CryoEM Services for Protein Structure Determination

What is CryoEM?

CryoEM (coupled with single particle analysis) has become a popular method for high resolution 3D protein structure determination. An aqueous biological sample is frozen rapidly and irradiated with a beam of electrons from a transmission electron microscope. A detector senses how the electrons are scattered and computerized image processing techniques are used to reconstruct the 3D-shape of the molecule.

Why do you need CryoEM Services?

X-ray crystallography and NMR have traditionally been the methods of choice for high resolution 3D protein structure determination, but cryoEM has emerged as a complementary technique. It is well suited for targets that are not suitable for X-ray crystallography or NMR. 

These are common situations where you could benefit from cryoEM services:

  • You have a small amount of material
  • The material is hard to crystallize
  • You have a large biomolecular complex
  • You're looking into the atomic details of the molecule
  • You want to observe the real "native state" structure

Useful Resources

Why choose NIS for CryoEM?

NIS is the first and largest CRO services provider to own and operate the necessary hardware and software for full structure enablement from sample to final map production. Our expanding state-of-the-art facilities, combined with best-in-class IT infrastructure and scientific know-how, has supported over 200 cryoEM projects resulting in more than 60 unique protein structures.

70% of these protein structures were determined at resolutions of 3.5Å or better and more than 50% have clearly resolvable ligands. From large protein complexes to small integral membrane proteins, we've successfully tackled a diverse set of projects that would not have been possible with X-ray crystallography or NMR.

Our CryoEM Workflow: From Sample to 3D Reconstruction

Aggregation Analysis
Homogeneity Analysis
Vitrification Optimization
CryoEM Grid Screening
High Res Data Acquisition
3D Reconstruction
Aggregation Analysis

Negative Stain Screening

The first step in the workflow is a cursory examination to determine whether the sample is sufficiently pure and homogeneous for further, more detailed study by electron microscopy. At this stage, the sample should show evidence of well-defined, separated, high contrast particles of the expected size, with minimal or no aggregation. We have a long-standing expertise in the field, and we will work with the client to identify possible issues and provide suggestions on how to improve the sample.

Homogeneity Analysis

Negative stain with 2D classification

Once the sample has shown no or minimal aggregation, a detailed negative stain analysis performed to verify that the overall size, structure, oligomerization state, domain organization and conformational heterogeneity of the particles are consistent with those expected for the macromolecule of interest.  Sample grid preparation at this stage is carefully optimized to maximize the quality and resolution of the images with sufficient data acquired to perform 2D class averaging. Several of the resulting class averages must be clearly interpretable, with crisp features, limited disorder and flexibility, and limited oligomerization states to be prioritized for subsequent cryoEM study.

Vitrification Optimization

CryoEM grid preparation and screening

To obtain high resolution structural information, data must be collected using vitrified samples.  NIS has extensive, proven experience in sample vitrification and has developed a proprietary workflow for generating optimal and reproducible cryoEM grids. Samples will be vitrified either with the Vitrobot (ThermoFisher Scientific), a semi-automatic plunger, or using the Chameleon prototype (SPT Labtech), depending on sample conditions and results. Sample quality is iteratively assessed using in-house Glacios electron microscopes to ensure maximal workflow efficiency.
CryoEM Grid Screening

Grid prioritization & low-resolution data acquisition

 Screening of the grids with the Glacios microscope is used to identify the presence (or absence) of these potential issues that might prevent the client from obtaining high resolution structures. Grids are prioritized to maximize the likelihood that a structure of suitably high resolution can be achieve from data acquired on the Titan Krios instrument.  Grid screening can be combined with acquisition of an overnice dataset on the Glacios, which is subsequently used to obtain 2D classes (and sometimes a 3D reconstruction) to assess sample quality. 

High Res Data Acquisition

Data acquisition on in-house Krios microscopes

A high-resolution data collection requires optimal instrument conditions and highly experienced operators. To ensure consistency and stability during the data collection period, our microscope is carefully maintained resulting in less than 10% annual downtime.  Our automated data collection software (Leginon) allows us to run the microscope 24/7, and we have a web-based application that allows the client to remotely monitor the data collection in real-time. Pre-processing steps run in parallel with data acquisition so that both raw images and fully pre-processed micrographs are available for the client on completion of the run.  These can be transferred to the client's home computer for further processing or the 3D reconstruction completed by NIS.
3D Reconstruction

Data processing & 3D reconstruction

NIS offers a complete workflow for data processing that leads to a 3D reconstruction. This workflow includes particle picking, 2D classification, 3D classification, and 3D reconstruction. The many particles present in the images are initially classified into 2D classes.  Each class provides a different projection view of the macromolecule of interest, and these views can be mathematically combined to generate a 3D map.  The reconstructed map provides information for the entire volume of the particle, both surface features and internal morphology, and can reach atomic-level resolution. 

Our Unique Service Offering

NIS provides a unique service offering that melds traditional CRO service packages with training and instrument access opportunities typically only accessible through academic and national laboratory facilities. In addition to project-based proposals, NIS offers custom service packages, onsite training in sample preparation and data acquisition, and data acquisition-only programs. Learn more through the Working With Us resource.

Related Services

New for 2020 - NIS is the starting of our development pipeline for macromolecular microED. Potential early adopters are encouraged to contact us for more information on our plans and to discuss opportunities for sample submission and analysis. 

We also provide services in Cryo-electron Tomography (CryoET). Visit this page to learn more about reconstructing a 3D volume of sample from a 2D projection images.

Request a CryoEM Consultation

Speak with a member of our scientific team about feasibility and sample considerations.