Protein Crystallization & Protein Crystallography Services
Introduction to Protein Crystallization & Protein Crystallography
Crystallization and protein structure analysis using protein crystallography is the oldest structure determination method at atomic resolution. It won't be an exaggeration to state that this method created the foundation of structural biology and its use in various areas of science, including drug discovery. After the discovery of X-rays by Conrad Röntgen (Nobel Prize in Physics, 1901), the physical principles of X-ray diffraction were discovered by Max von Laue (Nobel Prize in Physics, 1914), while the foundation for X-ray crystallography was laid by William Henry Bragg and William Lawrence Bragg (father and son, Nobel Prize in Physics, 1915).
Apart from X-ray crystallography, currently, both NMR spectroscopy (Nobel Prize to Felix Bloch and Edward Purcell, 1952, Richard Ernst, 1991 & Kurt Wüthrich, 2002) and cryo-electron microscopy (Nobel Prize to Jacques Dubochet, Joachim Frank & Richard Henderson, 2017) play an essential role in protein structure analysis and generally, in structural biology. The 2002 Prize, together with Kurt Wüthrich, was awarded to John B. Fenn and Koichi Tanaka. They contributed to developing the mass spectrometry method, which is also widely used in studying macromolecules and macromolecular complexes.
Here we give an overview of the typical workflow in protein crystallization and structure determination. These experiments are part of our gene-to-structure X-ray crystallography services, including antibody crystallization and structure analysis. You may contact us directly using the contact form to discuss your project.
Apart from X-ray crystallography, currently, both NMR spectroscopy (Nobel Prize to Felix Bloch and Edward Purcell, 1952, Richard Ernst, 1991 & Kurt Wüthrich, 2002) and cryo-electron microscopy (Nobel Prize to Jacques Dubochet, Joachim Frank & Richard Henderson, 2017) play an essential role in protein structure analysis and generally, in structural biology. The 2002 Prize, together with Kurt Wüthrich, was awarded to John B. Fenn and Koichi Tanaka. They contributed to developing the mass spectrometry method, which is also widely used in studying macromolecules and macromolecular complexes.
Here we give an overview of the typical workflow in protein crystallization and structure determination. These experiments are part of our gene-to-structure X-ray crystallography services, including antibody crystallization and structure analysis. You may contact us directly using the contact form to discuss your project.
Protein expression & characterization
The first step in any protein crystallography project is to get a sufficient amount of high-purity protein, also called crystallization-grade protein. The company’s recombinant protein expression and purification platform (see our catalog of high purity crystallization grade proteins) is adapted for obtaining high-purity and stable crystallization-grade proteins.
After cloning, expression, and purification, and before crystallization, an accurate biophysical characterization of the state of the recombinant protein in solution is performed. The purified protein in the solution must be well-folded, stable, and monodisperse at a given concentration and pH range. This means that the solution should not contain any denatured and aggregated material. This may be assessed, for example, by dynamic light scattering (DLS), which will quickly reveal the presence of any aggregated material.
If required, we may also use the following methods for the biophysical characterization of the protein:
Protein NMR spectroscopy may answer questions like whether the construct results in a folded, well-behaved protein. The HSQC fingerprint spectrum of the protein readily shows whether the protein is well-folded, unfolded, or in a ‘molten-globule’ state and if some parts of the protein are flexible.
We have developed protocols for characterization and crystallization to accelerate the process depending on the type of protein.
After cloning, expression, and purification, and before crystallization, an accurate biophysical characterization of the state of the recombinant protein in solution is performed. The purified protein in the solution must be well-folded, stable, and monodisperse at a given concentration and pH range. This means that the solution should not contain any denatured and aggregated material. This may be assessed, for example, by dynamic light scattering (DLS), which will quickly reveal the presence of any aggregated material.
If required, we may also use the following methods for the biophysical characterization of the protein:
- DLS (Dynamic Light Scattering)
- CD spectroscopy
- DSF, Differential Scanning Fluorimetry (also called thermal shift assay)
- Protein NMR spectroscopy
Protein NMR spectroscopy may answer questions like whether the construct results in a folded, well-behaved protein. The HSQC fingerprint spectrum of the protein readily shows whether the protein is well-folded, unfolded, or in a ‘molten-globule’ state and if some parts of the protein are flexible.
We have developed protocols for characterization and crystallization to accelerate the process depending on the type of protein.
A plate hotel at our crystallization facility is used for storing and monitoring crystallization plates, which are kept at a constant temperature. 96-well crystallization plate from Hampton Research.
Protein crystallization overview
Together with protein characterization, our services include developing a crystallization protocol for obtaining well-diffracting single crystals for protein crystallographic structure determination. The number of experimental parameters affecting protein crystallization can be huge. The most common protocols rely on the following parameters:
* Type of buffer and its pH
* Ionic strength
* The presence of various salts in the solution
* The presence of ligands (co-factors, substrate analogs, inhibitors)
* The type of precipitant used (polyethylene glycol (PEG) and ammonium sulfate are the most common)
Many conditions must be screened before a successful crystallization protocol can be formed. Hundreds and often thousands of conditions are tested until "good" crystallization conditions are identified. Commercial screens, like those from Hampton Research or Molecular Dimensions, are initially used. We also use our liquid-handling robots to design screens, usually for optimizing crystallization conditions identified in the initial screens.
Several different crystallization methods exist. The most common for water-soluble proteins is the method of sitting or hanging drops. For membrane protein crystallization usually, other methods are used. High throughput and high precision liquid handling and imaging robotics are also required for the best efficiency of the crystallization efforts. For example, 96 different conditions can be screened using robotics with as little as 15 microL of protein sample. This helps in saving both time and precious material. The crystallization plates are stored in plate hotels at a constant temperature to avoid temperature fluctuations in laboratory conditions.
Our guide for shipping samples may be helpful for information on sending samples. Of course, you may also contact us directly.
* Type of buffer and its pH
* Ionic strength
* The presence of various salts in the solution
* The presence of ligands (co-factors, substrate analogs, inhibitors)
* The type of precipitant used (polyethylene glycol (PEG) and ammonium sulfate are the most common)
Many conditions must be screened before a successful crystallization protocol can be formed. Hundreds and often thousands of conditions are tested until "good" crystallization conditions are identified. Commercial screens, like those from Hampton Research or Molecular Dimensions, are initially used. We also use our liquid-handling robots to design screens, usually for optimizing crystallization conditions identified in the initial screens.
Several different crystallization methods exist. The most common for water-soluble proteins is the method of sitting or hanging drops. For membrane protein crystallization usually, other methods are used. High throughput and high precision liquid handling and imaging robotics are also required for the best efficiency of the crystallization efforts. For example, 96 different conditions can be screened using robotics with as little as 15 microL of protein sample. This helps in saving both time and precious material. The crystallization plates are stored in plate hotels at a constant temperature to avoid temperature fluctuations in laboratory conditions.
Our guide for shipping samples may be helpful for information on sending samples. Of course, you may also contact us directly.
Good-quality protein crystals are required to record X-ray diffraction from a crystal for protein crystallography. The intensities of the spots measured from the collected diffraction images are used to calculate the electron density map into which a protein structure model is built.
Structure determination by protein crystallography
When suitable crystals are obtained, it is time for the first test in an X-ray beam. Then, if X-ray diffraction from the crystals is of good quality, data are collected. We use the MAX IV synchrotron radiation facility located a few kilometers from our Lund labs for X-ray data collection. Alternatively, we send the crystals to other European synchrotrons.
Suppose an experimental structure for the same protein is already available. In that case, the molecular replacement method can be used for solving the structure to obtain the so-called phases for subsequent calculation of an electron density map into which the protein model will be built. Sometimes is also possible to use models generated by the AlphaFold project for the initial phasing, which will substantially accelerate the project. Further refinement of the model will ensure that the fine details of the structure (positions of protein and ligand atoms) are well resolved in the electron density map.
Our educational site provides additional practical information on protein crystallography, including crystallization and structure determination.
Suppose an experimental structure for the same protein is already available. In that case, the molecular replacement method can be used for solving the structure to obtain the so-called phases for subsequent calculation of an electron density map into which the protein model will be built. Sometimes is also possible to use models generated by the AlphaFold project for the initial phasing, which will substantially accelerate the project. Further refinement of the model will ensure that the fine details of the structure (positions of protein and ligand atoms) are well resolved in the electron density map.
Our educational site provides additional practical information on protein crystallography, including crystallization and structure determination.
An example showing the side chain of tryptophan built into a well-resolved (good resolution) electron density map.