We are currently working on a variety of projects in the lab, all of which are inter-related. The recent Acta D Garman and Owen paper (Vol62, 2006) summarises several of these areas nicely. A very brief summary of our work is below, for more details see the relevant project page or our publications.

See also the lab tools section for tips and guides on experimental techniques. This now includes a RADDOSE guide.

Radiation damage to cryocooled (100K) protein crystals during X-ray diffraction experiments has become a limiting factor for the full utilisation of third generation synchrotron beam fluxes. Radiation damage focuses the mitigation of this damage and the structural changes caused by it.

In the Metripol section a new method for pre-determining the diffractive quality of protein crystals is described. The method uses a birefringence imaging microscope to analyse the optical properties of a protein crystal prior to X-ray irradiation opening up the possibiltiy of pre-sceening crystals in a crystallisation drop to select those which will diffract best.

Elemental analysis of protein crytals using MicroPIXE. This technique involves bombarding dried liquid protein or protein crystals with a beam of 3 MeV protons to induce X-ray emission from the elements present in the sample. A lithium drifted silicon X-ray detector with high energy resolution enables these characteristic X-rays to be identified with specific elements. The proton beam is scanned in X and Y to build up a 2-D 'contour map' of the individual elements in the sample.

In collboration with other groups we are working to determine the structure of human Fibronectin. Human Fibronectin is a large multidomain protein founded in several tissues and body fluids. It is involved in many cellular processes, including tissue repair, embryogenesis, blood clotting, and cell migration/adhesion.

In addition to the above, we have been working on a number of smaller Technical projects. These include the development of a new backstop, testing of new plastic loops and temperature measurements on ACTOR robot pucks and commonly used cryo vial arrangements. A users guide to the Oxford Xcell has also been included here. (Soon to be added to a lab tools section)

radiation damage

The picture above shows a 100K cryocooled protein crystal that has been subjected to data collection at three positions on an undulator beamline and then allowed to warm up. The release of secondary radiation products on warming has blackened the crystal at the beam spots.

We have made progress on three fronts: investigating radical scavengers to slow down the rate of radiation damage in cryo-cooled crystals, theoretical calculations on the X-ray absorption coefficients of protein crystals to look at the effect of X-ray wavelength and the presence of heavy atoms (including selenium) on the expected crystal lifetime in the beam, and lastly a study of unit cell and structural changes in crystals of holo- and apo-ferritin as a function of cryogen temperature and radiation load.

The results reported in Murray and Garman 2002 on ascorbate as a radical scavenger were obtained by looking at sequential datasets taken from HEWL crystals with and without ascorbate as a co-crystallistion additive. The rate of damage to disulphide bonds was monitored in electron density maps of refined structures, and the absorption spectrum of the crystal before and after irradiation was observed, as detected using an off-line microspectrophotometer at the ESRF: in particular the 400nm peak from a disulphide radical species. The spectral results were very difficult to reproduce due to problems with ice formation, both during transport round the storage ring to the off-line microspectrophotometer and with the multiple crystal handling required for an experiment. In addition, the alignment of the spectrometer light beam onto the part of the crystal which had been irradiated with the X-ray beam was time consuming and introduced an unecessary uncertainty into the measurements. We thus installed a microspectrophotometer on ID14-1 at the ESRF, Grenoble, funded by the Royal Society Equipment Fund. A convenient robust adjustable stand was designed and built at the ESRF. After initial commissioning and testing, this device (Figure 1) is in routine use during our radiation damage experiments. We are now in a position to conduct a broad survey of putative scavengers and the effects of various cryoprotective agents on the rate of damage. Fig 2 shows the absorption spectrum of cryoprotected cystine solution without (Fig 2a) and with (Fig2b) 0.5M ascorbate. It is clear that the 400nm di-sulphide species radical is suppressed in the presence of ascorbate.

On the theoretical side, the computer program RADDOSE, originally written by Raimond Ravelli (EMBL, Grenoble) was developed and extended to allow experimenters to enter the amino acid and heavier atom content of their crystals, the mM composition of the buffer, and the incident beam conditions, in order to calculate the estimated time to reach an absorbed dose of 2 x 107 Gy (J kg -1), the so called `Henderson limit' after which half the diffracting power of the crystal is predicted to be lost. This program is now available and a paper describing it and the implications of the predicted lifetimes has been submitted.

metripol

Images produced using the birefringence imaging microscope (Metripol)

A birefringence imaging microscope is a standard transmission microscope with additional optical components which allow measurement of the birefringence properties of a sample. Rotation of a polariser allows qualitative and quantitative measurements of the absorption, the anisotropy and the orientation of the sample.

Using an Oxford Cryosystems Metripol birefringence microscope, we have found a correlation between the width of the slow optical axis of Hen Egg White Lysozyme (HEWL) crystals and their diffraction quality (Owen et al. (2003)). Diffraction quality was assessed from Rmeas (Fig 4) and from the I/s(I) of the whole image as well as that of the highest resolution shell. We observe that the smaller the variation in the slow optical axis position (SOAP), the lower the Rmeas and the higher the I//s(I). However there appears to be no correlation between the optical axis width and the mosaicity of the crystals or their volume. This is an exciting finding which opens up the possibility of pre-screening crystals in a crystallisation drop to select those which should diffract best. Clearly the method cannot be used on cubic crystals. We are now extending the measurements to glucose isomerase crystals, both at room temperature and cryocooled to 100K.

mpixe

MicroPIXE elemental maps (1.75 mm x 1.75mm) of a dried 0.3┬Ál drop of FUR (ferric uptake regulator) protein
a) Sodium b) Bromine c) sulphur d) iron e) zinc

The Oxford Scanning proton microprobe moved to the National Ion Beam Centre at the University of Surrey, Guildford, in October 2002. The two year old accelerator there had never been used to produce proton beams and some extra source equipment was especially installed for this purpose in November 2002. While this modification was underway, we tried a new experiment on proteins: inducing X-ray emission using 1 MeV alpha particles (aIXE) instead of protons. The ionisation cross sections for alphas are lower than for protons, and although we were able to assess the suitability of aIXE for our purpose and check the calibration of the X-ray Si(Li) detector etc, it was clear that the technique does not hold long term promise for analysing proteins.

Following the production of a proton microbeam, we undertook a thorough search for a better backing material for use when analysing proteins (Yates 2003), since the mylar ‘spectrofilm™’ we have always used has trace contamination of calcium and phosphorus. We tested 0.9mm and 1.5mm thick PET (polyethylene terephthalate), 0.1mm silicon nitride membrane windows, sub-micron carbon film supported on a copper grid support, pioloform film (polyvinyl butyral) and formvar film (polyvinyl formal), as well as 2.5mm, 23.6mm and 6.3mm spectrofilm. Of these, the original 2.5mm thick spectrofilm is the most convenient for the majority of applications. However, the silicon nitride, although expensive, is advantageous for samples where the calcium concentration is required. Interestingly, the contaminant P/Ca ratio in spectrofilm was found to be constant over large areas of the film, and thus it is possible to calculate the calcium background as long as the sample contains no phosphorous, and vice versa.

Elemental analysis of a variety of liquid and crystalline protein samples (Garman 1999) was carried out on a large range of liquid samples. Calibration standards of solutions of pseudoazurin (1 copper per protein molecule) and cytochrome c550 (1 iron atom per protein molecule) were kindly supplied to us by James Allen (Biochemistry, Oxford) to allow us to check the new experimental arrangement at Guildford.

The large number of liquid samples measured this year has allowed us to refine our graph of the minimum protein concentration in mg/ml against the proportion of sulphur containing residues. The new graph is shown in Figure 3. We also exhaustively identified buffers which made protein samples unsuitable for PIXE measurements due to their sulphur content, including BES, DTT and HEPES.

microspectrophotometer

The on-line microspectrophotometer at ID14-4, ESRF.

....Microspectrophotometer information will be added here....

fibronectin frustrates

We are collaborating with Drs. Jennifer Potts and Ulrich Schwarz-Linek with the aim of determining the structure of the 2F1 and 3F1 human fibronectin domains, with and without a synthetic peptide (STATT1) from Staphylococcus aureus bound. We have used the in-house Tecan crystallisation robot to great effect to obtain crystals diffracting to at least 2.0Å in-house and better than 1.7Å at the ESRF of both native and peptide-bound protein.

The crystals appeared to be twinned as judged by some but not all of the standard twinning tests. Crystallisation conditions have been modified to try to minimise the chance of twinned crystals (e.g. the addition of MPD, glycerol and dioxane), but these strategies are rather anecdotal in the literature and their success often not open to rational interpretation.

X-ray diffraction datasets to high resolution limits of between 2.2Å and 1.7Å were collected from 7 different crystals: 4 in-house on the LMB rotating anode (3 native and 1 with STATT1), and 3 at synchrotron radiation sources: 1 native at the ESRF, Grenoble, France and 1 platinum derivative and 1 native at SRS, Daresbury, Cheshire. Following analysis of these data, some of which have very high multiplicity (up to 40), we now believe the crystals to be single and of space group P43212 or P41212.

Attempts to solve the structure by molecular replacement using an average of the 15 NMR structures of 2F1 (Schwarz-Linek et al. (2003)) have failed, probably due to a combination of factors including uncertainty in which model might be the most appropriate. All available molecular replacement software was sought and tried: CPP4 (Molrep), CNS, Phoenix, Beast, Phasor and combinations of these. Efforts are now being concentrated on using the platinum soaked dataset to solve the structure by the SAD method. We have also collected 540° of in-house native data for sulphur SAD. There are 8 cysteines and 2 methionines in 90 residues, so this should be feasible.

We have also started to search for crystallisation conditions for a construct containing all five F1 human fibronectin modules.

why fibronectin?

Reference:
Schwarz-Linek, U., Werner, J. M., Pickford, A. R., Kim, S. Gurusidda J. H., Pilka, E. S., Briggs, J. A. G., Gough, T. S., Hook, M., Campbell, I. D., Potts, J. R. (2003): Pathogenic Bacteria Attach to Human Fibronectin Through a Tandem Beta-Zipper. Nature 423 177

New loops Old loops

New (left) and old (right) loops in use in the lab.

As well as the research reported elsewhere, we have been active on a number of smaller technical projects (e.g. testing of new plastic cryo-loops and development of an improved backstop). With a view to improving the success rate with transporting cryocooled crystals to synchrotrons, we have performed temperature measurements on the ACTOR robot pucks and on commonly used cryovial arrangements. Four calibrated platinum resistance thermometers were mounted in top-hats to simulate crystals for the investigation. The results showed a) a temperature gradient existed within a dry transport dewar, but after 2 weeks of monitoring, was sufficiently small (7K: 78K at the bottom of a cane and 85K at the top position) not to affect the crystals, and b) and that when lifted out of the dry dewar, the pucks warmed up more slowly than the cryovials stored on canes, providing more time for crystal manipulation.

Xenon

In conjunction with Oxford cryosystems we have developed a xenon pressure chamber for use in easily producing xenon heavy atom derivatives. A short users guide for use in conjunction with the Oxford xcell is avaliable. More information can be found at Oxford Cryosystems.