X-Ray Crystallography: Limitations, Drawbacks and a Better Alternative

X-ray crystallography is a technique used to determine substances' atomic and molecular structure by analyzing the diffraction patterns produced by X-rays that pass through a crystalline sample. 

The technique has long been a cornerstone in understanding substances' atomic and molecular structures, playing a crucial role in structural biology and drug development. 

But despite its powerful applications, it presents several challenges to researchers and drug developers, including:

• The requirement for available crystalline samples
• Accurate model building
• Minimizing background noise
• Overcoming the costs and accessibility limitations associated with the technique

The implications of these challenges can be significant, potentially hindering progress in vital scientific endeavors.

Here, we briefly explore X-ray crystallography's current limitations and drawbacks — and present an innovative solution in Immuto Scientific’s platform. We’re addressing these challenges head-on, paving the way for more efficient and accessible structural analysis.

The limitations and drawbacks of x-ray crystallography

While X-ray crystallography has revolutionized the field of structural biology and enabled scientists to understand the properties and behavior of proteins better, drug developers need to consider several limitations and drawbacks.

1. Crystalline samples

The requirement for a crystalline sample is one of the most significant restrictions of x-ray crystallography. Especially with biomolecules, crystallizing proteins in the lab can be time-consuming and challenging because most proteins are not naturally crystalline.

Complex, big, or membrane-embedded proteins frequently cause them to fail. And even if crystals are obtained, their quality might not be high enough to generate a structure with high resolution.

Here at Immuto, we’ve developed platforms that use proprietary protein engineering approaches to generate highly stable proteins, which can be more easily crystallized and yield better quality structures than traditional methods.

2. Model building

Model construction is another x-ray crystallography limitation. There are ambiguities in the model since it’s often challenging to visualize the whole molecule inside the crystal due to the nature of crystal packing. Also, the diffraction data's limited resolution can result in unresolved areas or artifacts in the structure.

One solution to these limitations is to combine X-ray crystallography with other techniques, such as electron microscopy or nuclear magnetic resonance spectroscopy, to obtain a complete picture of the molecule.

3. Background noise

The diffraction patterns produced by x-rays passing through a crystal contain signal and noise, and separating the two can be challenging. This noise can lead to inaccuracies in determining the structure and make data analysis more complex. 

However, new software and algorithms are continually being developed to improve the structure determination's accuracy and make data analysis more efficient.

4. Cost and accessibility

Finally, the cost and accessibility of x-ray crystallography can be a limiting factor for many research groups. 

• Synchrotron sources, which generate high-intensity X-rays for crystallography, are limited and expensive. 
• Also, it can take several attempts to crystallize your protein and obtain helpful information from the scattering trials, increasing the expense associated with your program. 
• The x-ray crystallography equipment and software can also be costly.

However, some companies, such as Immuto, have developed cost-effective approaches to protein expression and crystallization, making the technique more accessible to researchers.

The key takeaway

X-ray crystallography has its limitations and disadvantages. Despite its drawbacks and weaknesses, x-ray crystallography is an effective method for figuring out the structures of proteins and other biological molecules. The precision and effectiveness of the technique are continually being enhanced by developments in protein engineering, software, and data analysis, making it an essential tool for drug discovery and structural biology. 

Drug developers should also be aware of its promise and the ongoing developments in the area that resolve these issues and not slow their progress. 

An innovative alternative

Immuto's platform is built to overcome the challenges around needing a crystalline sample, model-building artefacts and limitations, background noise and data analysis, and cost and accessibility.

PLIMB (Plasma Induced Modification of Biomolecules) is a novel and groundbreaking technology for performing hydroxyl radical protein footprinting–a technique that utilizes mass spectrometry to provide structural data of a protein in its native solution and conformation. PLIMB utilizes plasma to generate microsecond bursts of hydroxyl radicals, which modify and label solvent-accessible regions of a protein.

Immuto's platform offers numerous benefits that streamline the drug development process and enhance lead optimization.

• With a fast turnaround time of 2-3 weeks, our services enable rapid iteration, reducing cycle time and allowing multiple programs to run in parallel.
  
  
• This cost-effective approach can be applied to a wide range of lead candidates, resulting in better, more effective leads and reducing the risk of clinical trial failures.

Our services facilitate faster intellectual property filings backed by robust epitope claims. We provide native analysis, ensuring biologically relevant results by preserving the native structure and protein conditions. Our compatibility with challenging protein targets, such as membrane proteins and GPCRs, makes our services a versatile solution for a diverse array of research needs.

Resources and next steps

Download our free white paper—Hydroxyl Radical Protein Footprinting: A Breakthrough Technique for Epitope Mapping—to see exactly how your development lifecycle stands to gain from the latest techniques.

Explore our technology to learn more about how we’re revolutionizing the drug discovery process and helping our partners tackle previously incurable diseases, and promoting a healthier world. Contact us to learn more and schedule a discovery session.