Single Particle Analysis

Single particle analysis (SPA) is a group of related computerized image processing techniques used to analyze images from transmission electron microscopy (TEM). These methods were developed to improve and extend the information obtainable from TEM images of samples and now allow for high resolution structure determination of proteins, protein complexes and viruses.


NIS is the global leader in combining SPA with cryoEM to support high resolution, proprietary drug discovery projects. We provide services for all stages of single particle analysis needs, from grid preparation, to high resolution data collection, through processing and refinement of the 3D map. We were the first, and continue to be the only, CRO with dedicated in-house Titan Krios microscopes, maintaining and expanding state-of-the-art facilities in both San Diego and the Greater Boston area.

In the first 3 years of internal Titan Krios operation, NIS has collected data to support more than 200 SPA projects, solving over 60 unique structures in house. Of these, 70% of structures were determined at resolutions of 3.5A or better and more than 50% have clearly resolvable ligands.

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

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. 

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Speak with our experts about using Single Particle Analysis to support your drug discovery efforts.

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