the Shwachman-Diamond Syndrome. In solution, this protein coexists in an ensemble of three main conformations, with the N-and C-terminal ends adopting different orientations with respect to the central domain. The structure observed in the protein crystal corresponds to an average of those predicted by the SAXS flexibility analysis.

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A Novel Approach to Data Collection for Difficult Structures: Data Management for Large Numbers of Crystals with the BLEND Software

Crystals, 2017

The present article describes how to use the computer program BLEND to help assemble complete dat... more The present article describes how to use the computer program BLEND to help assemble complete datasets for the solution of macromolecular structures, starting from partial or complete datasets, derived from data collection from multiple crystals. The program is demonstrated on more than two hundred X-ray diffraction datasets obtained from 50 crystals of a complex formed between the SRF transcription factor, its cognate DNA, and a peptide from the SRF cofactor MRTF-A. This structure is currently in the process of being fully solved. While full details of the structure are not yet available, the repeated application of BLEND on data from this structure, as they have become available, has made it possible to produce electron density maps clear enough to visualise the potential location of MRTF sequences.

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MX data analysis developments at Diamond Light Source

Acta Crystallographica Section A Foundations and Advances, 2016

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Two-dimensional spherical-polar visualization of data completeness using equal-volume units of reciprocal space

Acta Crystallographica Section A Foundations and Advances, 2016

Modern microfocus X-ray sources define the state-of-the-art for most applications in X-ray diffra... more Modern microfocus X-ray sources define the state-of-the-art for most applications in X-ray diffraction. These sources are usually combined with multilayer X-ray mirrors which are excellent X-ray optical devices for beam shaping and preserving the brightness of the source. Microfocus rotating anode generators and liquid metal jet systems deliver flux densities in the range of 10 11 photons/s/mm 2 . However, these sources are expensive and need regular and sometimes time-consuming maintenance. Low power microfocus sealed tube sources, such as the Incoatec Microfocus source IμS, represent an interesting low-maintenance alternative to rotating anode generators. Power loads of several kW/mm 2 in anode spot sizes of < 50 μm deliver a small and bright beam. Flux densities of up to 10 10 photons/s/mm 2 can be achieved in a focused beam suitable for most protein crystals and poorly diffracting small molecule samples. The latest generation of the IµS, the IµS 3.0, is the first microfocus X-ray source that is optimized for X-ray diffraction resulting in a gain in intensity of about 30% compared to its predecessor. In this presentation, we will be reviewing the current performance levels of different microfocus X-ray sources. Further, we will be discussing the main features of the newest generation of the IμS. We will be presenting selected results to demonstrate the impact of these modern microfocus X-ray sources on the data quality for applications in chemical and biological crystallography.

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Applications of the BLEND Software to Crystallographic Data from Membrane Proteins

Advances in Experimental Medicine and Biology, 2016

X-ray diffraction from crystals of membrane proteins very often yields incomplete datasets due to... more X-ray diffraction from crystals of membrane proteins very often yields incomplete datasets due to, among other things, severe radiation damage. Multiple crystals are thus required to form complete datasets, provided the crystals themselves are isomorphous. Selection and combination of data from multiple crystals is a difficult and tedious task that can be facilitated by purpose-built software. BLEND, in the CCP4 suite of programs for macromolecular crystallography (MX), has been created exactly for this reason. In this chapter the program is described and its workings illustrated by means of data from two membrane proteins.

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cRy: a crystallographic toolbox in R

Acta Crystallographica Section A Foundations of Crystallography, 2011

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On the systematic scaling and merging of multiple datasets in macromolecular crystallography

Acta Crystallographica Section A Foundations of Crystallography, 2011

are primarily command-line driven, an emphasis has been placed on ease-of-use and automation. We ... more are primarily command-line driven, an emphasis has been placed on ease-of-use and automation. We have developed a graphical interface for the major components of PHENIX, which currently includes phenix. refine, phenix.xtriage, comprehensive validation tools based on the Molprobity web server, Phaser, and the AutoSol, AutoBuild, AutoMR, and LigandFit automation "wizards". The Python-based framework allows new GUIs to be generated semi-automatically while preserving all of the flexibility of the command-line programs, and supports both Macintosh and Linux. Python extensions for Coot and PyMOL facilitate real-time visualization of refinement and automated model-building, and convenient viewing of results. Transitions between separate modules within PHENIX are simplified or eliminated in the GUI, reducing the amount of manual input required and avoiding the use of command-line tools. Further automation is possible with definition of standard parameter sets and input files for individual programs. Future improvements will include greater use of multiprocessing and clusters, tools for handling multiple structures in parallel, and new automation pipelines.

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$Research paper thumbnail of Application of In Situ Diffraction in High-Throughput Structure Determination Platforms$

Application of In Situ Diffraction in High-Throughput Structure Determination Platforms

Methods in Molecular Biology, 2014

Macromolecular crystallography (MX) is the most powerful technique available to structural biolog... more Macromolecular crystallography (MX) is the most powerful technique available to structural biologists to visualize in atomic detail the macromolecular machinery of the cell. Since the emergence of structural genomics initiatives, significant advances have been made in all key steps of the structure determination process. In particular, third-generation synchrotron sources and the application of highly automated approaches to data acquisition and analysis at these facilities have been the major factors in the rate of increase of macromolecular structures determined annually. A plethora of tools are now available to users of synchrotron beamlines to enable rapid and efficient evaluation of samples, collection of the best data, and in favorable cases structure solution in near real time. Here, we provide a short overview of the emerging use of collecting X-ray diffraction data directly from the crystallization experiment. These in situ experiments are now routinely available to users at a number of synchrotron MX beamlines. A practical guide to the use of the method on the MX suite of beamlines at Diamond Light Source is given.

Solving Crystal Structures from Powder Data. IV. The Use of the Patterson Information for Estimating the |F|'s

Journal of Applied Crystallography, 1998

... J. Appl. Cryst. 29, 667--673. Altomare, A., Cascarano, G., Giacovazzo, C. & Guagliardi, A... more

Elucidations on the reciprocal lattice and the Ewald sphere

European Journal of Physics, 2008

The reciprocal lattice is derived through the Fourier transform of a generic crystal lattice, as ... more

On the allowed values for the triclinic unit-cell angles

Acta Crystallographica Section A Foundations of Crystallography, 2010

This short article questions and investigates the possible range of values for the three angles o... more This short article questions and investigates the possible range of values for the three angles of a unit cell in the triclinic system. Although no constraints are reported in manuals and tables for crystallography, the three angles are not really independent; the range of allowed values is calculated and presented in this paper. short communications Acta Cryst. (2011). A67, 93-95 Foadi and Evans Triclinic unit-cell angles 95

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In situmacromolecular crystallography using microbeams

Acta Crystallographica Section D Biological Crystallography, 2012

Despite significant progress in high-throughput methods in macromolecular crystallography, the pr... more Despite significant progress in high-throughput methods in macromolecular crystallography, the production of diffractionquality crystals remains a major bottleneck. By recording diffraction in situ from crystals in their crystallization plates at room temperature, a number of problems associated with crystal handling and cryoprotection can be side-stepped. Using a dedicated goniometer installed on the microfocus macromolecular crystallography beamline I24 at Diamond Light Source, crystals have been studied in situ with an intense and flexible microfocus beam, allowing weakly diffracting samples to be assessed without a manual crystal-handling step but with good signal to noise, despite the background scatter from the plate. A number of case studies are reported: the structure solution of bovine enterovirus 2, crystallization screening of membrane proteins and complexes, and structure solution from crystallization hits produced via a highthroughput pipeline. These demonstrate the potential for in situ data collection and structure solution with microbeams.

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