Gene to Structure - Protein Structure Analysis

HarkerBIO gene-to-structure pipeline is available to you for immediate deployment: all you need to do is provide us with the amino acid sequence of the gene(s) of interest, and we do the rest! 

 

Construct design


We use proprietary computational methods and decades of human experience to design constructs that enhance the probability of crystallization. For each customer sequence we typically design 8-24 constructs in order to enhance the target's crystallization potential.

DNA/RNA properties


We extensively optimize nucleotide sequence of the target in order to elicit high-level expression and folding of the protein.

Gene synthesis


We synthesize the genes for the native protein and the variants in parallel. Our process is optimized for minimal reagent cost and rapid turn-around.

Parallel cloning


We clone the target and its variants into an appropriate expression/shuttle vectors using our multiparallel molecular biology processes.

Parallel expression


The choice of expression host is target-based, however we will typically attempt to screen for expression in several bacterial species, a yeast species, and in insect cells -- depending on the level of complexity and difficulty of the target.

Parallel purification


All constructs that pass expression testing are purified and characterized in parallel. Typical quality criteria include DLS, size exclusion, thermal melt, and isoelectric focusing.

Crystallization


Purified protein samples enter the crystallization pipeline:

  • Primary Screening - High-Throughput, 1536 conditions, microbatch under oil
  • Secondary Screening - Medium-Throughput, all major screens plus in-house screens, sitting drops in 96-well plates
  • Optimization - 96-well sitting drops and 24-well hanging drops, counter-diffusion. Gradient screens, additives, drop ratio, temperature, and other parameters
 

Diffraction Screening


Frozen crystals are sent to one of several synchrotron facilities - commonly APS, ALS, or CLS where we screen them to determine crystal quality. We also screen larger crystals in-house, to cut down on the turn-around between crystallization and follow-up

Data collection


Diffraction data are collected for crystals that pass the screening.

Phasing


Depending on the nature of the target we can obtain phases by Molecular Replacement, Multiple/Single Isomorphous Replacement, or Multiple/Single Anomalous Diffraction. We have a deep and broad expertise in soaking heavy atoms into crystals (including iodination, phasing by soluble halides, etc.) and we can rapidly produce Selenium-labeled protein for (S/M)AD phasing.

Refinement


We refine crystal structures using state of the art software, until they meet globally acceptable quality criteria.

Analysis/Publication


Depending on the nature of the project, we provide detailed and in-depth analysis of crystal structures, including analysis of overall fold, olgomeric structure, ligand interactions, etc. We can prepare publication-ready manuscript sections and print-ready images.


Quick Contact

Monthly Crystallization Screening