High-Throughput Protein Crystallization Services

SARomics Biostructures offers protein crystallization services, including protein-ligand complex crystallization and structure determination by X-ray crystallography. Our services are offered to the pharmaceutical and biotech industry as well as to academic groups from university from all continents. Crystallization and crystallography are the oldest methods for three-dimensional structure determination at atomic resolution. After the discovery of X-rays by Conrad Röntgen (Nobel Prize in Physics, 1901), the basic principles of X-ray diffraction were discovered by Max von Laue (Nobel Prize in Physics, 1914) and the Braggs father and son (Nobel Prize in Physics, 1915). Currently, X-ray crystallography and protein NMR are the only two methods, which provide atomic resolution tertiary protein structures for structural biology. The majority of entries in the Protein Data Bank (PDB) have been determined by X-ray crystallography and NMR spectroscopy.


1. Protein expression, purification and characterization

The company’s custom protein and protein-ligand complex crystallization and high-resolution X-ray crystallographic structure determination services (fee-for-service crystallography) include all steps required for successful crystal structure analysis. Within our gene-to-structure services, after cloning, expression and purification, and prior to crystallization, an accurate characterization of the state of the protein in solution is performed. Probably the most important requirement is a monodisperse solution of the protein at a given concentration, which means the solution should not contain aggregated or denatured material. Monodispersity may be assessed using dynamic light scattering (DLS). The protein should also be stable in solution within a certain range of pH and ionic strength (these values will be varied during crystallization screening). By other words, a proper characterization of the protein, including its solubility in solution, is critical for the success of the whole project.

Our
services platform is built up around the most advanced biophysical protein characterization methods and includes:

  • DLS
  • CD spectroscopy
  • Differential scanning fluorimetry (DSF, similar to thermal-shift assay). DSF was first used at the Structural Genomics Consortium for protein characterization prior to crystallization.
  • Protein NMR spectroscopy is used as required. It may answer questions, like does the construct result in a folded, well-behaved protein? The HSQC fingerprint spectrum of the protein readily shows whether the protein is well-folded, unfolded or ‘molten-globule’ like, and whether parts of the protein are flexible.


2. Protein crystallization and structure determination

Crystallization
fter protein characterization, conditions for obtaining well-diffracting single crystals need to be identified (if the protein has not been crystallized before). The number of experimental parameters affecting protein crystallization can be very large. Among these one can find:

  • The type of the buffer and the pH of the solution

  • The ionic strength of the buffer

  • The presence of various salts in the buffer

  • The presence of various ligands (co-factors, substrate analogues, inhibitors).

  • The type of precipitant used, which is probably the most central components in the setup.


Among the most popular precipitants used in protein crystallization are polyethylene glycols of various lengths, ammonium sulfate, and some alcohols. To find the right combination of parameters, one needs to screen a large number of different conditions. Several hundreds, and often thousands of different conditions are screened before the protein crystallization conditions can be identified. Commercial screens, like those from Hampton Research or Molecular Dimensions, are initially used. However, we have the facilities to design our own screens, if required (usually done during optimization of the conditions found using the initial screens).

In protein structure determination, for the most efficient identification of the crystallization conditions we use high throughput and high precision screening and imaging robotics. Of the available crystallization methods, the sitting-drop method is the most commonly used with robotics equipment. Using such a robot, 96 different conditions can be screened with as little as 15 microL of protein sample using a Mosquito liquid handling robot (mosquito crystal). The use of robotics helps in saving both time and precious material. When an initial condition is found, it normally needs to be optimized further to yield crystals more suitable for X-ray crystallography.

Structure determination
When suitable crystals are obtained it is time for the first test in an X-ray beam. If the X-ray diffraction from the crystals is of good quality (good resolution and nice X-ray diffraction pattern), diffraction data are collected at some X-ray source. For this we use MAX IV synchrotron radiation facility, which is located in Lund, or take the crystals to other synchrotrons. If an experimental structure for the same protein is already available, the method of molecular replacement is used, an electron density map is calculated and interpreted, most often to identify the position of the bound ligand, its occupancy, etc. Further refinement of the model will ensure that the fine details of the ligand and its binding site are well visible in the electron density map.
Additional information on protein crystallization can be found on our educational site.

Crystallization techniques

96-well protein crystallization plate


96-well protein crystallization plate from Hampton Research for the use of sitting-drop vapor diffusion technique.

For the optimization of the crystallization and structure determination pipeline, SARomics Biostructures has developed standard operation procedures (SOP), which allow an efficient use of time and resources, also helping in keeping the operation costs as low as possible for our clients. These include:

• Developments of Freedom evo scripts and excel scripts for optimization of crystallization experiments
• Standard operating procedures (SOPs) for dynamic light scattering evaluation of protein solutions
• Development of SOPs for X-ray data collection
• Standard protocols for fast track structure determination