If your sample is not challenging you, then your sample might not be challenging (enough)!
ProteoPlex MacroDSF for protein optimization:
Sample hundreds of conditions in the time required to screen one sample by EM
Patented algorithm automatically interprets melting curves of multi-subunit protein complexes
With recent technological advances in cryo-EM and X-ray crystallography, the specimen itself has often become the limiting factor. In EM - for example - high-end, automated transmisson electron microscopes, powerful software and direct electron detectors greatly help determine structures of large and/or dynamic macromolecules in their native state.
Astoundingly, we do not yet see the same technological advancement in the most crucial step ahead - sample preparation. Great microscopes and detectors cannot make up for a bad specimen.
The optimal treatment of macromolecular complexes during purification and EM grid preparation plays an extremely important role in the maximum achievable resolution for cryo-EM structures, especially for fragile and/or transient macromolecular complexes. The stability of multisubunit specimens is strongly influenced by the buffering conditions in which they are purified.
Although it is possible to resolve cryo-EM sample heterogeneity computationally, processing an image dataset that contains broken particles and/or has extremely high heterogeneity is very time consuming and allows for high-resolution structure determination of only the most rigid parts, while losing structural information on the more mobile parts. The final resolution is also likely to be suboptimal. Therefore, the main goal for sample preparation is to preserve the intactness and to reduce the heterogeneity of the sample while not reducing the image contrast later.
ProteoPlex MacroDSF, an advanced adaptation of the DSF technique, systematically explores stabilizing buffer conditions by using a sparse-matrix approach. An important advantage of ProteoPlex in comparison to DSF is that it allows not only for screening of small-molecule stabilizers but also for screening of proteinaceous cofactors and binders such as nanobodies. ProteoPlex MacroDSF provides unique guidance to biochemists; it can sample hundreds of conditions in the time required to screen one sample by EM.
ProteoPlex MacroDSF efficiently guides you to more stable proteins and thus improves specimen quality - even and especially for complex proteins. Its findings can dramatically improve your purification results and allow high quality structure determination - so, e.g., your protein might regularly crystallize after MacroDSF improvement. Due to its unique biophysical algorithm, ProteoPlex MacroDSF is the only technology that can cope with protein complexes that show multi-state unfolding behavior.
Core parts of ProteoPlex MacroDSF
Suggested further reading
Chari, A.; Haselbach, D.; Kirves, J. M.; Ohmer, J.;
Paknia, E.; Fischer, N.; Ganichkin, O.; Moller, V.; Frye, J. J.; Petzold, G. et al.: ProteoPlex: Stability optimization of macromolecular complexes by sparse-matrix screening of chemical space.
Nature Methods 12 (9), S. 859-865 (2015)
User manual ProteoPlex analytical instrument
Important remarks, caveats & limitations
Fig. 1: Comparison to conventional data interpretation. Comparison of melting-curve evaluation by ProteoPlex and conventional DSF. Results obtained by both methods perfectly agree (green) only for complexes that require no further optimization and are already present in a stabilized form. In early stages of a project, there is no overlap between DSF and ProteoPlex results.
Still want to read more?
Joint press release with NovAliX, 27th March 2018
Press release MPG,
7th Nov 2017:
Press release EMBL,
4th Aug 2016:
8th Aug 2016: