Laboratory of Molecular Biophysics Laboratory Journal 2001 Facilities

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Minakshi Ghosh and Elspeth Garman

X-ray Facility

A number of improvements have been made to the X-ray facilities this year.

New Pirani gauges have been installed between the turbo molecular and the rotary pumps on both the generators in T3 and T5. These facilitate efficient monitoring of the backing vacuum, particularly during pump-down. The rotary pump on the T3 generator has been replaced by a new one. Similarly, a new rotary pump will be bought for the T5 generator during the next year.

On the T3 generator, we have had a recurrent misalignment problem between the copper target and the target motor since October 2000. The fault has finally been remedied by replacing the old copper anode by a new one. The old anode had been in use for 10+ years and had become unbalanced. At the same time a new target motor was installed as the insulation of the old motor was worn out. After these changes, the T3 generator is now again running reliably.

Several repairs have been carried out on the mar345 Image Plate in T5 during the past year. The Mar shutter had to be replaced as its top lead pin had come loose. An add-on PCB was fitted to the mar345 scanner to correct a small change in the phase between the two signals of the rotation encoder. Lastly, the reading head spindle of the scanner had become dry and had to be greased, involving significant disassembly of the detector. The erase bulbs were changed at the same time.

Recently the Osmic optics on the mar345 detector were carefully re-aligned with the exit nozzle of the mar slits, with a resulting improvement in the quality of the data. Earlier in the year, an MSC engineer serviced the Yale mirrors on the left-hand port of the T3 generator. These mirrors had remained stable and well tuned since their reinstallation in 1999.

A new 700 series Cryostream cooler has been purchased and commissioned. Initially it was set up in T3 to stream freeze crystals for storage, but it has now been swapped with the 600 series Cryostream cooler and is being used on the mar345 detector in T5.

The glove box, which was located on the east side of T3 and used for anaerobic crystallisation experiments, has now been relocated to S9, the crystallisation room.

In T5, a new L-shaped worktop has been installed to replace the old separate benches. This now provides one continuous and convenient working surface, where all the necessary instruments are available for users. A new high power microscope, a Leica MZ6, has been purchased for T5. A wider base, designed and built in our workshop, has been attached to the existing WILD microscope to make the handling of crystallisation trays safe and convenient while selecting and cryoprotecting crystals for X-ray analysis.

A new enlarged top plate and support have been installed on the T5 generator to provide more working space around the mar345 detector. This too was designed and installed by our workshop. In the next phase of the modifications, a new set of failsafe switches will be installed on the radiation shelter doors and the existing doors will be modified to allow easier access to the crystal position.

A scheme to completely reorganise the existing air-conditioning system used in the T3 detector room is at an advanced planning stage. A VRV condenser will be installed on the roof replacing the existing separate condensers for the units in T3. It will have a capacity of 28kW to start with, and will provide for the two units in T3, in addition to those in other laboratories in the LMB and MRC Immuno-Chemistry Unit. This large unit will also have potential for adding extra modules in the future, if necessary.

During the past year, members of LMB as well as researchers from other laboratories have used the image plate detectors. Dr. Tom Davies of LMB collected a 2.5Å dataset from crystals of a fully active CDK2-CyclinA complex with bound inhibitor. This dataset was the first one from a crystal of this complex which has been collected in-house. The structure refined to R_cryst = 24% (R_Free= 33%). This is part of a project aiming to develop potent CDK2 inhibitors as a possible anti-cancer treatment. Dr. Elspeth Garman's group collected both room temperature and 100K control datasets from HEWL and N9 neuraminidase crystals as a part of their radiation damage studies. James Murray screened D62G mutant salmonella typhimurium neuraminidase crystals soaked in substrate at different temperatures and concentrations to try to find conditions for binding. He also installed a modified MAR data collection programme which allows up to 32 data segments to be entered at once and then collected in sequence. Dr. Martin Noble and James Sandy collected data-sets from native Arylamine N-acetyltransferases (NATs) crystals as well as from cocrystals with derivatives of acetyl Co-enzymeA, a carboxymethylCoA (CMCoA) and butyrylCoA (BuCoA). The aim of the project is to elucidate which amino-acids are required for substrate and co-factor binding sites as well as improving on the resolution of the native structure.

A variety of test data on crystals of Proclavaminate Amidinohydrolase have been collected by Prof Chris Schoefield's group. Initially the crystals diffracted to only 2.6Å, but there was a dramatic improvement with the use of an alternative cryoprotection protocol. Using the improved conditions, it has been possible to achieve diffraction to 2.0Å resolution with a good signal to noise ratio. Work on a plant enzyme, Anthocyanidin synthase from Arabidopsis thaliana is in progress by the same group. The X-ray facility was used to screen for suitable cryoprotectant conditions and also for heavy metal and substrate bound enzyme. Complete data sets collected included a xenon derivative to 2.4Å (R_merge = 0.077) and, on alpha-ketoglutarate containing structure to 2.7Å (R_merge = 0.067).

In the summer, Prof. Nikos Oikinomakos and his team from Athens collected data sets from crystals of glycogen phosphorylase bound to indirubin analogues, in a project to extend the understanding of glycogen phosphorylase regulation.

The Xentronics area detector has not been used this year.

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