For many of our clients, there is no time or sample to waste when high value, challenging targets arrive at our lab for analysis. Frequently arriving fresh from the column or needing fast handling following freeze-thaw, it’s important to make the most of each sample that we receive.
Inbound samples can be precious for several reasons:
- The protein or complex is highly labile, aggregating or quickly dissociating.
- The sample may be challenging to reproduce, suffering from batch-to-batch variability.
- The sample may be expensive and/or slow to iterate either due to upstream CRO turnaround times or internal production queues.
Before issuing a proposal, our clients meet with members of our scientific team to review target information, construct design, biochemical and biophysical data for any constructs of interest. Typically, each sample will have been extremely well characterized, exhibiting high levels of purity, monodispersity, thermostability and activity (if applicable). Together, we identify any obvious cause for concern, plan carefully for the sample that NIS will receive, and roadmap possible subsequent iterations. For high resolution projects, time and effort invested in sample quality and optimized sample preparation pays off in terms of dataset size, computational complexity, and the overall time and cost of all subsequent structures when supporting biologics engineering and medicinal chemistry efforts.
After receipt at NIS, the sample is assessed by our team of structural biologists and microscopists working in tandem, leveraging both by negative stain and vitrification workflows. In this model, successful outcomes are maximized relative to sequential studies, as working quickly and collaboratively ensures sample integrity and maximal sample characterization. Negative stain, in conjunction with 2D classification, is used to assess sample homogeneity. It also provides a baseline for behavioral features that can help identify if issues arising during vitrification are due to the sample quality or the vitrification process.
The NIS vitrification workflow comprises a large set of iterations in sample supports, sample dilutions, additives and freezing devices. Typically, it is not possible to exhaustively assess all of these variables in the this first stage, but extensive sample prep and screening in the Starter Package phase often results in a grid suitable for overnight data acquisition on one of our Glacios microscopes. This is followed by 2D classification, and sometimes 3D ab initio map calculations, to assess the suitability of this cryo-EM grid condition, as well as the sample itself, for ongoing efforts, such as preparing for a high-resolution Krios data collection, all with the objective of creating a 3D reconstruction of the protein.