Cipriani Team
Diffraction Instrumentation Team
BioSAXS sample changer at the ESRF/EMBL beamline ID14-3.
Previous and current research
Our team develops instruments and methods for X-ray scattering experiments in collaboration with the synchrotron crystallography team (page 96) and the ESRF structural biology group, as well as contributing to the development of the EMBL@PETRA3 beamlines (see page 100). Part of our work is also to support and improve equipment at the ESRF beamlines. Most of our technologies are transferred to industry and available to other synchrotrons worldwide via industrial companies.
ESRF’s macromolecular crystallography (MX) beamlines still rely on our MD2/MD2M diffractometers, SC3 sample changers and C3D crystal centring software to process several hundred crystals per day. In Hamburg, the EMBL@PETRA3 beamline MX1 will soon be equipped with a MD2 diffractometer. To fully benefit from the micron-sized beam of MX2, we are currently developing a vertical Kappa diffractometer (MD3) based on a prototype spindle tested on the ESRF ID23-2 micro-focus beamline. The sub-micron stability obtained at crystal position, showed that it will be possible to re-orient and process crystals down to 2-3 micrometers in size.
The diffraction quality of large unit cell crystals, such as macromolecular complexes, is often a limiting factor to structure determination.The newly developed HC1 crystal dehydration device is now routinely used to help enhance crystal quality at beamlines by reducing the amount of solvent contained in the crystals matrix. It is also useful for testing crystals at room temperature. To satisfy growing user demand, a second HC1 machine has been put into operation. Our aim is to integrate dehydration control in MxCube and eDNA. A dehydration experiment database and associated tools have been developed to collect statistics on the results obtained from dehydration protocols in relation with crystallisation parameters. Enriched by HC1 users at the ESRF, Max-LAB and Diamond, this tool should help on new dehydration experiments in predicting initial dehydration levels and in proposing protocols successfully used with similar crystal parameters.
The automated BioSAXS sample environment project developed in collaboration with EMBL Hamburg and the ESRF is almost complete. At the ESRF ID14-3 beamline, several hundred samples and buffers stored in SBS plates can be exposed automatically to X-rays in a glass capillary at controlled temperature (see figure). Sample volumes down to 5μ l of solution can be transferred reliably to the exposure cell. In-line sample concentration measurement and pipetting functions are available for in situ sample dilution and additions. A second system will be installed early next year at EMBL@PETRA3. In collaboration with the HTX team, we have developed the proof-of-concept on a technology to automatically harvest crystals grown in specifically designed crystallisation supports, on which we have filled a patent. The affordable number of samples processed at automated MX beamlines is partly limited by the design of the Spine sample holders. To overcome this problem, we have started studies for a compact and precise sample holder that should allow high-density crystal storage and reduced crystal alignment time. On this basis, a future sample holder standard should be developed in collaboration with European synchrotrons.
Future projects and goals
Next year, EMBL@PETRA3 should receive a BioSAXS automated sample environment, and we expect the MD3 dffractometer introduced above to be operational at MX2. In the context of the ESRF MASSIF upgrade programme, our main focus will be on the development of an automated crystal harvester and new sample holder standard, with the ambition to build a pilot automated integrates MX screening unit jointly with the HTX and synchrotron crystallography teams.