Biophysics and crystallisation

LP3 has been granted beamtime at BioMAX (the X-ray macromolecular crystallography beamline of MAX IV). This allows users to have their crystals rapidly evaluated for diffraction and LP3 can also offer the possibility to process data and solve structures.

Equipment

Crystallisation

Mosquito LCP nanolitre pipetting robot (SPT Labtech)

Allows setting up crystallisation drops with volumes as low as 50 nl protein + 50 nl reservoir and 1–3 protein samples per condition, thus 96–288 experiments can be done in one plate. Automatic LCP (lipidic cubic phase) experiments are also possible. A typical vapour diffusion experiment might consist of two general 96-condition screens (e.g. PACT Premier and JCSG+) conducted at two temperatures (typically 20°C and 4°C). A typical drop size consists of 100 nl of protein and 100 nl of precipitant solution. Each screen requires 20 µl of macromolecule. At 10 mg/ml this represents only 0.20 mg of sample per screen.

Dragonfly crystal (SPT Labtech)

Using nanoliter volumes this liquid handling system pipets your optimisation screen directly into the crystallisation plate (pipetting range: 200 nL – 4 mL). Our Dragonfly is equipped with five (disposable) tips, so you can have 4 ingredients plus water in your screen (if using more you can pipette twice). The screen is easily designed with the software.

Plate incubation and automatic imaging system (Rigaku)

Performs automatic imaging of crystallisation experiments at 20 or 4°C using visible and UV light. The latter allows the identification of even very small crystals hidden in precipitate, or discrimination of protein crystals from salt crystals.

Biophysical characterisation

Prometheus NT.48 (Nanotemper)

Differential scanning fluorimetry (DSF) that measures the intrinsic fluorescence from your protein (Tryptophans and Tyrosines), hence no reporter dye is needed. Stability of your protein in various environments and unfolding events can be analysed.

Zetasizer APS dynamic light scattering system (DLS) (Malvern)

Used to study the aggregation state of proteins.

Omnisec (Malvern Panalytical)

Gel Permeation Chromatography (GPC) / Size Exclusion Chromatography (SEC) system (with integrated multi-detector module) for the measurement of absolute molecular weight, molecular size and sample composition. Allows a better characterisation of synthetic and natural polymers and proteins in solution. Available SEC columns are:

• Superose 6 Increase 10/300 GL (Cytiva): 5 kDa-5 MDa size range
• Superdex 200 Increase 10/300 GL (Cytiva): 10-600 kDa size range
• Superdex 75 Increase 10/300 GL (Cytiva): 3-70 kDa size range
• SRT-SEC 300 (silica, Sepax): 5 kDa-1.25 MDa size range