Protein Crystallization & Crystallography Services: Methods and Technologies
Over many years, we have perfected our project workflow to best serve our clients at all stages of the process and ensure efficient project execution.
Services workflow
Established in 2006, SARomics Biostructures quickly established itself as a leading provider of protein crystallization and crystallography services, fragment screening, and integrated drug discovery offered to a wide range of pharmaceutical and biotech companies and academic institutions. With our extensive expertise in working with different protein classes, we excel in delivering high-quality results for various structural biology and drug discovery projects.
Outlined below is the typical workflow for our protein crystallization and crystallography services. Our project types include gene-to-structure X-ray crystallography, antibody and antibody-antigen crystallization, structure determination and analysis, and protein-ligand co-crystallization and structure determination. Leveraging our libraries of off-the-shelf structures, we accelerate our client’s projects by offering hundreds of protein targets with validated expression, purification, and crystallization protocols.
If you are ready to benefit from our exceptional crystallography services, please don’t hesitate to reach out to us via the provided contact form for further details.
Before protein crystallization: Sample preparation
Several essential requirements must be fullfiled to obtain crystallization-grade protein and to ensure the success of the crystallization experiment. These requirements are general, we provide them here in case a client decides to send own sample for the project.
First, the protein must be well-folded, stable and soluble within a given temperature, concentration and pH range. The solution must be monodisperse, i.e. does not contain any denatured or aggregated material. The protein solution may be characterized using the following biophysical methods:
- Dynamic Light Scattering (DLS)
- Circular dichroism (CD) spectroscopy
- Differential Scanning Fluorimetry (DSF, also called thermal shift assay)
- Protein NMR spectroscopy
DLS is used to assess the protein’s monodispersity. CD spectroscopy is used to verify secondary structure content, while DSF is used to assess the stability of the protein in different buffer solutions and at different temperatures. In NMR spectroscopy, the HSQC fingerprint spectrum shows whether the protein is well-folded, unfolded at a given temperature, or if some parts of the molecule are highly flexible. Our staff follows specific protocols during all the experiments to maximize their efficiency. The results of the measurements are subsequently used to decide whether the protein is suitable for crystallization or if some modifications in the cloning and purification protocols are required.
Screening for crystallization conditions
We can start protein crystallization if we are satisfied with the protein’s state in the solution. However, many parameters may potentially affect crystallization. For this reason, we need to run crystallization and optimization screens to identify the best conditions for obtaining well-diffracting crystals.
Our services workflow starts by running a large number of crystallization screens. Commercial screens, like those from Hampton Research or Molecular Dimensions, are initially used. In addition, the initial crystallization conditions usually need to be optimized to obtain well-diffracting single crystals for subsequent structure determination. The large number of buffer conditions used in the screening reflects the large number of parameters and combinations of the parameters that may affect the crystallization. The most common protocols rely on the variation of the different parameters, the most common being:
- 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), etc.
When we crystallize water-soluble proteins, we typically use sitting or hanging drops. To set up the initial crystallization screens and optimize conditions, we use high-throughput and high-precision liquid handling and imaging robotics. With robotics, we can screen 96 conditions using as little as 15 microL of protein sample, saving time and precious material. The crystallization plates are stored in plate hotels at a constant temperature to avoid fluctuations, and they can be monitored using a computerized optical system. If you prefer to send your prepared protein, our shipping and handling samples guide can provide helpful information on preparing and sending samples. If you need more information on sample preparation, please feel free to contact us directly through the contact form.
Crystals in the beam: X-ray data collection and structure determination
Once we have obtained high-quality crystals, the next step is to test them in an X-ray beam. We carry out X-ray data collection at the BioMax beamline at MAX IV Laboratory, one of the world’s best synchrotrons, conveniently located just a few kilometers from our labs in Lund. Alternatively, we can also send the crystals to other synchrotrons in Europe.
To determine the crystallographic structure, we require phase information, which is lost in X-ray crystallography experiments. If there is an existing experimental structure of the same protein, we utilize the molecular replacement method to obtain the phase information. This approach allows us to calculate an electron density map, which is then used to build the protein model. In certain instances, we can also utilize models generated by the AlphaFold project for initial phasing. Molecular replacement significantly accelerates the protein crystallography process.
In cases where a reliable model is not available, we use experimental methods to obtain phase information. We then refine the model to ensure that the finer details of the structure, such as the positions of protein and ligand atoms, are well defined. Our services platform is optimized to efficiently carry out all these processes in a timely manner.
Please visit our services pages for details of our protein X-ray crystallography services.