Deliverables
At the EMBL Hamburg Outstation and Elettra devices are available to the user community, which allow flash cooling of crystals pressurized under xenon and krypton. Experience and preliminary tests using porcine pancreatic elastase (PPE) crystals have been carried out.
We found that a procedure using 40 bar pressurization for around 1 minute to be generally effective for xenon derivatisation. A number of the beamline support staff has been trained in the use of the devices so that access for users is now routinely available. Over the two years that the devices have been available to the user community there has been regular usage of the facilities, usually on projects where all else has failed.
The Cryostream Cooler from Oxford Cryosystems was modified for use with helium gas. The nitrogen gas produced in the heat exchanger is released into the atmosphere and substituted by an appropriate (8L/min) flux of helium. Similarly to the nitrogen, the helium is cooled down to 100K. This is then suitable for feed to the He purged beampath.
An instrument to generate derivatives of protein crystals with gases, such as Xenon and Krypton, and to rapidly freeze-trap these crystals in order to prevent desorption of the bound gas has been constructed. The instrument was designed to be fully computer-controlled to allow easy operation and to permit reproducible gas-charging parameters (pressure and time of exposure, profile of pressure build up and release, etc).
Moreover, no pressurised rare-gas should be required, and the gas should be fully confined and recyclable in order to permit work with very expensive or very toxic gases. The instrument consists essentially of a piston to pressurize the gas up to 100bar. The protein crystal is picked-up from its cryo-protectant with a standard cryo-loop mounted in a special pin. This pin is attached to a pin holder, which snaps into the instrument. From that point on, operation is under computer control until the crystal is flash-cooled.
The detector developed during the project period will present a breakthrough in the field of X-ray detectors for protein crystallography. Marresearch played an important role in the development of CCD detectors, currently used almost exclusively on third generation synchtrotron beamlines, but it was clear from the beginning that a SOLID STATE system based on the DIRECT CONVERSION of X-rays into charges will be the system of the future. This is exactly the detector, which has been developed in this project.
The properties of this system are so outstanding that it will undoubtedly play an important role in the high throughput protein structure research at synchrotrons around the world. Not only synchrotrons, but also home laboratories will benefit of the high sensitivity and the low noise of the new system. We have confidence that the detector will prove a useful addition to the tools available for structural biology research.
The construction of the helium purged beam path (HePBP) at Elettra was driven by the fact that the scattering and absorption of helium is much lower than air, which can be assessed by their scattering and absorption cross sections at different wavelengths.
The HePBP described here matches these requirements by enclosing the complete path of the primary beam (from the last beamline Kapton window to the crystal sample), and of the secondary beams region (from the sample to the detector surface), avoiding any window along the beams path. Moreover, it allows the use of conventional cryo-cooling instrumentation (see other results) for the data collection and sample mounting.
It is pivotal to minimise every source of background scatter in diffraction experiments at long wavelengths. In particular, a large benefit can be gained by careful design of the beamstop. The main X-ray beam scatters from the air between the collimator and the crystal, and between the crystal and the beamstop. Thus a beamstop which can be positioned as close as possible to the crystal, but which is small enough not to cast a large central shadow on the detector, is highly beneficial in increasing the signal/noise ratio of counts on the detector, thereby making the small MAD and SAD signals easier to measure. In addition, a beamstop, which can be easily centred and is robust to knocks by experimenters is highly desirable, since through proper centering, asymmetric scatter of X-rays from the beamstop can be eliminated. Thus a new beamstop was designed, fabricated and extensively tested.
The new beamstop has the following features, and is innovative for macromolecular crystallography equipment in combining all these:
- It is adjustable on x and y translations on easy to turn knurled screws (no tool required).
- It is cut away at one side so that it can be positioned under the goniometer, allowing it to be placed much nearer the crystal, so cutting down the air path.
- It has a 1.5mm diameter collection cup for the beam as opposed to the 2mm or 2.5 mm diameter cups used previously, so minimising the size of the shadow cast on the detector and thus allowing much lower resolution data to be collected.
- A thin metal strip with its plane parallel to the beam direction attaches the cup to one side of the beamstop support. This arrangement is much more robust than the strips of mylar foil stretched across the support which were used previously, making the whole beamstop less prone to being knocked and misaligned by experimenters.
- It attaches to the existing MAR beamstop carriage with no modifications being necessary.
Global Phasing has developed the autoSHARP package, incorporating version 1.4.0 production of SHARP and version 3.0. of its graphical user interface SUSHI. This was released in early January 2002.
An upgrade to SHARP 2.0.1 and SUSHI 3.0.15 were released in January 2003. It has now been downloaded by over 800 academic institutions, and these academic users are given the same level of support as commercial users of the same software.
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