Policing Diversity: Lessons from Lambeth

This report describes a local initiative, Policing Diversity in Lambeth (PDL), which was developed to address the challenges of policing a highly ethnically diverse population. The report is timely. The report of the Macpherson Inquiry into the death of Stephen Lawrence has substantially redrawn the policy landscape in relation to policing and race issues. The Home Secretary’s priorities for policing in 2000-1 emphasise the need for improved community relations. The follow-up report by Her Majesty’s Inspectorate of Constabulary, ‘Winning the Race Revisited’, further stressed the need for urgent action. Parallel with these developments, the 1998 Crime and Disorder Act imposed a duty on police and local authorities to work to reduce crime in partnerships with local communities, and to engage more fully in consultation with local communities. The report provides a snapshot of PDL at a particular stage in its development, and this report may seem of historical interest only. However, the analysis of underlying relations between the police and the community will have wider applicability for both the police and crime and disorder partnerships, as do the lessons that can be drawn from PDL’s first year

From crystal to structure with CCP4

An introduction to the 2017 CCP4 Study Weekend Special Issue

Structural Evidence for a Copper-Bound Carbonate Intermediate in the Peroxidase and Dismutase Activities of Superoxide Dismutase

Copper-zinc superoxide dismutase (SOD) is of fundamental importance to our understanding of oxidative damage. Its primary function is catalysing the dismutation of superoxide to O2 and H2O2. SOD also reacts with H2O2, leading to the formation of a strong copper-bound oxidant species that can either inactivate the enzyme or oxidise other substrates. In the presence of bicarbonate (or CO2) and H2O2, this peroxidase activity is enhanced and produces the carbonate radical. This freely diffusible reactive oxygen species is proposed as the agent for oxidation of large substrates that are too bulky to enter the active site. Here, we provide direct structural evidence, from a 2.15 Å resolution crystal structure, of (bi)carbonate captured at the active site of reduced SOD, consistent with the view that a bound carbonate intermediate could be formed, producing a diffusible carbonate radical upon reoxidation of copper. The bound carbonate blocks direct access of substrates to Cu(I), suggesting that an adjunct to the accepted mechanism of SOD catalysed dismutation of superoxide operates, with Cu(I) oxidation by superoxide being driven via a proton-coupled electron transfer mechanism involving the bound carbonate rather than the solvent. Carbonate is captured in a different site when SOD is oxidised, being located in the active site channel adjacent to the catalytically important Arg143. This is the probable route of diffusion from the active site following reoxidation of the copper. In this position, the carbonate is poised for re-entry into the active site and binding to the reduced copper. © 2012 Strange et al

Perspective: Structure determination of protein-ligand complexes at room temperature using X-ray diffraction approaches

The interaction between macromolecular proteins and small molecule ligands is an essential component of cellular function. Such ligands may include enzyme substrates, molecules involved in cellular signalling or pharmaceutical drugs. Together with biophysical techniques used to assess the thermodynamic and kinetic properties of ligand binding to proteins, methodology to determine high-resolution structures that enable atomic level interactions between protein and ligand(s) to be directly visualised is required. Whilst such structural approaches are well established with high throughput X-ray crystallography routinely used in the pharmaceutical sector, they provide only a static view of the complex. Recent advances in X-ray structural biology methods offer several new possibilities that can examine protein-ligand complexes at ambient temperature rather than under cryogenic conditions, enable transient binding sites and interactions to be characterised using time-resolved approaches and combine spectroscopic measurements from the same crystal that the structures themselves are determined. This Perspective reviews several recent developments in these areas and discusses new possibilities for applications of these advanced methodologies to transform our understanding of protein-ligand interactions

A histidine residue and a tetranuclear cuprous‐thiolate cluster dominate the copper loading landscape of a copper storage protein from Streptomyces lividans

The chemical basis for protecting organisms against the toxic effect imposed by excess cuprous ions is to constrain this through high‐affinity binding sites that use cuprous‐thiolate coordination chemistry. In bacteria, a family of cysteine rich four‐helix bundle proteins utilise thiolate chemistry to bind up to 80 cuprous ions. These proteins have been termed copper storage proteins (Csp). The present study investigates cuprous ion loading to the Csp from Streptomyces lividans (SlCsp) using a combination of X‐ray crystallography, site‐directed mutagenesis and stopped‐flow reaction kinetics with either aquatic cuprous ions or a chelating donor. We illustrate that at low cuprous ion concentrations, copper is loaded exclusively into an outer core region of SlCsp via one end of the four‐helix bundle, facilitated by a set of three histidine residues. X‐ray crystallography reveals the existence of polynuclear cuprous‐thiolate clusters culminating in the assembly of a tetranuclear [Cu4(μ2‐S‐Cys)4(Νδ1‐His)] cluster in the outer core. As more cuprous ions are loaded, the cysteine lined inner core of SlCsp fills with cuprous ions but in a fluxional and dynamic manner with no evidence for the assembly of further intermediate polynuclear cuprous‐thiolate clusters as observed in the outer core. Using site‐directed mutagenesis a key role for His107 in the efficient loading of cuprous ions from a donor is established. A model of copper loading to SlCsp is proposed and discussed

Challenges and solutions for the analysis of<i>in situ</i>,<i>in crystallo</i>micro-spectrophotometric data

Combining macromolecular crystallography within crystallomicro-spectrophotometry yields valuable complementary information on the sample, including the redox states of metal cofactors, the identification of bound ligands and the onset and strength of undesired photochemistry, also known as radiation damage. However, the analysis and processing of the resulting data differs significantly from the approaches used for solution spectrophotometric data. The varying size and shape of the sample, together with the suboptimal sample environment, the lack of proper reference signals and the general influence of the X-ray beam on the sample have to be considered and carefully corrected for. In the present article, how to characterize and treat these sample-dependent artefacts in a reproducible manner is discussed and theSLS-APEin situ,in crystallooptical spectroscopy data-analysis toolbox is demonstrated.</jats:p

Serial synchrotron and XFEL crystallography for studies of metalloprotein catalysis

Research into the earliest development of inhibitory control is limited by a lack of suitable tasks. In particular, commonly used inhibitory control tasks frequently have too high language and working memory demands for children under 3 years of age. Furthermore, researchers currently tend to shift to a new set of inhibitory control tasks between infancy, toddlerhood, and early childhood, raising doubts about whether the same function is being measured. Tasks that are structurally equivalent across age could potentially help resolve this issue. In the current report, a new response inhibition task, the Early Childhood Inhibitory Touchscreen Task (ECITT), was developed. This task can be minimally modified to suit different ages, whilst remaining structurally equivalent. In the new task, participants have to overcome a tendency to respond to a frequently rewarded location on a touchscreen and instead make an alternative response. The ECITT was validated in three independent studies (with additional data, N = 166, reported in Supporting Information). In Study 1 (N = 81), cross-sectional data indicated that inhibitory performance on the task improved significantly between 24 and 30 months of age. In Study 2 (N = 38), longitudinal data indicated steady improvement in inhibitory control between 18, 21 and 24 months, with significant stability in individual performance differences between each consecutive age in terms of accuracy (but not in terms of reaction time). Finally, in Study 3 (N = 64), inhibitory performance on a faster-paced version of the same task showed a similar developmental course across the lifespan (4–84 years) to other response inhibition tasks and was significantly correlated with Stop-signal performance. The ECITT extends the assessment of response inhibition earlier than previous tasks–into early toddlerhood. Because the task is simple and structurally equivalent across age, future longitudinal studies should benefit from using the ECITT to investigate the development of inhibitory control in a consistent manner across the toddler years and beyond

Serial femtosecond crystallography approaches to understanding catalysis in iron enzymes.

Enzymes with iron-containing active sites play crucial roles in catalysing a myriad of oxidative reactions essential to aerobic life. Defining the three-dimensional structures of iron enzymes in resting, oxy-bound intermediate and substrate-bound states is particularly challenging, not least because of the extreme susceptibility of the Fe(III) and Fe(IV) redox states to radiation-induced chemistry caused by intense X-ray or electron beams. The availability of novel sources such as X-ray free electron lasers has enabled structures that are effectively free of the effects of radiation-induced chemistry and allows time-resolved structures to be determined. Important to both applications is the ability to obtain in crystallo spectroscopic data to identify the redox state of the iron in any particular structure or timepoint

Detecting massive gravitons using pulsar timing arrays

Massive gravitons are features of some alternatives to general relativity. This has motivated experiments and observations that, so far, have been consistent with the zero mass graviton of general relativity, but further tests will be valuable. A basis for new tests may be the high sensitivity gravitational wave experiments that are now being performed, and the higher sensitivity experiments that are being planned. In these experiments it should be feasible to detect low levels of dispersion due to nonzero graviton mass. One of the most promising techniques for such a detection may be the pulsar timing program that is sensitive to nano-Hertz gravitational waves. Here we present some details of such a detection scheme. The pulsar timing response to a gravitational wave background with the massive graviton is calculated, and the algorithm to detect the massive graviton is presented. We conclude that, with 90% probability, massles gravitons can be distinguished from gravitons heavier than $3\times 10^{-22}$ eV (Compton wave length $\lambda_{\rm g}=4.1 \times 10^{12}$ km), if biweekly observation of 60 pulsars are performed for 5 years with pulsar RMS timing accuracy of 100 ns. If 60 pulsars are observed for 10 years with the same accuracy, the detectable graviton mass is reduced to $5\times 10^{-23}$ eV ($\lambda_{\rm g}=2.5 \times 10^{13}$ km); for 5-year observations of 100 or 300 pulsars, the sensitivity is respectively $2.5\times 10^{-22}$ ($\lambda_{\rm g}=5.0\times 10^{12}$ km) and $10^{-22}$ eV ($\lambda_{\rm g}=1.2\times 10^{13}$ km). Finally, a 10-year observation of 300 pulsars with 100 ns timing accuracy would probe graviton masses down to $3\times 10^{-23}$ eV ($\lambda_{\rm g}=4.1\times 10^{13}$ km).Comment: 13 pages, 5 figures, Accepted by Ap

An Aromatic Dyad Motif in Dye Decolourising Peroxidases Has Implications for Free Radical Formation and Catalysis

Dye decolouring peroxidases (DyPs) are the most recent class of heme peroxidase to be discovered. On reacting with H2O2, DyPs form a high‐valent iron(IV)‐oxo species and a porphyrin radical (Compound I) followed by stepwise oxidation of an organic substrate. In the absence of substrate, the ferryl species decays to form transient protein‐bound radicals on redox active amino acids. Identification of radical sites in DyPs has implications for their oxidative mechanism with substrate. Using a DyP from Streptomyces lividans, referred to as DtpA, which displays low reactivity towards synthetic dyes, activation with H2O2 was explored. A Compound I EPR spectrum was detected, which in the absence of substrate decays to a protein‐bound radical EPR signal. Using a newly developed version of the Tyrosyl Radical Spectra Simulation Algorithm, the radical EPR signal was shown to arise from a pristine tyrosyl radical and not a mixed Trp/Tyr radical that has been widely reported in DyP members exhibiting high activity with synthetic dyes. The radical site was identified as Tyr374, with kinetic studies inferring that although Tyr374 is not on the electron‐transfer pathway from the dye RB19, its replacement with a Phe does severely compromise activity with other organic substrates. These findings hint at the possibility that alternative electron‐transfer pathways for substrate oxidation are operative within the DyP family. In this context, a role for a highly conserved aromatic dyad motif is discussed