1,721,071 research outputs found
Methods development for structural biology
Get PDFTwo research questions are investigated here: the first, major, section addresses the problem of uneven distributions of dose (absorbed energy per unit mass) in crystals used for macromolecular crystallography (MX), and the second presents the develop- ment of a high-throughput metalloprotein characterisation technique, HT microPIXE.
In MX, the advent of X-ray microbeam data collection has led to uneven distributions of dose within the crystal volume becoming increasingly common. In these cases, the rotation method creates a highly damaged central region of crystal that stays within the beam throughout exposure, and less damaged outer regions, which are introduced during rotation. This thesis presents a new software program, raddose-3d, which performs a full 3D simulation of the profile of absorbed energy (the dose state) within a crystal during X-ray exposure. In order to utilise this time resolved, 3D picture of the dose state of the crystal, a new metric â Diffraction Weighted Dose â is proposed. This metric is then experimentally validated, and is found to summarise the dose state into a single dose value, which reflects the damage state of the crystal. Simulations are performed using raddose-3d and Diffraction Weighted Dose to compare possible dose spreading strategies, and generalised recommendations for MX experimentalists are offered.
Uniquely identifying the species and stoichiometry of bound metals in protein sam- ples is a significant challenge for biophysical characterisation. Low throughput mi- crobeam Proton Induced X-ray Emission (microPIXE) provides an unambiguous anal- ysis of these properties, but has a limited throughput of ∼10 samples per day. As a consequence, its applicability has been restricted to niche cases. This thesis presents significant progress, including proof of principle experiments, on developing sample preparation methods, data acquisition systems, and data analysis protocols to increase this throughput by an order of magnitude, opening up major new applications for the technique.</p
Investigation of X-ray induced radiation damage in proteins, nucleic acids and their complexes
Get PDFMacromolecular X-ray crystallography (MX) is currently the dominant technique for the structural eluci- dation of macromolecules at near atomic resolution. However, the progression and deleterious effects of radiation damage remains a major limiting factor in the success of diffraction data collection and subsequent structural solution at modern third generation synchrotron facilities. For experiments conducted at 100 K, protein specific damage to particular amino acids has been widely reported at doses of just several MGy, before any observable decay in average diffraction intensities. When undetected, such artefacts of X-ray irradiation can lead to significant modelling errors in protein structures, and ultimately the failure to derive the correct biological function from a model. It is thus vital to develop tools to help MX experimenters to detect and correct for such damage events. This thesis presents the development of an automated program, RIDL, which is designed to objectively quantify radiation-induced changes to electron density at individual atoms, based on Fobs,n â Fobs,1 Fourier difference maps between different dose states for a single crystal. The high-throughput RIDL program developed in this work provides the ability to systematically investigate a wide range of macromolecular systems.
To date, damage to the broad class of nucleic acids and nucleoprotein complexes has remained largely uncharacterised, and it is unclear how radiation damage will disrupt the validity of such models derived from MX experiments. This thesis presents the first systematic investigations on a range of nucleic acid, protein-RNA and protein-DNA complex case studies. In general, it is concluded that nucleic acids are highly robust to radiation damage effects at 100K, relative to control protein counterparts across the tested systems.
For protein crystals at 100K, cleavage of the phenolic CâO bond in tyrosine has disseminated through the MX radiation damage literature as a dominant specific damage event at 100K, despite the absence of any energetically favourable cleavage mechanism. To clarify the radiation susceptibility of tyrosine, this thesis presents a systematic investigation on radiation damage to tyrosine in a wide range of MX protein radiation damage series retrieved from the Protein Data Bank. It is concluded that the tyrosine C â O bond remains intact following X-ray irradiation, however the aromatic side-group can undergo radiation-induced displacement.
This thesis also presents further applications of the RIDL program. A protocol is introduced to calculate explicit half-dose values for the electron density at individual atoms to decay to half of their initial value at zero absorbed dose. In addition, a methodology is developed to detect radiation-induced changes to electron density occurring over the course of the collection of a single MX dataset of diffraction images, all of which are required for structural solution. These protocols aim to advise experimenters of when previously-undetected site-specific damage effects may have corrupted the quality of their macromolecular model.
Overall, the work in this thesis is highly applicable to both the future understanding of radiation damage in macromolecular structures, as well as of interest to the wider crystallographic community.</p
Structural studies of the PIR and RIFIN families from malaria-infected erythrocytes
Get PDFMalaria, caused by the Plasmodium parasite, is responsible for the deaths of close
to half a million people a year. During the course of infection, the parasite invades
and remodels the host red blood cell, often requiring the export of a number of
proteins to the erythrocyte surface. These include the RIFIN family of Plasmodium
falciparum, which have been shown to directly modulate the host immune response
through interactions with inhibitory immune receptors such as LILRB1 and LAIR1.
Additionally, the PIR family of a number of human- and rodent-infective Plasmodium species have been implicated in the determination of parasite virulence and
the outcome of infection, along with cytoadhesion of infected erythrocytes to the
vasculature. Here, I present the structure of the variable region of a RIFIN in
complex with LILRB1. This is the first structure of any member of the RIFIN
family and reveals that the RIFIN utilises a conserved mode of binding, which can
be blocked through a single mutation in all RIFINs tested, and which mimics the
interaction of MHC class I, the native ligand of LILRB1. The RIFIN is also able
to mimic the effect of MHC binding both on signalling through LILRB1, and on
inhibition of NK cell activation at the immunological synapse. I also present the
structure of the extracellular domain of a PIR protein from Plasmodium chabaudi,
the first for a member of this family, revealing a novel α-helical fold. Sequence
analysis and homology modelling show conservation of this fold across PIRs from 4
human- and 3 rodent-infective Plasmodium species, as well as excluding the RIFINs
and STEVORs from membership of the PIR family. These findings provide both a
molecular mechanism for RIFIN function, along with a structural definition of the
PIRs, giving valuable insight into 2 major families of Plasmodium variant surface
antigens
Quantifying radiation damage in X-ray diffraction experiments in structural biology
Get PDFQuantitative studies of global radiation damage are presented for two different types of experiments in structural biology: macromolecular crystallography (MX) and small angle X-ray scattering (SAXS)
MX is the most common technique to elucidate the atomic resolution structures of biological macromolecules.
However, these molecules undergo radiation induced changes during the experiment that undesirably affect the data.
Global radiation damage, which is characterised by an overall loss in the diffracted intensity of Bragg reflections, limits the amount of useful data that can be collected from a single crystal in an experiment.
Furthermore, for experimental phasing experiments, the radiation induced intensity changes can be so significant that the phasing signal becomes undetectable, thereby hindering successful structure determination.
This thesis investigates methods to track and correct the diffraction data that are affected as a result of global radiation damage.
First, extensions to the diffraction weighted dose (DWD) metric are investigated for the ability of DWD to track the overall intensity decay of reflections.
This metric then is combined with a new mathematical model of intensity decay to perform zero-dose extrapolation.
An additional probabilistic extrapolation approach is incorporated into the traditional regression based approach to allow extrapolation of low multiplicity reflections.
As an alternative approach, a new hidden Markov model representation of the data collection experiment is developed that allows the time-resolved calculation of structure factor amplitudes, with error estimates calculated explicitly.
This method gives comparable refinement statistics to that obtained from data processed with the current data reduction pipeline, and improvements to the algorithm are proposed.
SAXS, on the other hand, is a complementary structural technique that results in low resolution information about macromolecules.
However it still requires the probing of the macromolecules with ionising radiation, so radiation induced changes are still a problem.
Unfortunately the tools for assessing radiation damage in SAXS experiments are not mature enough for them to be used routinely.
This thesis presents extensions to RADDOSE-3D to perform dose calculations for SAXS samples.
Additionally, a free, open source Python library has been developed to allow the exploration and visualisation of the results of a similarity analysis of frames within a dataset.
These tools are then used to determine the efficacy of various radioprotectant compounds at different concentrations to mitigate radiation damage effects.</p
Arylamine N-Acetyltransferases from mycobacteria: investigations of a potential yarget for anti-tubercular therapy
Get PDFReactivation of latent infection is the major cause of tuberculosis (TB). Cholesterol is a critical carbon source during latent infection. Catabolism of cholesterol contributes to the pool of propionyl-CoA, a precursor that is incorporated into cell-wall lipids. Arylamine N-acetyltransferase (NAT) is encoded within a gene cluster that is involved in the sterol-ring degradation and is essential for intracellular survival. NAT from M. tuberculosis (TBNAT) can utilise propionyl-CoA and therefore was proposed as a target for TB-drug development. Deleting the nat gene or inhibiting the NAT enzyme prevents intracellular survival and results in depletion of cell-wall lipids. NAT inhibitors, including the piperidinol class, were identified by high-throughput screening. The insolubility of recombinant TBNAT has been a major limitation in pursuing it as a drug target. Subcloning tbnat into a pVLT31 vector resulted in a yield of 6-16 mg/litre-bacterial-culture of pure-soluble recombinant TBNAT. The increased yield allowed for extensive screening for crystallisation conditions. However, since a structure was not obtained, the model NAT from M. marinum (MMNAT) was employed to further understand NAT as a target. Screening against a panel of Acyl-CoA cofactors showed that MMNAT can also utilise propionyl-CoA. The MMNAT structure in complex with the high affinity substrate hydralazine was determined (2.1 Å) and the architecture of the arylamine pocket was delineated. A novel mechanism for the acetylation reaction of hydralazine has emerged. It is proposed that the acetyl group is transferred from acetyl-CoA to the heterocyclic aromatic nitrogen of hydralazine, which explains the immediate cyclisation of the acetylated metabolite into an N-methyltriazolophthalazine. By employing mass spectroscopy, enzyme assays, computational docking and structural studies, a covalent mechanism of inhibition by the piperidinol class was established, and the inhibitor-binding pocket was identified. Inhibitors with new scaffolds were identified using the in silico 3D-shape screening and thermal shift assay
Structural and functional studies on a prokaryotic homologue of the mammalian SLC7 cationic amino acid transporters
Get PDFThe Amino acid/Polyamine/Cationic transporter superfamily is among the largest of the sec-ondary active transporters. APC family members use either a proton or sodium gradient to drive substrate translocation across the membrane. Members of the APC superfamily are responsible for the uptake of amino acids and are found in all kingdoms of life. Amino acids, and their derivatives, are crucial for both prokaryotic and eukaryotic cell biology, being the building blocks of a variety of macromolecules and play important roles in metabolism and cell signalling. Therefore, the systems responsible for their transport are important targets of further study, in particular to gain greater insight into how these proteins recognise and discriminate between different types of amino acids. The transport mechanism of several APC members has been elucidated using a variety of structural, biophysical and functional approaches. However, these studies have focussed on sodium coupled transporters due to their function as neurotransmitter transporters. Here, the crystal structure of the first pro-ton-coupled amino acid transporter of the APC superfamily, from Geobacillus kaustophilus, GkApcT, is presented. GkApcT closely related to the mammalian cationic amino acid trans-porters (CATs) of the SLC7 family, responsible for the transport arginine and lysine into the cell. The mechanism of transport was further investigated using liposome-based functional assays to characterise the substrate specificity and kinetics. The crystal structure was used to guide a detailed site-directed mutagenesis study, which identified several conserved res-idues involved in substrate binding and proton-coupling. The current structure serves as a template to further understand proton coupled amino acid transport in bacteria and to un-derstand mammalian amino acid transport via the SLC7 and SLC36 families
Radiation damage in small molecule crystallography: fact not fiction
Get PDFTraditionally small molecule crystallographers have not usually observed or recognised significant radiation damage to their samples during diffraction experiments. However, the increased flux densities provided by third generation synchrotrons have resulted in increasing numbers of observations of this phenomenon. The diversity of types of small molecule systems means it is not yet possible to propose a general mechanism for their radiation induced sample decay, however characterisation of the effects will permit attempts to understand and mitigate it. Here systematic experiments are reported on the effects that sample temperature and beam attenuation have on radiation damage progression, allowing qualitative and quantitative assessment of their impact on crystals of a small molecule test sample. To allow inter-comparison of different measurements, radiation damage metrics (diffraction intensity decline, resolution fall-off, scaling B-factor increase) are plotted against the absorbed dose. For ease of dose calculations, the software developed for protein crystallography, RADDOSE-3D, has been modified for use in small molecule crystallography. It is intended that these initial experiments will assist in establishing protocols for small molecule crystallographers to optimise the diffraction signal from their samples prior to the onset of the deleterious effects of radiation damage
Radiation damage in protein crystallography: susceptibility study
Get PDFProtein structure models obtained from X-ray crystallography are subject to radiation damage. The resulting specific alterations to protein structures can be mistaken for biological features, or may obscure actual protein mechanisms, leading to misidentification or obscuration of biological insight. The radiation chemistry behind this site-specific damage is not well understood. Radiation damage processes progress in proportion to the dose absorbed by the crystal in the diffraction experiment. Doses can be estimated using existing software, but these assume idealised experimental conditions. To simulate complex diffraction experiments, including treatment of imperfect X-ray beam profiles and inhomogeneous dose distributions, a new program, RADDOSE-3D, was developed. RADDOSE-3D can be integrated into beamline software to provide convenient, more accurate, comparative, and publishable dose figures, also facilitating informed data collection decisions. There is currently no method to automatically detect specific radiation damage in protein structure models in the absence of an 'undamaged' reference model. Radiation damage research therefore generally relies on detailed observation of a few model proteins. A new metric, BDamage, is designed and used to identify and quantify specific radiation damage in the first large-scale statistical survey of 2,704 published protein models, which are examined for the effects of local environments on site-specific radiation damage susceptibility. A significant positive correlation between susceptibility and solvent accessibility is identified. Current understanding of radiation damage progression is mostly based on a few consecutive structure model 'snapshots' at coarse dose intervals. The low sampling rate considerably limits the ability to identify varying site susceptibility and its causes. Real space electron density data are obtained for crystals of different mutants of a RhoGDI protein with very high sequence identity, to determine sensitising and stabilising factors for radiation induced structural changes. Utilising a newly developed data collection and analysis protocol, these changes could be tracked with unprecedented time resolution
Radiation damage to macromolecules: kill or cure?
Get PDFInternational audienceRadiation damage induced by X-ray beams during macromolecular diffraction experiments remains an issue of concern in structural biology. While advances in our understanding of this phenomenon, driven in part by a series of workshops in this area, undoubtedly have been and are still being made, there are still questions to be answered. Eight papers in this volume give a flavour of ongoing investigations, addressing various issues. These range over: a proposed new metric derived from atomic B-factors for identifying potentially damaged amino acid residues, a study of the relative damage susceptibility of protein and DNA in a DNA/protein complex, a report of an indication of specific radiation damage to a protein determined from data collected using an X-ray free-electron laser (FEL), an account of the challenges in FEL raw diffraction data analysis, an exploration of the possibilities of using radiation damage induced phasing to solve structures using FELs, simulations of radiation damage as a function of FEL temporal pulse profiles, results on the influence of radiation damage during scanning X-ray diffraction measurements and, lastly, consideration of strategies for minimizing radiation damage during SAXS experiments. In this short introduction, these contributions are briefly placed in the context of other current work on radiation damage in the field
The solution and crystal structures of a module pair from the Staphylococcus aureus-binding site of human fibronectin - A tale with a twist
No full textAn important goal of structural studies of modular proteins is to determine the inter-module orientation, which often influences biological function. The N-terminal domain of human fibronectin (Fn) is composed of a string of five type I modules (F1). Despite their small size, to date F1 modules have proved intractable to X-ray structure solution, although there are several NMR structures available. Here, we present the first structures (two X-ray models and an NMR-derived model) of the (2)F1(3)F1 module pair, which forms part of the binding site for Fn-binding proteins from pathogenic bacteria. The crystallographic structure determination was aided by the novel technique of UV radiation damage-induced phasing. The individual module structures are very similar in all three models. In the NMR structure and one of the X-ray structures, a similar but smaller interdomain interface than that observed previously for (4)F1(5)F1 is seen. The other X-ray structure has a different interdomain orientation. This work underlines the benefits of combining X-ray and NMR data in the studies of multi-domain proteins. (c) 2007 Elsevier Ltd. All rights reserved
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