I02-Macromolecular Crystallography
Data acquisition
Diagnostics
Health Physics
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Diamond Proposal Number(s):
[8492]
Abstract: Transaldolase B (TalB) and D-fructose-6-phosphate aldolase A (FSAA) from Escherichia coli are C-C bond-forming enzymes. Using kinetic inhibition studies and mass spectrometry, it is shown that enzyme variants of FSAA and TalB that exhibit D-fructose-6-phosphate aldolase activity are inhibited covalently and irreversibly by D-tagatose 6-phosphate (D-T6P), whereas no inhibition was observed for wild-type transaldolase B from E. coli. The crystal structure of the variant TalBF178Y with bound sugar phosphate was solved to a resolution of 1.46 Å and revealed a novel mode of covalent inhibition. The sugar is bound covalently via its C2 atom to the [epsilon]-NH2 group of the active-site residue Lys132. It is neither bound in the open-chain form nor as the closed-ring form of D-T6P, but has been converted to [beta]-D-galactofuranose 6-phosphate (D-G6P), a five-membered ring structure. The furanose ring of the covalent adduct is formed via a Heyns rearrangement and subsequent hemiacetal formation. This reaction is facilitated by Tyr178, which is proposed to act as acid-base catalyst. The crystal structure of the inhibitor complex is compared with the structure of the Schiff-base intermediate of TalBE96Q formed with the substrate D-fructose 6-phosphate determined to a resolution of 2.20 Å. This comparison highlights the differences in stereochemistry at the C4 atom of the ligand as an essential determinant for the formation of the inhibitor adduct in the active site of the enzyme.
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Apr 2016
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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
Detectors
Diagnostics
Health Physics
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Abstract: To establish successful infection, a retrovirus must insert DNA replica of its genome into a host cell chromosome. This process is catalysed by integrase (IN), the viral enzyme that synapses ends of viral DNA forming a highly stable nucleoprotein complex, intasome. The structure of full-length IN, either separately or in complex with viral DNA, has been lacking. Furthermore, although clinically useful inhibitors of HIV IN have been developed, their mechanism of action remained speculative. Using data acquired at Diamond beamlines I02 and I04, we determined the long-sought-after crystal structure of a functional retroviral intasome, revealing a tetramer of IN assembled on viral DNA ends. Soaking intasome crystals in the presence of clinical HIV IN inhibitors Raltegravir and Elvitegravir elucidated their common mechanism of action. Binding within the active site, the drugs cause dislocation of the reactive 3’ viral DNA end, disarming the viral nucleoprotein complex. These results represent a quantum leap in understanding of the retroviral DNA integration process and provide a platform for rational design of IN inhibitors.
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Oct 2010
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Accelerator Physics
Controls
Diagnostics
Health Physics
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Abstract: Diamond Light Source is a 3 GeV electron storage ring, which has been successfully operating in top-up mode since October 2008, having previously operated in decay mode only. Although in the UK there is no legal requirement to submit a safety case to the relevant authority (the Health and Safety Executive) when implementing top-up operation for the first time, it is required to keep doses ALARP and within the 1 mSv annual dose limit which Diamond has set for all staff, users and visitors. Prior to operating Diamond in top- up mode, a study of the radiological safety implications was carried out to ensure that these requirements could be met. The study involved calculation using FLUKA of dose rates arising from accidental beam losses. These losses took the form of either continuous losses in a front end arising from poor injection, or loss of a single injected electron pulse into a beamline optics hutch. In addition to the calculations, dose rate measurements were made outside beamline hutches under conditions of deliberately engineered beam losses in front ends, intended to compare as closely as possible with those modelled. As a result of this study, a number of changes to Diamond’s radiation monitoring regime were proposed and implemented before top-up operation was permitted. The study also helped to define the limits within which top-up would be permitted to operate.
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Dec 2009
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Accelerator Physics
Controls
Diagnostics
Health Physics
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R.
Walker
,
R.
Bartolini
,
P.
Bonner
,
F.
Burge
,
Y.
Chernousko
,
C.
Christou
,
J.
Dobbing
,
M.
Heron
,
V.
Kempson
,
G.
Rehm
,
R.
Rushton
,
S.
Singleton
,
M. C.
Wilson
,
I.
Martin
Abstract: It is planned to start top-up operation in Diamond in the near future. In this report we summarise the various activities that have led up to this point, including radiation safety considerations, preparation of hardware interlocks and control software, and injection optimisation.
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Jun 2008
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Health Physics
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Jun 2007
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Health Physics
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Abstract: The European Directive 96/29/Euratom of 13/05/1996, based on the ICRP 60 Recommendations, defines basic safety standards for the protection of the health of workers and the general public against the dangers arising from ionising radiation. Member states of the European Union have had to integrate these basic standards in their national legislation. Synchrotron radiation facilities are affected by this new radiation protection legislation. In the UK, the Daresbury Synchrotron Radiation Source (SRS) operates under the Ionising Radiations Regulations 1999, and the third generation Diamond Light Source (DLS) project is largely benefiting from their experience. In France, the ESRF case is presently being used to define detailed prescriptions, that will also apply to future sources in this country, for example the third generation light source Synchrotron Soleil, presently under construction. This paper summarises general policies and practices of these four SR facilities: SRS, European Synchrotron Radiation Facility (ESRF), DLS and Synchrotron Soleil.
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Dec 2006
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