Single Particle Analysis
We provide services for all levels of single particle analysis needs, from grid preparation to high resolution data collection, and reconstruction and refinement. Each step can be independent. For example, a client may only need grid preparation capabilities or extra data collection time on any of our instruments. For fully outsourced projects, we offer a complete cryoEM workflow. Learn more about our modular approach to supporting cryoEM adoption for high resolution structure determination in our Working With Us resource.
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.
Get to know our team
Speak with our experts about using Single Particle Analysis to support your drug discovery efforts.