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
Why choose NIS for CryoEM?
NIS is the first and only 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
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.
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.
CryoEM grid preparation and 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.
Data acquisition on in-house Krios microscopes
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.
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.
Get to know our team
Speak with our experts about using cryoEM to support your drug discovery efforts.