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Without the need for expensive lab infrastructure costing $5M+ here is a quick way to get up and running with experimental protein structure using established CRO partnerships:
The table below outlines the process, some of my favorite CRO friends, and a pathway to quickly get a first structure!
- Clone the gene encoding the target protein into an expression vector
- Express the protein in a suitable host system (e.g., E. coli, yeast, insect, or mammalian cells) to ensure proper folding and post-translational modifications
- Optimize expression conditions to maximize yield and solubility
- Purify the expressed protein using chromatography techniques like affinity, ion-exchange, and size-exclusion chromatography
- Assess purity (>95%) using SDS-PAGE and Dynamic Light Scattering (DLS) to confirm monodispersity
- Conduct crystallization trials using vapor diffusion (e.g., hanging drop or sitting drop) with a range of precipitants, salts, and pH conditions
- Optimize crystal growth conditions to produce high-quality crystals suitable for diffraction
- Collect diffraction data from well-formed crystals at a synchrotron facility or lab-based X-ray source
- Use cryoprotectants to prevent radiation damage and freeze crystals in liquid nitrogen before data collection
- Process diffraction data using software like CCP4 or XDS to generate electron density maps
- Solve the structure via molecular replacement or experimental phasing (e.g., selenomethionine labeling)
- Refine the atomic model iteratively to achieve a high-resolution structure (≤2 Å)
| Step | Timeline | Estimated Cost | CRO resources |
|---|---|---|---|
| Gene Cloning & Expression | 2-4 weeks | $5k-$10k | Saromics Helix Biostructures WuXi Genscript Proteos |
| Protein Purification | 1-2 weeks | $3k-$8k | Proteos |
| Crystallization | 2-6 weeks | $5k-$15k | Acelero Bio Structures Saromics |
| Data Collection | 1-2 weeks | $5k-$20k | SSRL APS NSLS DIAMOND MAXIV |
| Structure Determination | 1-2 weeks | $5k-$50k | - Me! 🙂 |
- Challenges: Protein solubility, crystallization reproducibility, and data quality are common bottlenecks
- Cost Variability: Costs depend on protein complexity (e.g., size or modifications) and whether a de novo structure is required
- CRO Expertise: Selecting CROs with integrated services (e.g. SARomics) can streamline the process by combining protein production with crystallography