CrystalDirect can be installed into protein crystallization platforms or directly installed on MX beamlines.
CrystalDirect® uses EMBL well plates commercially available from MiTeGen (CrystalDirectTM plates) which fits to the standard 96-well plates. They are composed of a plastic frame and a thin membrane on which the samples are deposited (“hanging drop” or « sitting drop » type).
The CrystalDirect® robot cuts the membrane around one or more than one selected crystals by means of laser photoablation. (Cipriani et al.,2012). Thereafter the crystal support is fixed on a SPINE sample pin and immediately cryo-cooled (gaseous stream of 100 K cold nitrogen).
Then, cryo-cooled samples can be transferred into a Dewar either manually or by means of a sample changer robot.
CrystalDirect® is a fully-automated system designed for high throughput crystal harvesting that fits perfectly to modern crystallization platforms: CrystalDirect® needs about 1 minute per harvest and reaches this performance with a success rate superior to 99 %.
In addition to harvesting crystals at high rate, addition of cryoprotectants can also be automatized with CrystalDirect®. The system goes further: CrystalDirect® enables the removal of the mother liquor surrounding protein crystals. In this way, risks of mechanical stress to the crystal during the cryo-cooling process are strongly reduced. (Pellegrini et al., 2011)
CrystalDirect Plate - inside CrystalDirect robot
Crystal selection - software view - CrystalDirect
Protein crystal harvesting by laser photoablation with CrystalDirect - Arinax
Cryo-cooled crystals - harvested with CrystalDirect - Software view
Thanks to the laser photoablation technology, CrystalDirect® combines precision and flexibility giving possibilities to harvest single protein crystal and multiple crystals without a minimal size limit.
Complex crystals such as single needle crystal can also be harvested by CrystalDirect®. It separates nested crystals by laser cutting from crystal agglomerates (crystal surgery).
Protein crystals harvested by this technique do not get deteriorated, neither does their diffraction power as could be demonstrated by numerous experiments. (Zander et al.,2016)
Single protein crystal harvested by CrystalDirect - Software view
Multiple protein crystals harvested from a crystal cluster - software view
single needle crystal harvested by CrystalDirect - picture extracted from Zander et al. (2016)
From the left to the right: single crystal selection, multiple crystal selection and single needle crystal selection
CrystalDirect® offers a large amount of cut shapes for the laser photoablation process. The different shapes adapt to different protein crystal distributions and orientations on the native membrane. Consequently, CrystalDirect® can select and harvest single crystals, batches of crystals, micro-crystals and single needles from clusters.
CrystalDirect® is aimed to be integrated into a fully automated environment, ranging from pre-crystallization steps to diffraction data analysis. Such an environment is generally handled via specialized management software. CrystalDirect® is able to work hand-in-hand with such crystallization management systems.
The EMBL has developed a web based application called CRIMS (CRystallization Information Management System) which is fully compatible with CrystalDirect®.
CRIMS takes in charge data management of the entire crystallization line, giving web functionalities for each step:
CRIMS records data related to the samples (name, type, concentration and others), designing crystallization experiments and it tracks all experimental parameters (crystallization conditions, temperature, etc.) for each well. At any time users can access to these data and modify them. CRIMS links these data with the next step, the crystallization screening.
CRIMS can collect pictures generated by Crystal Farms (e.g. Rock imagers). The lab’ manager and expert users can inspect and score all of these pictures for each crystallization well. This “image viewing & scoring” function allows the expert user to analyses and annotate the results for each well.
The EMBL has developed the automated protein crystal harvester CrystalDirect® to take full advantage of the features offered by CRIMS.
Based on the crystallization screening results the expert user can select the crystals to be harvested and defines the cut shape and its orientation for each harvesting process. Then, CRIMS transfers the information to CrystalDirect® which will perform the harvest as set.
CrystalDirect® cryo-protects harvested crystals, takes pictures of the cryo-cooled samples and transfers these images to CRIMS. The expert user can check these images by means of CRIMS and hence, gets visual feedback on the success of the harvesting process and the final sample quality.
Following the harvest, CrystalDirect® makes sample pins available to a sample changer robot which stores the cryo-protected crystals in liquid nitrogen.
Once the collection of diffraction data is finished, CRIMS retrieves a summary of the data quality from the synchrotron’s diffraction database, e.g. ISPyB (ESRF Synchrotron database, France).
CRIMS is a development of the EMBL Grenoble – HTX laboratory (Márquez Team)
Cipriani et al., Acta Cryst. (2012). D68, 1393-1399 « CrystalDirect: a new method for automated crystal harvesting based on laser-induced photoablation of thin films »
| Performances | |
| Harvesting time | 1 harvest/min |
| Success rate | >99 % |
| Laser cutting | No heat transfer Micrometric precision Multiple cut profiles (pre- & user-defined) |
| Crystal soaking process | 500 µL reservoirs film piercing: photo ablation |
| Software | |
| User interface | Windows / JAVA |
| Web based crystal selection | CRIMS or other solution |
| Multi-device servers | TINE, TANGO, EPICS |
| Sample changer robot | Robot hardware & software integration |
| Power | |
| 230 VAC; 50 Hz | <3680 W |
| 110 VAC; 60 Hz | <3680 W |
| Dimensions (X,Y,Z) | |
| 1.0 x 1.0 x 2.3 m3 | |
CrystalDirect plates can be ordered on MiTeGen website
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