National Facility for Structural Biology

To ensure maximum productivity and research quality, the NF for StrucBio benefits from state-of-the-art equipment. The list of technologies available for external user to access the Cryo-Electron Microscopy Unit (IU1) includes:

• Thermo Scientific Titan Krios G4i 300kV TEM equipped with Thermo Scientific Falcon 4i direct electron detector, Thermo Scientific Selectris X energy filter, Thermo Scientific CETA 16M and Volta phase-plate;
• Thermo Scientific Spectra 300kV STEM equipped with a Thermo Scientific CETA 16M with speed enhancement fully dedicated to electron tomography workflows;
• Thermo Scientific Glacios 200kV TEM equipped with Thermo Scientific Falcon 4i, Thermo Scientific Selectris X energy filter, and CETA-D and Volta phase-plate;
• Thermo Scientific Talos L120C 120kV TEM equipped with Thermo Scientific CETA 16M to allow both room temperature and cryogenic imaging by Gatan ELSA cryo-holder;
• Leica Stellaris 5 confocal microscope equipped with white-light laser and cryo-stage to perform cryo-CLEM experiments. Leica Thunder widefield microscope equipped with cryo-stage to perform cryo-CLEM experiments;
• Ancillary equipment for sample preparation including: plunge freezing devices (Thermo Scientific Vitrobot Mark IV), glow discharger units (Pealco EasyGlow and Quorum GloQube), plasma cleaner (Gatan Solarus II), carbon coating system (Leica ACE600), and other sample preparation tools for high-pressure freezing (Leica EM ICE) and freeze substitution (Leica AFS2).

The Biomass Production Unit provides a wide set of cell cultivation equipment, facilitating protein expression across various scales and ensuring scalability for bioprocesses, including large-scale productions.

Cell Cultivation Equipment:

• Shakers: nine dedicated shakers specific for the cultivation of yeast, bacteria, mammalian, and insect cells.
• 15L Fermenter: compact fermenter suitable for yeast and bacteria, with a maximum working volume of 10L. Ideal for small-scale production and bioprocess testing and scale-up, supporting batch and fed-batch processes. Equipped with controls for pH, pO2, temperature, and turbidity.
• 150L Fermenter: larger fermentation unit for yeast and bacteria, with a maximum working volume of 100L. Designed for large-scale productions, offering batch and fed-batch processes, and featuring controls for pH, pO2, temperature, turbidity, and pressure.
• 5L Bioreactor: small bioreactor tailored for mammalian cells, useful for testing the cell in bioreactor and setting the cultivation condition with a maximum working volume of 400mL. Equipped with controls for pH, pO2, and temperature, along with a Perfusion Module – BioSep.
• 10L Bioreactor: bioreactor dedicated to mammalian cell production, supporting up to 10L of maximum working volume. Features controls for pH, pO2, and temperature, along with a Perfusion Module – BioSep.

Cell Collection Equipment:

• Super Speed Centrifuge: a high-speed centrifuge capable of processing samples with a capacity of up to 6L.
• High-Speed Tubular Centrifuge: a large-scale semi-continuous centrifuge offering flows ranging from 25 to 330 L/h. Enables centrifugal separation within a range of 2’800 g to 15’500 g.

Cell Lysis Equipment:

• High-Pressure Cell Disruptor: utilizes high pressure to disrupt the plasma membrane of cells, with a continuous flow processing capacity of up to 6L per hour. Maximum process pressure reaches 40kpsi (2700 bar).

Freezer Mill: disrupts cells by cooling them to cryogenic temperatures, followed by mechanical disruption using a magnetic steel impactor. The cryogenic temperatures are maintained throughout the processing, preventing any thermal degradation. Capable of grinding samples ranging from 0.1 to 100 grams.

The Biophysics Unit manages instrumentation allowing the biophysical characterization of macromolecular samples and the determination of their affinities of interaction. The equipment available at the Unit includes:

• Refeyn OneMP photometer (mass photometry), allowing the determination of the mass of macromolecules in solution and the homogeneity of the sample;
• Sartorius Octet R8 (bio-layer interferometry – BLI), providing the ability to monitor binding between macromolecules, their specificity, rates of association and dissociation, and allowing quantification of known ligands in complex mixtures;
• Nanotemper Monolith (microscale thermophoresis), equipped with pico RED and labelfree detectors, allowing the determination of affinity constant using extremely low amounts of samples;
• MicroCal PEAQ-ITC (isothermal titration calorimetry), allowing, through the measure of the heat exchanges occurring upon interaction of macromolecules, determination of the affinity, stoichiometry, and enthalpy of the interaction;
• Wyatt OptiLab and DAWN, coupled to Cytiva Äkta Micro system, (SEC-MALS), allowing the determination of molar mass of macromolecules separated in size exclusion chromatography, providing information on the mass and homogeneity of macromolecular samples;
• Xtal Concepts Spectrolight 610 (dynamic light scattering) allowing measurement of the size and size distribution of macromolecules in nanodrop volumes;
• Nanotemper Prometheus (nano differential scanning fluorimetry) allowing measuring the denaturation profile of a protein monitoring its intrinsic fluorescence, and detecting its aggregation in the process.

The structural proteomics unit deals with everything from protein extraction to digestion, peptide fractionation, mass spec acquisition and data analysis, with a focus on crosslinking MS. The instruments include:

Mass spectrometer:

• Thermo Scientific Orbitrap Eclipse Tribrid mass spectrometer, ETD-equipped. Also equipped with high-field asymmetric ion mobility (FAIMS) attachment.
• Thermo Vanquish Neo HPLC in-line with the mass spec, equipped with column oven;

Sample preparation:

• Akta Micro FPLC equipped for size exclusion chromatography, strong cation exchange, hydrophilic anion exchange chromatography;
• Bandelin RK-100 sonicator bath;
• Eppendorf Speed-vac concentrator;
• Access to offline Thermo Vanquish Duo HPLC for additional peptide chromatography;
• Equipment for in-gel or in-solution digestion with StageTip, Sep-Pak, SP3, iST cleanup or phospho enrichment

Dynamic Single Molecule (DSM) provides tools to visualise biological processes in real time with single molecule sensitivity. The platform is based on two cutting-edge instruments, which combine optical tweezers with various fluorescence and label free detection modules:

• Dymo700: up to four optical traps (Q-trap), confocal imaging with 3 detection channels, additional STED module for 1D super resolution kymographs. The Dymo is predominantly used for imaging ‘in solution’.
• Edge350: standard two optical traps, 3 colour fluorescence detection (single molecule sensitivity) based on either wide field or TIRF illumination, additional label free imaging based on IRM, which makes this instrument ideal for surface-based assays.

While single molecule detection allows direct observation with high spatial and temporal resolution, optical tweezers have unique capability to manipulate/perturb biological systems by applying pN forces in highly controlled manner. This integrated approach provides complementary information to high resolution structures, unravelling dynamics underlying molecular processes.

More information can be found on: https://lumicks.com/dsm-overview/