The Biocrystal Facilty activities focus on high-troughput crystallization screening using an Art Robbins Crystallization Robot and it supports the users throughout the crystal optimization process. Researchers of the Facility provide support to users for the whole process of structure determination (choice of the construct, protein production, crystallization, data collection and analysis). The laboratory is endowed with equipment for the analysis of protein sample quality such as light-scattering, analytical gel filtration, CD and UV-vis spectroscopy and thermal stability evaluation. The Facility was set up to stimulate the access to macromolecular crystallography, a key technology in structural biology, and to large international facilities which are not fully exploited by the Italian scientific community, especially in view of the strategic European focus on large and distributed facilities. Its aim is also to widen the user community of structural biology large facilities by training and dissemination.
The structural biologists from both IBPM and the researchers of the Department of Biochemistry of the “Sapienza” University of Rome carry out the evaluation of the feasibility of the requests and, if necessary, provide advice to potential users on how to bring macromolecular samples to crystallization quality.
Platforms available for access through Instruct-ITALIA
Instrumentation
Liquid handling robot Phoenix (Art Robbins) to perform crystallization trials with nanovolums of protein (Sapienza University). The crystallization robot is placed in a temperature-and humidity-controlled room (20 ° C and 55%, respectively), dedicated exclusively to crystallization.
Leica stereoscope Z12 cold light equipped with digital camera and sliding platform, for the non-automated control of the crystallization trials.
Cabinets thermostated at 21° C, 4° C and 37° C, with vibration-proof motors (Angelantoni).
Apparatus to test the temperature effect on crystallization conditions, TG40 (Centeo), capable of simultaneously test 5 different temperatures between 2 ° and 60 ° C in 40 wells miniscreen.
Techniques
High Throughput Crystallization
The high throughput crystallization platform at the Biocrystal facility avails itself of an ARI (Art Robbins Phoenix Protein Crystallography Dispenser). This crystallization robot is ideal to perform 300 crystallization trials in few minutes. The screening will be performed using the sitting drop technique.
The ARI takes less than 50 second to dispense protein and the screen to the small well and membranes. This crystallization robot is equipped with 96 syringes able to dispense from 100 nanoliters to 100 microliter of screen solutions and a nano-dispenser able to dispense from 100 nanoliters to 10 microliters of protein solution.
The crystallization trials will be controlled every two days by the qualified personnel of the facilities using the dedicated microscopes.
Crystals optimization and Data Collection
Crystals optimizazion by hanging drop or sitting drop techniques will be performed by the users under the supervision of the qualified personnel of the biocrystal facility. The data collection will be performed at the synchrotron radiation light sources by users under the supervision of the biocrystal facility qualified personnel.
The facility support the users for beamtime application at the European synchrotron radiation facilities of ELETTRA (Trieste, Italy), ESRF (Grenoble, France), DIAMOND (Didcot, England) for the acquisition of crystal diffraction data sets.
