Single-Particle Cryo-EM and Cryo-Electron Tomography

Overview

The platform provides sample preparation, grid optimization, screening, and data-acquisition services for structural studies by single-particle cryo-electron microscopy and cryo-electron tomography.

Single-particle cryo-EM is used to determine the three-dimensional structure of purified macromolecules, macromolecular complexes, and viruses preserved in vitreous ice. When sample quality and particle distribution are suitable, the method can provide near-atomic-resolution structural information.

Cryo-electron tomography provides three-dimensional information from individual pleomorphic particles, viruses, organelles, bacteria, cells, and cryo-FIB-milled cellular lamellae. Depending on the specimen and scientific objective, tomographic data may be used for direct visualization, three-dimensional segmentation, or subtomogram averaging.

The platform supports projects from initial sample assessment and vitrification through grid screening, optimization, and high-resolution data collection. Routine preparation and screening are performed at IBS, while selected projects may access the CM02 Titan Krios 300 kV cryo-electron microscope operated by IBS as an ESRF Collaborative Research Group beamline.

Both on-site and remote data-collection sessions are possible.

Main Applications

The service supports structural studies including:

• Single-particle analysis of purified proteins and macromolecular complexes
• Structural analysis of viruses and virus-like particles
• Analysis of flexible, heterogeneous, or multi-component assemblies
• Molecular cryo-electron tomography of purified particles
• Subtomogram averaging of repeated macromolecular structures
• Tomography of vesicles, organelles, bacteria, viruses, and cellular samples
• Cryo-electron tomography of cryo-FIB-milled cellular lamellae
• Preliminary screening before high-resolution data collection on a 300 kV microscope
• Tilted single-particle acquisition for samples displaying preferential particle orientation

Single-Particle Cryo-Electron Microscopy

Single-particle cryo-EM is used to study purified macromolecules or complexes embedded in a thin layer of vitreous ice.

A large number of two-dimensional particle images are acquired from different orientations. Computational alignment, classification, and reconstruction can then be used to determine a three-dimensional structure. Suitable specimens range from relatively small proteins, typically above approximately 60 kDa, to large macromolecular assemblies and viruses.

Successful single-particle projects generally require a purified and sufficiently homogeneous sample, an appropriate particle concentration, good particle distribution within the ice, and a broad range of particle orientations.

Typical Single-Particle Workflow

1. Sample Quality Assessment

Before vitrification, sample quality should be evaluated using complementary methods whenever possible. Negative staining electron microscopy, mass photometry, size-exclusion chromatography, SDS-PAGE, and other appropriate biochemical or biophysical techniques can provide important information about purity, homogeneity, aggregation, concentration, and oligomeric state.

Quality control by negative staining is strongly recommended before beginning an extensive cryo-EM optimization campaign.

2. Grid Preparation and Vitrification

The purified specimen is applied to an electron microscopy grid, blotted to produce a thin liquid film, and rapidly plunged into liquid ethane. This procedure vitrifies the sample and preserves it in a hydrated state without forming crystalline ice.

Vitrification can be performed using a Vitrobot Mark IV or a Leica EM-GP2 plunge-freezing system.

3. Grid Screening and Optimization

Initial grids are screened to assess:

• Ice thickness and ice quality
• Particle concentration and distribution
• Particle integrity
• Aggregation or adsorption at interfaces
• Particle orientation
• Grid and support-film behavior
• Overall suitability for automated data collection

Grid preparation is usually iterative. Sample concentration, buffer composition, grid type, blotting conditions, waiting time, temperature, humidity, and other parameters may need to be adjusted before a suitable condition is identified.

4. Data Collection

Once suitable grids have been identified, automated movie acquisition can be performed on the Glacios 200 kV microscope or, for selected projects, on a 300 kV Titan Krios.

The Glacios can be used for both detailed sample evaluation and high-resolution data collection. Depending on the specimen, grid quality, and data-processing results, datasets acquired on the Glacios may support three-dimensional reconstructions in the approximately 2.3–4 Å range.

For particularly demanding projects, zero-loss imaging on a 300 kV microscope such as CM02 may be recommended after sample suitability has been demonstrated through screening and a preliminary data collection.

Cryo-Electron Tomography

Cryo-electron tomography is used to reconstruct a three-dimensional volume from a series of images acquired while the specimen is tilted inside the microscope.

The technique is suitable for specimens that cannot be adequately described by conventional single-particle averaging, including pleomorphic particles, irregular macromolecular assemblies, viruses, organelles, bacteria, cellular regions, and cryo-FIB-milled lamellae.

Molecular Cryo-Electron Tomography

Molecular cryo-electron tomography can be performed on purified macromolecules, complexes, viruses, or other particles embedded in vitreous ice.

Tilt series are aligned and reconstructed to generate tomograms. Repeated particles or structural features may subsequently be extracted, classified, and averaged using subtomogram-averaging approaches.

Molecular cryo-ET may be particularly useful for:

• Pleomorphic or structurally variable particles
• Irregular macromolecular complexes
• Host–virus assemblies
• Helical or repetitive structures
• Generating initial three-dimensional models
• Studying specimens for which conventional single-particle analysis is difficult

Cellular Cryo-Electron Tomography

Cellular cryo-ET provides three-dimensional information about macromolecular and cellular organization in a close-to-native frozen state.

Relatively thin specimens, such as small bacteria or peripheral regions of cells, may sometimes be imaged directly. Thicker specimens generally require preparation of electron-transparent lamellae by cryo-focused ion beam milling.

Fluorescence microscopy may be used before or after milling to identify a protein, organelle, or region of interest. The resulting lamellae can then be analyzed by cryo-TEM and cryo-electron tomography.

Projects requiring cellular vitrification, cryo-correlative microscopy, or cryo-FIB/SEM lamella preparation are coordinated with the platform’s cellular electron microscopy service.

Sample Requirements

Sample suitability is one of the main factors determining the success of a cryo-EM experiment. Users are strongly encouraged to contact the platform before preparing or shipping samples.

Purity and Homogeneity

Samples should be as pure, homogeneous, stable, and monodisperse as possible. Purity should normally be documented, for example by SDS-PAGE or another suitable analytical method.

Whenever possible, users should provide results from negative staining, mass photometry, size-exclusion chromatography, dynamic light scattering, or other relevant biochemical and biophysical analyses.

Sample Quantity and Concentration

As a general starting point, the platform requests:

• Approximately 30 µL of purified sample
• A concentration of approximately 1–3 mg/mL
• Approximately 100 µL of the corresponding buffer for dilution or condition optimization

The optimum concentration is sample-dependent and may differ substantially from these initial recommendations.

Buffer Composition

Detergent concentrations should be kept as low as reasonably possible. Glycerol should preferably be avoided because it can interfere with vitrification, ice quality, image contrast, and sample distribution.

High salt concentrations, free affinity tags, excess ligands, aggregates, precipitates, and other buffer components may also affect grid quality. Buffer exchange or additional purification may therefore be recommended before vitrification.

Molecular Size

Particles above approximately 60 kDa may be suitable for direct observation under favorable conditions. Smaller particles are generally more challenging because of their reduced image contrast and may require particularly homogeneous samples, optimized grids, and advanced acquisition strategies.

Larger complexes are not automatically easier to analyze: flexibility, heterogeneity, aggregation, dissociation, symmetry, and preferential orientation can all limit structure determination.

Biosafety and GMO Compliance

All samples must comply with the applicable IBS and ESRF biosafety, biohazard, transport, and GMO regulations.

Users must disclose any biological or chemical hazards before samples are accepted. Samples may only be handled after the required institutional declarations and authorizations have been completed.

Requirements for CM02 Data Collection

Evidence of sample quality and suitability must be provided when requesting access to the CM02 Titan Krios.

For single-particle experiments, the application should include relevant preliminary results such as:

• Representative micrographs
• Two-dimensional class averages
• Preliminary three-dimensional reconstructions
• Grid atlases
• Screening or test-acquisition results

For molecular cryo-electron tomography, an atlas and representative high-magnification images should be provided.

For cellular cryo-electron tomography, the application should include an atlas and medium-magnification images showing the cells or lamellae and the regions proposed for tilt-series acquisition.

Before a CM02 session, grids must have been screened and clipped into autogrids compatible with the microscope autoloader. Up to four grids can normally be loaded for a session.

Data-Collection Strategies

Standard Single-Particle Acquisition

Typical single-particle sessions on CM02 last 48 hours. A 24-hour session may be allocated on request, while sessions of up to 72 hours may be considered for projects with a clearly justified need.

Multigrid Acquisition

Several grids may be included in a single session using a multigrid acquisition strategy, provided that the same acquisition parameters are appropriate for all selected grids.

Tilted Acquisition

Tilted data acquisition, generally up to approximately 40°, may be considered when previous high-resolution datasets reveal strong preferential particle orientation that could not be resolved by further sample or grid optimization.

Tomography Acquisition

Tomography sessions generally last between 48 and 72 hours, depending on the number of grids, number of tilt series, specimen type, acquisition geometry, and scientific objective.

Instruments and Equipment

Thermo Fisher Scientific Glacios

The Glacios is a 200 kV field-emission-gun cryo-transmission electron microscope equipped with:

• A Falcon 4i direct electron detector
• Automated EPU acquisition software
• Cryogenic sample handling and autoloader capabilities

The microscope is used for grid screening, sample optimization, preliminary acquisitions, and high-resolution single-particle data collection.

FEI Tecnai F20

The Tecnai F20 is a 200 kV field-emission-gun transmission electron microscope that can be used for cryogenic sample observation, screening, and selected data-acquisition applications.

CM02 Titan Krios G4

CM02 is a Thermo Fisher Scientific Titan Krios G4 operated by IBS as an ESRF Collaborative Research Group beamline.

The microscope is equipped with:

• 300 kV accelerating voltage
• Cold field-emission gun
• Fringe-free illumination
• Falcon 4i direct electron detector
• Selectris X energy filter
• Volta phase plate
• Spherical aberration coefficient of approximately 2.7 mm
• EPU software for automated single-particle acquisition
• Tomo software for automated tomography acquisition

CM02 is part of the France Cryo-EM national infrastructure funded through the French “Plan d’Investissement d’Avenir 3” programme. The infrastructure supported the installation of three state-of-the-art 300 kV cryo-electron microscopes in France.

CM02 is maintained and operated by a dedicated IBS/ISBG team in Grenoble.

Routes of Access to CM02

Users are encouraged to contact the CM02 team before submitting a proposal.

National CRG Access

Up to two-thirds of the available CM02 beam time is allocated through the national CRG access route for French academic laboratories.

Applicants should complete the current proposal template and send it to:

cm02.contact@ibs.fr

Current eligibility, evaluation, scheduling, and charging conditions should be confirmed with the CM02 team before submission.

ESRF Access

Approximately one-third of CM02 beam time is allocated through ESRF access.

Applications are submitted through the ESRF User Portal as structural biology rolling-access proposals. Applications may be submitted throughout the year. Dedicated cryo-EM Block Allocation Groups are available and are distinct from macromolecular crystallography BAG proposals.

Contact:

cryo-em@esrf.fr

For accepted ESRF proposals, travel support is subject to the current ESRF user-access rules.

Instruct-ERIC Access

International users may apply for access through Instruct-ERIC. Depending on the applicable call and eligibility rules, support may include instrument access, sample shipment, and travel expenses.

Further information:

https://instruct-eric.org/centre/instruct-centre—france-2

Industrial Access

Industrial users should contact the platform to discuss feasibility, confidentiality, scheduling, and quotation.

Remote and On-Site Sessions

Both remote and on-site sessions can be arranged.

The appropriate format is determined according to the experiment, access route, user experience, biosafety requirements, and the level of intervention expected during data collection.

A local contact provides microscope operation and scientific or technical support according to the agreed scope of the session.

Sending Samples

National CRG and Instruct-ERIC Access

Dry shippers should be sent to:

IBS
[Local contact name] – MEM Group
71 avenue des Martyrs
CS 10090
38044 Grenoble Cedex 9
France

Users must inform their local contact before shipment and provide the courier name and tracking number.

A completed return document and all information required for the return shipment should be included with the dry shipper.

ESRF-Allocated Access

Users accessing CM02 through ESRF should follow the current ESRF procedures for importing samples and equipment:

https://www.esrf.fr/UsersAndScience/UserGuide/Preparing/ImportingEquipment

Users are responsible for ensuring that all transport, customs, biosafety, and dry-shipper documentation is complete.

Data Retrieval

Data acquired on CM02 can be downloaded from the ESRF computing infrastructure.

Instructions for off-site data transfer are available at:

https://www.esrf.fr/Infrastructure/Computing/ComputingOffsite/FtpWindows

Users should confirm data-retention periods, account access, transfer procedures, and expected dataset size with their local contact before the session.

Acknowledgments

Publications, posters, presentations, and other communications using data collected on the platform should acknowledge the relevant facility, funding programme, and local support.

National CRG and In-House Experiments

Suggested acknowledgment:

“The cryo-electron microscopy data were acquired at the CM02 CRG beamline operated by IBS at the European Synchrotron Radiation Facility in Grenoble. The purchase of this microscope was funded by the EquipEx+ France Cryo-EM project (ANR-21-ESRE-0046). We thank [local contact] for performing the experiments and providing local support.”

ESRF-Allocated Beam Time

Suggested acknowledgment:

“We acknowledge the European Synchrotron Radiation Facility for provision of beam time on the CM02 CRG beamline. The purchase of this microscope was funded by the EquipEx+ France Cryo-EM project (ANR-21-ESRE-0046). We thank [local contact] for performing the experiments and providing local support.”

The following sentence may also be added where appropriate:

“We thank Guy Schoehn for the establishment of the cryo-EM facility.”

Users are encouraged to acknowledge the platform not only in publications, but also in conference talks, posters, preprints, reports, and other scientific communications.

Main Contacts

CM02 access and project enquiries:
cm02.contact@ibs.fr

ESRF cryo-EM access enquiries:
cryo-em@esrf.fr

Project-specific scientific and technical contacts are assigned after initial discussion with the platform.

Location

IBS – Institut de Biologie Structurale
Methods and Electron Microscopy Group
Ground floor
71 avenue des Martyrs
Grenoble, France