SP2; Structure Determination Platform

NorStruct's SP2 "Structure determination" services include protein sample evaluation tasks of quality control and stability/solubility optimization, crystallization trials, data collection, using either the home X-ray source or synchrotron radiation, followed by structure determination and refinement.

The standard services are divided into 5 tasks which can be ordered separately or together. However, individual customization is generally recommended.

Download SP2 Structure determination order form

^aAcademic users can obtain access to the same services via collaboration whereby a NorStruct staff member is hired part-time on the project. In such cases, the researcher(s) involved must be credited in publications in accords with standard rules and practice.

SP2.1 Sample preparation and quality control
NorStruct collaborators and customers are encouraged to provide NorStruct with all relevant information regarding protein behavior (pH stability, cofactor or metal binding, stability before and after freezing, DLS, etc.); an SDS-PAGE gel of the purified protein sample is mandatory and an MS analysis is highly recommended. The ideal protein sample will have been purified with size exclusion chromatography (gel-filtration), has a concentration of at least 10 mg/ml (and higher if the protein is highly soluble) and comprise a quantity of more than 3 mg that is aliquoted in several tubes. For this amount of protein, NorStruct will be able to perform crystallization trials, optimization of crystallization conditions for appearing protein crystals, buffer optimization (if needed) and redo the crystallization trials one time in the new buffer (if needed). The quality control, preferably done for all protein samples, will be performed using Dynamic Light Scattering (DLS) and MS, and optionally, differential scanning calorimetry (DSC) to obtain information about protein stability, aggregation and folding properties. DCS need more protein and will therefore be customized.

SP2.2 Initial crystallization screening
The preliminary sample characterization aims at choosing the best protein concentration for the subsequent 480 crystallization trials, and includes 96 crystallization conditions with 3 different protein:reservoir ratios (i.e 100:100, 200:200, 500:500 nanoliters).

SP2.3 Robotic nanolitre crystallization screening
Nanolitre crystallization trials with our Phoenix RE robot for a minimum of 480 different conditions will be set up, along with subsequent optimization of crystallization conditions using the liquid handling Alchemist™ II robot. The crystallization plates will be stored in our Gallery Plate Hotel/ Desktop Minstrel imager, which automatically record images of crystallization experiments and enables detailed evaluation of the crystallization conditions. The service is appropriate for crystallization of proteins, complexes and membrane proteins if soluble protein is provided. For the latter, automated trials with membrane crystallization screening kits will be offered, and the customer may also choose a more time consuming customized lipid cubic phase approach. The service will also cover determination of cryogenic conditions and diffraction tests for obtained crystals. When SAD/MAD (Single/Multiple Anomalous Dispersion) techniques are required for determining the structure, NorStruct will consult with the customer regarding strategies for incorporation of relevant heavy atoms and/or ligands.

SP2.4 Buffer optimization
If no suitable protein crystals are obtained, this might be due to poor stability, microheterogeneity, or inhomogeneous oligomerization of the protein. Buffer and additive optimization has been found to increase the crystallization success rates significantly and will therefore be offered by NorStruct using a thermofluor assay procedure in high-throughput format. The thermal shift assays will be performed using a fluorescent dye in a real-time PCR machine to determine the melting temperature (Tm), typically from 20-90 °C, for 24 buffers (pH-range from 4-9) and 12 different additives. The protein will susequently be transferred into the buffer and/or additive with the highest Tm, followed by a second round of crystallization trials. If no crystals are obtained in the second round of robotic crystallization, further actions will be discussed with the customer.

SP2.5 X-ray data collection and structure determination
Once diffraction quality protein crystals are obtained, data will be collected on our state-of-the-art in-house X-ray system. We expect that our capacity is sufficient for all users in Norway. When synchrotron radiation is needed for high quality data or for structure solution, application for synchrotron radiation beamtime will be sent to the European synchrotrons that we routinely use (ESRF, BESSY, SLS). If the project has sufficient impact to be granted beamtime, data will be collected by the NorStruct staff as a part of the service. Structure determination will be included in the service with the methods of molecular replacement and SAD/MAD (if anomalous scattering is sufficient in the crystal) as a standard within the package structure determination package. In cases where heavy atoms or anomalous scatterers need to be additionally incorporated for structure determination, e.g. Se-Met protein production or heavy atom soaking, the work task must be customized.

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