Available Services

This service is designed to cover imaging on large volumes and includes sample preparation by either high-pressure freezing or chemical fixation, freeze substitution, resin embedding, section preparation by ultramicrotomy and S/TEM imaging at 300kV. The User can provide specimen at any stage, so being already fixed, stained, embedded as well as already sectioned and made ready for EM acquisition. A maximum of 2 specimens can be processed per unit of service, and 4 samples maximum (i.e., replicates) can be prepared per specimen. High-pressure freezing will be performed on a Leica EM ICE. Freeze substitution will be performed on a Leica AFS2, and a User might specify a substitution protocol of preference, which will be utilized if compatible with NF practices. Similarly, for chemical fixation, the User is allowed to specify their protocol of preference, which will be adopted if compatible with NF practices. For resin-embedding, the User could provide a resin of preference, or alternatively, the currently available resin be used. Sections of resin-embedded sample will be prepared on Leica UC7. A maximum of 1 resin-embedded sample can be processed for unit of service, to be sectioned in ribbons, and to be applied to a maximum of 4 TEM support grids. User might specify thickness of final sections, and these recommendations will be followed if compatible with the NF practices. User might provide TEM grid support of preference, otherwise the currently available grid support will be used. Imaging will be performed an a Thermo Scientific Spectra 300 kV either in TEM, equipped with CETA 2 camera, or STEM mode in BF/DF/HAADF. Imaging conditions, if requested by the Usermust be compatible with NF practices. Microscope Access is granted for a maximum of 5 days per unit of service, including all steps from sample loading to alignments and collection, and according to NF staff availability. All steps of this service will only be performed by NF staff. To maximise efficient use and Access to high-end TEM instruments, priority will be given to applicants specifically requesting parts of this service (e.g. only sectioning, or only S/TEM imaging of already prepared sections).

Many workflows available in other Infrastructural Units of the National Facility for Structural Biology may require a rapid sample quality control verification (if not performed by the users) or a step of sample optimisation, for instance via size-exclusion chromatography just prior to experiments. These services, which differ from the full characterisation of the macromolecular sample because of their ancillary role with respect to other IU workflows, may only be requested in combination with such workflows.

This service comprises 8 hours of equipment time on the Orbitrap Astral LC-MS. The configuration of the system is: Vanquish NEO HPLC in trap and elute configuration. Mobile phase A is 0.1% formic acid in water, mobile phase B is 80% acetonitrile in water with 0.1% formic acid. The MS is set up with an EasySpray source. The MS is operated for high- throughput, with EasySpray pepMap columns (15cm, 150µm diameter, 10nm pore size). It is possible to use the FAIMS filter to improve sensitivity or fractionate samples online. Overhead on measurements due to sample loading and column washing is about 2 minutes per injection. Typical gradient length 15 minutes (no gains are typically observed beyond this gradient length due to the speed of the Astral analyser and the operation in high flow). This configuration allows for up to 100 samples per day. Both DIA and DDA proteomics workflows are available.  The service does not comprise sample preparation from proteins to peptides.

This service comprises 8 hours of equipment time on the Orbitrap Astral LC-MS. The configuration of the system is: Vanquish NEO HPLC in trap and elute configuration. Mobile phase A is 0.1% formic acid in water, mobile phase B is 80% acetonitrile in water with 0.1% formic acid. The MS is set up with an EasySpray source. The MS is operated for high- load, using EasySpray pepMap NEO columns (50cm, 75µm diameter, 10nm pore size). It is possible to use the FAIMS filter to improve sensitivity or fractionate samples online. Overhead on measurements due to sample loading and column washing is about 10 minutes per injection. Typical gradient length 30 minutes (no gains are typically observed beyond this gradient length due to the speed of the Astral analyser). Both DIA and DDA proteomics workflows are available. Each proposal can request up to 9 sessions for a total of 3 days of MS time. The service does not comprise sample preparation from proteins to peptides.

We offer access to expert support in isolating and purifying proteins from a variety of biological sources. We offer customisable workflows, including cell lysis via sonication or homogenization, depending on any given sample’s specific requirements. The service can accommodate both purification from standard small and medium-volume lysates, or isolation from large-volume sample preparations, ensuring flexibility for different experimental needs. Our purification techniques include affinity, ion exchange, and size-exclusion chromatography, delivering (when possible) high-quality proteins suitable for downstream applications such as structural analysis or functional assays.

The service offers dedicated pipelines for small- and large-scale cell cultures and expressions:

• Small scale: 15 litres fermenter useful for initial trial test before moving to larger scale fermenter or small expression using both bacteria and yeast cells. Typical culturing strategies are available such as batch and fed batch (up to 2 feeds). Max working volume of 10 litres.
• Large scale: 150 litres fermenter dedicated to large cultivation and protein production. Typical cultivation strategies are available, including batch and fed batch (up to 2 feeds). Max working volume of 100 litres.

In both cases, cells will be collected and separated from the supernatant using dedicated superspeed centrifuges or a High-Speed Tubular Centrifuge. Upon request, the cells can be lysed using a continuous flow cell disruptor or by a high-capacity cryogenic grinder.

The service offers dedicated pipelines for small- and large-scale cell cultivation and protein expressions:

• Small scale: utilizing commercial Expi293F cells, transient transfection is conducted with a plasmid containing the gene/s of interest (potentially with a fluorescent marker), provided by the applicant. Cell cultivation and transfection take place in Erlenmeyer flasks within dedicated shaker incubators; typically, 500 ml of cell culture per 2-liter flask is utilized up to a total cultivation volume of approximatively 10 litres. Transfection of an Expi293F cell culture is performed at approximately 2 to 3 x10^6 cells/ml, following a modified protocol with polyethylenimine (PEI). While no expression test will be performed, the efficiency of transfection can be readily assessed if the plasmid includes a fluorescent marker. Transfected cells are subsequently harvested by centrifugation, and either the frozen pellet or the refrigerated supernatant will be provided.
• Large scale: bioreactor cultivation and protein expression are performed either with commercial cell lines (e.g., Expi293F cells) transiently transfected with applicant-provided plasmids, or with any mammalian cell line adapted to suspension cultivation supplied by the applicant. Preliminary tests are normally performed using a 0.5 litres scale bioreactor to define and optimize bioreactor cultivation conditions. Subsequently, a 10 litres scale bioreactor enables large-scale cell cultivation and protein expression, employing both transient transfection protocols and stable cell line expressions. Moreover, the implementation of perfusion, utilizing an acoustic retention device, may be explored to increase the biomass production and, consequently, protein expression. Similar to small-scale operations, no expression test will be performed. Instead, monitoring of transfection efficiency for transient expression can be undertaken if the plasmid carries a fluorescent marker or fluorescently tagged protein of interest.

The service aims to cover the entire process of Baculovirus protein expression, starting from a Bacmid. Initially, commercial Sf9 cells are transfected with the Bacmid, supplied by the applicant, which contains the gene of interest and a fluorescence marker. The initial stage results in a low-titre Baculovirus suspension (P1 generation), followed by a second amplification (P2 generation) to produce a high-titre suspension. The P2 Baculovirus suspension could be directly used for protein expression or another round of Baculovirus amplification is performed before infecting commercial High Five insect cells (at 0.5×10^6 cells/ml). The virus amount used depends on the construct and might need optimization in initial small-scale infections. Protein expression is monitored indirectly by checking for the fluorescence marker (carried by the Bacmid) and cell viability. Finally, cells are harvested via centrifugation, and the pellet or supernatant is made available.

We offer sample preparation by negative stain EM followed by TEM imaging at 120 kV. A maximum of 4 service units (i.e. 4 days of screening and sample preparation on 1 specimen or 4 different ones) can be requested. For each session a maximum of 8 grids can be prepared and processed. Imaging will be provided for a maximum of 8 continuous hours per unit of service. An optional “polishing” size-exclusion chromatography (SEC) step can be performed on thawed material by the Biophysics Unit, as and if considered necessary by NF Staff. 400 mesh copper grids with amorphous carbon film layer will be used as support. Glow discharging will be performed by a Pelco EasyGlow device. Staining will be performed with a 2% (w/v) uranyl acetate aqueous solution. Imaging in TEM mode at 120 kV will be performed on a Thermo Scientific Talos L120C equipped with CETA 16M camera. Imaging conditions requested by the User, if provided, must be compatible with NF practices. This service will only be performed by NF staff.

The Biophysics Unit is equipped with instruments designed to determine the strength and biophysical properties of macromolecular or protein-ligand interactions, including association and dissociation constants, as well as stoichiometry of binding. Available techniques include isothermal titration calorimetry, microscale thermophoresis, and bio-layer interferometry (which can also be utilized for quantifying known components in complex mixtures).

The Structural Proteomics unit will perform integrative structural modelling combining medium/ low resolution (from 7 to 30 Å) cryo-EM densities and crosslinking MS data acquired at our NF. In case of very large systems, negative stain data may also be used. This does not refer to model building in cryo-EM densities, but to calculations of localization of subunits or of areas not observed in EM maps. This task can be performed on data from services SB-IU4-A or SB-IU4-B only. Example software: DisVis, integrative modelling platform (IMP), AlphaLink/ AlphaLink2. This service will be performed by NF staff, but training will be available if requested, provided basic bioinformatics skills of the User (bash terminal, python).

This service provides high-resolution cryo-TEM data collection of vitrified specimens. To ensure efficient usage of high-end microscope time this service is exclusively dedicated to EM grid ready for data collection at the time of application (i.e. user cannot request this service together with service SB-IU1-B – Cryo-EM Screening). A maximum of 2 samples for data collection can be requested per application. For each sample a maximum of 48hr of microscope time can be requested per application. According to instrument availability and experimental needs, data collection will be carried out either on 200kV or 300kV microscope systems. The User can provide cryo-TEM grids either unmounted or mounted on a Thermo Scientific cartridge. In case of an unmounted grid, clipping of the specimen in Thermo Scientific cartridges will be performed by NF staff. In case of User-provided already-clipped grids, these will be inspected by NF staff prior to acceptance. Imaging at 200 kV will be performed on a Thermo Scientific Glacios while imaging at 300 kV will be performed on a Thermo Scientific Titan Krios G4, both equipped with a Falcon 4i direct electron detector and a Selectris X energy filter. Imaging conditions (i.e., dose, pixel size, magnification, etc.), if requested by the User, must be compatible with the NF best practices. Microscope time includes all steps from clipping to loading and TEM alignments and according to NF staff availability. For single-particle acquisition, User might opt for beam-image shift assisted data collection (~ 450 – 600 movies/hour) or stage movement for each hole (~ 100 – 250 movies/hour). This service will only be performed by NF staff.

This service is dedicated to projects where specific single molecule assays have been already established or, alternatively, should be combined with ‘assay development’ service as a simple ‘package’. Examples of experiments currently performed routinely by the unit: (i) detecting changes to DNA structure upon protein binding e.g., melting, looping, compaction; (ii) characterizing protein behaviour upon binding to DNA (diffusion, direct motion, interactions with other proteins). This service can be performed by the NF staff or by a trained User. The training can be provided by the unit as part of this service. Full training typically requires 3-5 days. The User can spend up to 10 days at the NF. The work can be split into two visits. Original data will be handed to the User as .h5 files. The unit will assist the User in Accessing the files using Pylake. In case of kymographs, NF staff can provide a short training on existing tools such as Lakeview.

This service provides cryo-Fluorescence Microscopy imaging including sample preparation by plunge-freezing and grids clipping. A maximum of 2 specimens can be processed per unit of service, maximum 4 grids (i.e. replicates) can be prepared per specimen. Glow discharging will be performed by either a Pelco EasyGlow, a Quorum GloQube, or Solarus II plasma cleaner device. Plunge-freezing will be performed on a Thermo Scientific Vitrobot Mk IV or a Leica EM GP2. Applicants may provide TEM grid supports of preference, otherwise the NF staff will use the available TEM grid supports. Widefield imaging will be performed on a Leica Thunder cryo-CLEM system. Confocal imaging will be performed on a Leica Stellaris 5 cryo-CLEM system equipped with white light laser. Both microscopes are equipped with a 50x / 0.9 NA lens. This service is provided for a maximum of 8 continuous hours per unit of service, including plunging, clipping, and imaging. In case of User-provided already-clipped grids (in Thermo Scientific cartridges), these will be inspected by NF staff prior to acceptance. This service will only be performed by NF staff.

This service provides sample preparation by plunge-freezing, grid clipping for autoloader system and cryo-TEM imaging at 200 kV. User can either provide the purified sample in solution, the sample already vitrified on EM grid, or the vitrified sample on an already clipped (i.e. Thermo Scientific autoloader compatible) EM grid. User might request cryo-EM screening on a maximum of 4 different samples per application. For each sample, a maximum of 3 Cryo-EM Screening sessions can be allocated. For each Cryo-EM screening session a maximum of 8 grids can be prepared and processed. An optional “polishing” SEC step can be performed on thawed material by the Biophysics Unit, as and if considered necessary by NF Staff. Glow discharging will be performed by either a Pelco EasyGlow, a Quorum GloQube, or Solarus II plasma cleaner device. Plunge-freezing will be performed on a Thermo Scientific Vitrobot Mk IV or a Leica EM GP2. Applicants may provide TEM grid supports of preference, otherwise NF staff will use holey TEM grid supports available. Imaging will be provided for a maximum of 8 continuous hours per unit of service. In case of grids showing optimal particle distribution and ice quality, this service can be extended for overnight data collection, if compatible with NF staff working hours. Cryo-TEM imaging will be performed only in EF-TEM mode at 200 kV on a Thermo Scientific Glacios equipped with a Falcon 4i direct electron detector and Selectris X energy filter. Imaging conditions requested by the User, if provided, must be compatible with NF practices. In case of User-provided already-clipped grids, these will be inspected by NF staff prior to acceptance. This service will only be performed by NF staff. This service does not cover for buffer screening (i.e. detergents, pH, salt, additives screening or else) or any other grid modifications (i.e. graphene oxide, pegylation, affinity grids or else).

The Structural Proteomics unit will perform crosslinking MS on a purified protein complex to identify protein-protein interactions and map residue distances. This service does not include preliminary crosslinking reaction optimisation, which should be performed by the User at their own institution (in consultation with NF staff). This service will only be performed by NF staff.

The Structural Proteomics unit will perform crosslinking MS on a purified protein complex to identify protein-protein interactions and residue distances. This service includes preliminary crosslinking reaction optimisation and proteomics acquisitions. These can be performed by our NF staff provided the protein sample, or by a visiting User with our assistance.

The Structural Proteomics unit will perform crosslinking MS to characterize the interactome of a particular target protein in situ. Crosslinking reactions can be performed on cells, in lysate or after competitive or otherwise non-denaturing (native) elution. As this experiment requires a careful and at times tricky design, preliminary results will be shared with the User and guidance provided to set or optimize the experimental conditions. This service will be performed by NF staff if the crosslinking reaction occurs at the home institution with guidance from us, or by a visiting User under our direct supervision if the entire workflow is requested.

The Structural Proteomics unit will perform crosslinking MS to characterize the topology of the protein-protein interactome of a particular target protein or enriched cellular fraction in situ. Examples include: interactome of pulldowns with an endogenously tagged protein of interest; interactome of vesicles/ cellular compartments; interactome of bacterial cells or virus/ host interactions after pulldown of specific virulence factors. Crosslinking reactions can be performed on cells, on lysates or after competitive or otherwise non-denaturing (native) elution. As these experiments require careful design, the User will be granted Access to preliminary acquisitions to characterise the sample and set the correct reaction conditions, or guidance to perform the optimization at their home institution. This service will be performed by NF staff in collaboration with a visiting User.

Structural biology and biochemical assays often require sample optimization to achieve stability and homogeneity, while understanding the oligomerisation state and precise composition of macromolecular complexes is crucial for unravelling their architecture. To address these needs, the Biophysics Unit offers a service leveraging various instruments to determine the molecular weights of species in solution and their thermal stability parameters. Techniques utilized include mass photometry, SEC-MALS, dynamic light scattering, and nanoDSF.

This service enables researchers to explore the potential of single-molecule techniques and develop new protocols and workflows. Prior experience with dynamic single-molecule (DSM) approaches is not required.

The unit is equipped with two state-of-the-art instruments:

1. C-Trap ‘Dymo’ utilizes confocal scanning and is primarily used for imaging in solution.
2. C-Trap ‘Edge’ offers the flexibility to switch between wide-field fluorescence and TIRF imaging. Additionally, it allows label-free imaging via IRM, making it ideal for studies close to the surface.

The unit provides support for a wide range of experimental approaches.

This service will only be performed by NF staff, ideally accompanied by the User. As part of this service, the NF staff will check sample quality, troubleshoot, and define optimal conditions for data acquisition. If possible, an automation protocol for the experiment can be developed to streamline future data collection. The User can spend up to 10 days in the NF, testing various samples. The work can be split into two visits. The outcome of this service is an established protocol that can be used for ‘data acquisition’ service.