Overview
Target identification & preparation
Protein constructs for crystallisation have different requirements than for biochemical assays. It is pivotal to reduce protein flexibility and heterogeneity in the constructs to enable crystal packing. The C2f facility can help in designing proper constructs for crystallisation trials. We use homology modelling and various in silico analysis methods to predict the most-promising constructs for crystallography. Even after initial trials, further, optimisation might necessary. We can help in with surface entropy reduction and alternative construct generation.
Protein expression & purification
Protein production and Purification
Prokaryotic expression
Together with the research group of Prof. Baumann proteins can be expressed in various E. coli strains, ideally using the pET family. Multi-component complexes can be co-expressed with the combination of maximal 3 pDuet vectors allowing the expression of up to 6 proteins in E. coli. Typically, expression of eukaryotic proteins in E. coli needs some optimisation. Over 40 different E. coli strains are available and can be combined with various temperature regimes and growth medium alteration, to finally obtain soluble proteins.
Eukaryotic expression
For proteins unsuitable for E. coli expression, two eukaryotic expression systems are established. Especially for extracellular proteins, expression in HEK293 cells using the pCEP-vector systems is the common standard for high-level expression. Cost-effective production is realised in roller bottles in the dedicated cell culture available through our host research group of Prof. Baumann.
Intracellular proteins and proteases are often more effectively expressed in the Baculovirus expression system using sf21- cells. In comparison to the HEK system typically higher yields can be obtained with less material.
Purification
For crystallisation attempts, proteins should be of the highest purity. A two-step purification with an initial affinity chromatography followed by a size-exclusion chromatography is the minimum. The research groups of Prof. Baumann own various chromatographic system for assisting in purification and the C2f can helps in planning, establishing and refining a proper purification protocol.
Crystallisation
Structure solution
We can help to solve your crystal structure!
The biggest problem (after getting crystals) in crystallography is the phase problem. We can typically solve this problem either by molecular replacement (a similar structure is already known) or by experimental phasing. Latter normally needs additional experiments and more crystals.
In typical cases, our facility staff will discuss potential ways of solving your crystal structure even before starting crystallisation trials. We are happy to help you in getting the right data for solving your structure, and – if necessary – will perform the whole structure determination and refinement for you.
Molecular replacement:
In molecular replacement, identification of a proper "search model" is the key to success. As a rule of thumb, a PDB with at least 30% sequence identity should be existent.
Experimental phasing:
In experimental phasing, additional diffraction experiments are used to calculate the phases based on the observer diffraction intensities. Typically a heavy atom is needed in the structure.
Software support
We routinely use diverse software packages to solve, build and refine crystal structures and we are happy to support our Cologne community.
We typically use XDS or DIALS for data reduction, phase with Phaser or HKL2MAP (ShelX pipeline), built with Coot and refine with Phenix. Analysis and graphical representation are performed with PyMol or Chimera. Besides the typical servers we use Modeller for homology modelling and Yasara for small molecular dynamics analysis. If you need any help in installing, running and using these software, feel free to ask.
For typical processes we have easy to use protocols, which might help the adept user to remind specific steps.
Structure prediction
AlphaFold
The advent of AlphaFold has revolutionized structural prediction, making it increasingly accessible and prevalent. The C2f has embraced this technology, integrating AlphaFold into our routine processes to enhance the resolution of crystal structures and to help forming hypotheses about functions and interactions. We operate our own specialised hardware setup, enabling us to conduct more complex (and longer) predictions, than the standard “ColabFold” run. Additionally, we have a more direct control about the process itself.
We also offer guidance in interpreting AlphaFold's outputs, assisting researchers in distinguishing between viable and less promising predictions. Recently, we have expanded our use of AlphaFold to explore de novo protein interactions. Although this application is still emerging, it has shown potential in generating valuable hypotheses.
By leveraging our expertise in structural predictions and computer engineering, we provide comprehensive support for researchers looking to harness the power of AlphaFold in their scientific inquiries.
If you are still unsure what Alphafol does and how it might help you, you'll find multiple videos at YouTube and one at Media.cc of our facility head, explaining the algorithm for a broader audience. We have embedded it below for your convenience