X-ray Crystallography & High-Throughput Protein Crystallization Services

SARomics Biostructures is a CRO company that offers custom protein crystallization and X-ray crystallography services, including access to its FastLane off-the-shelf protein libraries, protein-ligand complex crystallization, and gene-to-protein structure determination.

FastLane premium library for accelerated protein crystallography

FastLane™ Premium off-the-shelf crystallization-grade proteins

FastLane™ Standard off-the-shelf proteins

Why Choose SARomics Biostructures CRO Services?

Our crystallization and X-ray crystallography services offer access to off-the-shelf FastLane protein libraries, featuring over 600 verified drug targets ready to be crystallized with your small molecule ligands.
If the protein of your interest is not in our library, you can benefit from our custom gene-to-protein structure services.
Your project will be handled by a leading CRO team with extensive expertise in drug discovery, protein crystallization, and crystallographic structure determination services across many protein families.
Our publications demonstrate our company’s proven expertise in many areas of structural biology. Download a list of publications featuring case studies with antibody and antibody-antigen crystallographic structure determination.

See also our blog post discussing a publication from the AstraZeneca team on the use of synchrotron X-ray crystallography in drug design.

State-of-the-Art Protein Crystallization & X-ray Crystallography Services Platform

SARomics Biostructures’ high-throughput technology platform is tailored to provide custom CRO services in protein crystallization and crystallographic structure determination, supporting all stages of our clients’ integrated drug discovery projects. Our services include:

Custom protein crystallization, X-ray crystallography analysis, and drugability assessment services.
Small-molecule ligand complex crystallization & binding site mapping. Over 600 FastLane off-the-shelf drug target proteins.
Crystallographic fragment screening services, atomic-level analysis of initial hits’ binding with the target protein.
Structure-based lead optimization.
Crystallization and X-ray crystallographic structure determination services for challenging targets through our custom gene-to-protein structure services.
Crystallization and X-ray structure determination of antibodies and antibody-antigen complexes. High resolution epitope mapping. We also offer a library of extracellular proteins that can be rapidly co-crystallized with your monoclonal antibodies.
Biosimilar higher-order structure (HOS) comparability analysis using X-ray crystallography and NMR spectroscopy.

Our services platform is enhanced by high-precision, high-throughput liquid-handling systems and imaging robotics, which enable us to establish initial crystallization conditions and rapidly optimize them. A typical project workflow for a protein structure determination service project is presented below.

We regularly use the state-of-the-art high-intensity BioMax protein crystallography beamline for X-ray data collection. Our location close to the MAX IV synchrotron laboratory provides flexible access to beamlines, ensuring fast and safe sample delivery. Additionally, it allows us to offer our clients serial protein crystallography services at the MicroMax beamline.

Björn Walse & Raymond Kimbung discussing a protein crystallization experiment

Custom Gene-to-Protein-Structure Services

Our comprehensive protein crystallization and X-ray structure determination services platform is adapted to provide custom gene-to-protein-structure services, which include:

Cloning, expression, purification, and biophysical characterization of the protein.
Polydispersity assessment by dynamic light scattering (DLS).
Folding assessment by circular dichroism (CD) spectroscopy.
Stability assessment by differential scanning fluorimetry (DSF).
If required, additional assessments may be conducted using protein NMR spectroscopy services.
Protein crystallization screens, which include thousands of conditions, are used to identify initial crystallization conditions.
Synchrotron X-ray data collection, followed by crystal structure determination of the protein using molecular replacement or SeMet-labeled protein (see method discussion below).

Protein Crystallization and Protein Crystallography Services Workflow

To provide an overview of our leading protein crystallization and crystal structure determination services, we outline below the typical process of a protein X-ray crystallography project, which includes sample preparation and characterization, high-throughput protein crystallization, and subsequent crystallographic work, including X-ray data collection, data processing, electron density map calculation, model building, and refinement. A concise summary of the process is available in our guide on sample preparation, shipping, and handling.

Obtaining good-quality protein for crystallization and structure determination is the first step in any X-ray crystallography project. Our crystallization services start with thorough sample preparation and characterization. After expressing and purifying the protein, or upon receiving a client’s sample, we employ various biophysical techniques to analyze it. This assessment includes:

Evaluating the protein’s state in solution, e.g., the presence of aggregates, and monodispersity.
Assessing stability in various buffers and at different temperatures.

For the above, we use:

Differential scanning fluorimetry (DSF) to determine optimal conditions for protein stability before crystallization.
Size-exclusion chromatography (SEC) can be used to remove aggregates, which are known to interfere with protein crystallization.
In addition, dynamic light scattering (DLS) and mass spectrometry (MS) can be used to confirm the sample’s monodispersity. Protein sample monodispersity has also been shown to be a requirement for successful protein crystallization.
To ensure the protein is well-folded, we may also use NMR spectroscopy to verify its folding status via HSQC fingerprint analysis. This helps us detect partially unfolded regions at a specific temperature or identify regions of the molecule that exhibit high flexibility, which may prevent the formation of high-quality crystals.

Accelerating the Process

Our staff adheres to specific protocols during all experiments to maximize the speed and efficiency of the crystallization and subsequent X-ray crystallographic structure determination. Based on these studies, we can determine whether the sample is suitable for crystallization and X-ray crystallography, or whether modifications to the cloning and purification protocols are necessary. As an example of the required sample quality, you may download a product sheet for one of our crystallization-grade Protein Shop proteins. More examples can be found on the same page.

Our protein crystallization and crystallography services workflow always starts with screening and subsequent optimization of conditions after the initial crystallization conditions are identified. The screening and optimization of the protein crystallization conditions can take as little as 3-6 weeks, though some samples may require more time. Even if crystallization conditions for the protein are known from previous structural studies, it is often necessary to set up new screening experiments to obtain high-quality crystals suitable for protein crystallographic analysis or for crystallographic fragment screening. Hundreds of screens, such as those available at Hampton Research, are used initially. Typically, approximately 5 mg of protein at a concentration of around 10 mg/ml is required at this stage. Lower protein concentrations can be used if solubility issues arise.

The large number of buffer conditions used in the screening reflects the numerous factors and their various combinations that may affect protein crystallization. The most common protocols rely on the variation of factors, such as:

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.

Liquid-Handling Robotics

Our protein crystallization and crystallography services routinely use high-throughput, high-precision liquid-handling and imaging robotics to set up initial screens and optimize newly identified conditions. With robotics, we can screen 96 conditions using as little as 15 microL of protein sample, saving time and precious material. Temperature is a vital parameter for crystallization. In the lab, we control it by storing the crystallization plates at different temperatures in plate hotels equipped with an imaging system, such as the Formulatrix Rock Imager.
On our educational site, you may find more information on the basics and techniques of protein crystallization.

Once we have obtained suitable and stable crystals, we test them in an X-ray beam and collect X-ray diffraction data for our custom protein X-ray crystallography services. Crystal testing and X-ray data collection are conducted at the crystallography facilities at the MAX IV synchrotron in Lund, one of the world’s premier synchrotrons, conveniently located just a few kilometers from our laboratories. Sometimes we send the crystals to other synchrotrons in Europe for X-ray data collection. Crystals are typically delivered to the beamline frozen in liquid nitrogen. To minimize radiation damage from the high-intensity synchrotron X-ray beam, data collection is performed at cryogenic temperatures. However, there are instances in which room-temperature X-ray crystallography can be advantageous. For more information on the advantages of the method and the technical solutions implemented at synchrotrons, please refer to our blog article, “Protein-Ligand Interactions: Room Temperature or Cryo-Crystallography? “

Phase Determination

Protein crystallography and protein structure determination require phase information to calculate the electron density map into which the protein model is built. However, the phases cannot be obtained directly from the X-ray diffraction image, which only registers intensities of the scattered X-rays. A common way of obtaining phases is by using the molecular replacement method. In this case, an experimental structure of a close homolog (generally with a sequence identity above 40-50%) from the Protein Data Bank (PDB) or an AlphaFold model can be used for initial phasing, electron density calculation, and model building. If molecular replacement fails, “de novo” structure determination has to be applied. Typically, a selenomethionine (SeMet)-labeled protein sample, which we can produce upon the client’s request, enables structure determination using synchrotron radiation and anomalous X-ray scattering.
Please visit our educational site for more information on the technique of protein X-ray crystallography.

The Benefits of Synchrotrons in X-ray Crystallography

The success of protein structure determination by single-crystal X-ray diffraction relies heavily on the technology used to record X-ray diffraction images, including the ability to generate high-intensity, focused X-ray beams, to record diffraction patterns, and to process the data. Additionally, crystal handling, especially in a high-intensity X-ray beam, is crucial. The low-intensity X-ray sources available during the early stages of protein crystallography made collecting quality diffraction data very slow, and for small, weakly diffracting crystals, entirely impossible.

It would not be an exaggeration to say that the introduction of synchrotrons has sparked a technological revolution in X-ray crystallography and, consequently, in structural biology. It is estimated that, compared to laboratory sources, at the early synchrotrons, the X-ray flux increased by about 200-fold, and by 80,000-fold already at the second-generation dedicated synchrotrons (Dauter et al. 2010). Currently, the brightness of the most modern fourth-generation synchrotrons, among which is MAX IV Laboratory in Lund, has exceeded the brightness of second-generation synchrotrons by many orders of magnitude.

Data Collection Techniques at Synchrotrons

Alongside synchrotrons, the development of new data collection techniques was vital to advancing protein crystallography. One of them was the emergence of electronic X-ray detectors, the first of which was the imaging plate, developed by Jules Hendrix and Arno Lentfer and first tested at the DESY synchrotron in Germany in 1989. Until then, X-ray data were collected on film, which had to be developed and scanned with an optical film scanner to obtain the diffracted X-ray intensities. The process involved loading film cassettes with packs of three films and, during experiments, developing, fixing, and washing hundreds of films in nearly complete darkness, often spanning over a day and night.

Compared to these procedures, the collection of a complete X-ray data set within a couple of minutes at modern synchrotrons feels like science fiction! Recent technical advancements also enabled new types of experiments, including crystallographic fragment screening, serial crystallography, and room-temperature crystallography.

Highlights of Synchrotron X-ray Crystallography

Among the highlights of synchrotron crystallography was the determination of the ribosome’s three-dimensional structure. Three researchers, Ada Yonath, Venki Ramakrishnan, and Thomas Steitz, were awarded the Nobel Prize in Chemistry in 2009 for their work on the structure of the ribosome. The Nobel Foundation report on the scientific background of the prize stresses the role of new technologies, such as the “introduction of CCD area-detectors for precise and automated analysis of x-ray diffraction patterns and tunable synchrotron radiation sources for optimal use of anomalous scattering for phase determination” in the project’s success.

Thanks to advances in technology, synchrotron radiation is now routinely utilized by all companies that do X-ray crystallography. A recent study conducted by the AstraZeneca team on the impact of synchrotron radiation on drug discovery emphasized the shift from using a “combined laboratory X-ray source-synchrotron” to adopting a “synchrotron-only” approach for the company’s drug discovery projects.

Some of the main benefits of modern synchrotron X-ray crystallography include:

No more time-consuming crystallization conditions optimizations, the high beam brilliance allows X-ray data to be collected from much smaller crystals than before.
A brighter, more focused beam yields higher-resolution data than laboratory X-ray sources.
Tunable wavelengths can be used for phasing by multiple-wavelength anomalous scattering (MAD).
Time-resolved studies and serial crystallography are only possible at synchrotrons.
The revival of room-temperature crystallography has essential applications in structure-based design.

See also the video presentation of the BioMax beamline by Dr. Anna Gonzales.

Case Study: Do We Need Protein X-ray Crystallography in AI Drug Discovery?

A recent excellent paper, “Prospective de novo drug design with deep interactome learning,” by the group of Professor Gisbert Schneider at ETH Zurich, with a contribution from SARomics Biostructures, demonstrates that X-ray crystallography remains necessary for ligand-binding verification.

Prospective de novo drug design with deep interactome learning.
Atz K, Cotos L, Isert C, Håkansson M, Focht D, Hilleke M, Nippa DF, Iff M, Ledergerber J, Schiebroek CCG, Romeo V, Hiss JA, Merk D, Schneider P, Kuhn B, Grether U, Schneider G (2024). Nat Commun. 15, 3408.

In this work, new ligands targeting the binding site of the human peroxisome proliferator-activated receptor subtype gamma (PPARgamma, a protein from our FastLane™ Premium library ) were generated. The ligand’s binding mode was subsequently confirmed by the crystal structure of the protein-ligand complex provided by the SARomics team. The structure shows that the ligand effectively interacts with the receptor in a canonical binding mode.