If your sample is not challenging you, then your sample might not be challenging (enough)!

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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. 

 

 

Installations

ProteoPlex 001

MPIBPC,

Goettingen,

Germany

ProteoPlex 002

NovAliX SAS,

Illkirch, France

ProteoPlex 003

University of Minnesota

The Hormel Institute,

Austin, MN, USA

ProteoPlex 004 - MaxStAF

Nanyang Technological University

Singapore

 

"The ProteoPlex system allowed me to screen for alternative pH conditions that stabilized my protein complex in preparation for Cryo-EM. The ease of the software in sorting and analyzing results is a powerful tool in finding or rejecting conditions, way before reaching the grid preparation stage."

ProteoPlex 005 - MaxStAF

VBCF - Vienna Biocenter Core Facilities

Research Institute of Molecular Pathology (IMP)

Max F. Perutz Laboratories

Gregor Mendel Institute of Molecular Plant Biology

Institute of Molecular Biotechnology

Vienna, Austria

ProteoPlex 006 - MaxStAF

Stock-listed pharmaceutical company

Cambridge, MA, USA

 

"We acquired Proteoplex (MaxStAF) software to drive our biophysical and structural biology studies. The service provided by the Proteoplex team is outstanding. They worked closely with us to ensure the software is optimized to our needs [..]. They adapted the user interface to analyze the data obtained from 384-well plates, enabled processing and analysis of multiple data sets together, significantly enhancing the data analysis throughput.

 

The product is user-friendly, fast to execute, facilitating faster assay development and protein biochemistry studies. The Proteoplex technology is capable of accurately fitting the experimental data. The results obtained have helped us tremendously in identifying novel conditions and optimize them for successful execution of multiple projects." 

 

 

Core parts of ProteoPlex MacroDSF

  • Liquid handling robot
  • Realtime PCR
  • Well plate sealing machine (optional)
  • Patented ProteoPlex MacroDSF software for multi-state unfolding analysis

 

Functionality

  • Dilution screen: screen for optimal dilution to achieve low protein consumption and best results
  • Buffer screen: screen 88 buffer conditions simultaneously
  • Additive screen: screen 88 additives simultaneously
  • Obtain ranking of best results

 

Advantages

  • Speed: screen 88 buffer conditions in less than 2 hours
  • Reproducibility: rule out most significant sources of errors (pipetting volume, sample contamination, air bubbles, suboptimal dilution, etc.)
  • Multiple unfolding analysis: only technology that can correctly interpret multi-state unfolding (and of course also two-state unfolding) --> particularly in early purification stages no overlap between ProteoPlex and DSF results!

 

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)
Download: http://www.nature.com/nmeth/journal/v12/n9/full/nmeth.3493.html

 

User manual ProteoPlex analytical instrument

ProteoPlex MacroDSF User Manual v06
20180515 ProteoPlex MacroDSF Instruction[...]
PDF-Dokument [6.8 MB]

MacroDSF Flyer

Flyer MacroDSF
20170113 ProteoPlex flyer v01.pdf
PDF-Dokument [621.2 KB]

 

 

Important remarks, caveats & limitations

  • ProteoPlex MacroDSF is not applicable to membrane proteins​ as the micelles distort the signal
  • Buffer improvement does not always mean to get better images. There are other parameters that can destabilize or even destroy a protein complex. Thus, buffer optimization is not a panacea but one of many levers to be checked; it can be a limiting condition - or not.​
  • Yes, there are cases where Thermofluor / DSF yield similar results as ProteoPlex MacroDSF. But, as a matter of fact, Tm is a physically wrong criterion for complexes, which might in some cases coincidentally appear to be "right". In general, the results from MacroDSF and Thermofluor / DSF converge with increasing protein optimization, i.e. results of both methods agree for complexes that require no further optimization and are already present in a stabilized form. In the early stages of a project, there should hardly be any overlap between the two methods and that is where ProteoPlex can be an extraordinary asset, as shown in Fig.1.

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

"NovAliX enters into cooperation agreement with ProteoPlex"

Press release EMBL,

4th Aug 2016:

"Every atom counts"

EMBL Science,

8th Aug 2016:

"Blocking the cell's waste disposal unit"