The Cipriani team develops instruments and methods for X-ray scattering experiments and works with industry to make them available to scientists worldwide.
Micro-crystals in-situ data collection – CD plate mounted on a MD3 diffractometer, at the EMBL@PETRA3 P14 beamline.
Previous and current research
The core activity of our team is to develop instruments and methods for X-ray scattering experiments in collaboration with the ESRF Structural Biology group, EMBL@ PETRA3 and the McCarthy and Márquez team teams. Strongly involving EMBLEM, the EMBL partner for technology transfer, an important part of our mission is to make our instruments available to the scientific community worldwide. The MD3 Kappa diffractometer developed in collaboration with EMBL@PETRA3 is now available to the users of the P14 beamline (led by Gleb Bourenkov). The combination of a small and intense beam with a fast X-ray detector and MD3 is ideal for collecting diffraction data from micron-sized needles and batches of micro-crystals using 4D scans or serial rastered data collection methods.
CrystalDirect™ (CD), a system jointly conceived with the Márquez team, is now available to users through the high throughput crystallisation (HTX) laboratory. Crystals grown in CD plates can be marked remotely using CRIMS, the crystallisation laboratory information management system of the HTX lab, and automatically harvested and frozen in the CD lab. The automated removal of the solution surrounding the crystals is often sufficient to safely freeze the crystals, but a cryo-protective treatment can also be automatically applied before harvesting if necessary. More than 1500 crystals from 50 different proteins have been successfully harvested so far.
A basic setup to diffuse chemical solutions in crystallisation drops was recently integrated and the first ligand soaking experiment has been very encouraging. The intrinsic low background of the CD plates makes them ideal for in situ X-ray data collection. Specific goniometer supports were developed to process CD plates on the MD2 and MD3 diffractometers. In situ data collection is now offered on request at the EMBL/ESRF BM14 beamline. The first in-situ rastered data collections were made on the MD3 of P14, with batches of 5-20 micron-sized crystals of model proteins (see figure). Several hundred crystals in the same crystallisation drop were shot, producing thousands of diffraction images, from which high resolution structures were obtained. Similar experiments were successfully performed with crystals harvested in batches using CD and frozen, showing both the potential of serial data collection and the benefit of CD for this method. In parallel, studies are ongoing to develop a compact and precise sample holder for frozen crystals. Prototypes are available and a robotised test setup is installed on BM14. This collaborative project called ‘NewPin’, is part-supported by BioStruct-X.
Future projects and goals
A second CD harvester with extended crystal treatment capabilities will soon be built for projects involving ligands or derivatives. Our long-term ambition is to bridge the HTX lab and the BM14 beamline via CD as a pilot project for an automated and integrated MX facility. In the context of the ESRF MASSIF upgrade programme, we will equip the ESRF ID30B beamline with a MD2 diffractometer and new 6-axis robotic sample changing solution. The ultimate goal is to offer the ESRF user community a serial in situ and frozen data collection environment in a flexible robotised manner ready for the next generation of sample holders.