Unraveling the Mysteries of the Molecular World: Structural Biology Techniques

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May 30, 2023

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3D reconstruction of beta amylase protein structure determination by high resolution cryo-EM | Structural Biology | Nano Imaging Services
colorful 3D reconstruction of human adenosine A1 receptor-Gi2-protein complex (6D9H) from high resolution cryo-EM | Structural Biology | NanoImaging Services
3D Reconstruction of human adenosine A1 receptor-Gi2-protein complex (6D9H).

For centuries, scientists have been on a mission to unlock the mysteries of the molecular world by trying to understand the intricate and delicate machinery that powers all life. From the initial applications of X-ray crystallography to the emergence of nuclear magnetic resonance (NMR) spectroscopy, the tools and techniques of structural biology have evolved rapidly, providing increasingly detailed glimpses into the molecular landscape. 

Among these techniques, cryo-electron microscopy (cryo-EM) has recently emerged as a transformative technology, offering unparalleled insights into the structures and functions of molecules. In recent years, cryo-EM has captured the attention of researchers around the world, opening up new frontiers in drug discovery, biotechnology, and beyond. 

Cryo-EM has emerged as a versatile and powerful tool for structural analysis of large, dynamic, and membrane-associated biomolecules that have been challenging to study until now.

Using X-ray Crystallography to Elucidate Small Molecules 

X-ray crystallography is one of the oldest and most commonly used techniques for studying and determining the 3-D structure of molecules. In its early days, researchers were limited by the availability of high-quality crystals, but advances in technology and methodology have made it possible to study ever more complex structures.

This technique has been used to determine the structures of many important molecules, including insulin, myoglobin, and hemoglobin, and has played a critical role in the development of new drugs and therapies. Despite the advent of newer techniques, X-ray crystallography remains an important tool for structural biologists thanks to its ability to keep up with the fast pace of the drug research and development industry. 

Even though X-ray crystallography continues to be a powerful tool for structural biologists, there are some limitations to the technique. One of the main challenges is obtaining well-diffracting crystals, which requires a significant amount of trial and error. Additionally, the technique is not ideal for studying large, dynamic, or membrane-associated molecules. While it may not be the go-to method for every target, its impact and influence on the field cannot be denied, and its legacy will continue to endure for years to come.

Nuclear Magnetic Resonance (NMR) for Solving 3D Structures

Cartoon illustration of nuclear magnetic resonance for structure based drug design | structural biology | nano imaging services
Ligand-observed NMR in hit compound screening and/or validation: (a) Cartoon illustration of the Carr-Purcell-Meiboom-Gill pulse sequence (CPMG) experiment. Validation of Hsp82:SOMCL-16-171 interaction by using the CPMG technique is shown as an example. (b) Cartoon illustration of the difference spectrum in the saturation transfer difference (STD) experiment. Validation of Hsp90:1-E6 interaction by using the STD technique is shown as an example. (6)

Another key asset in the structural biology toolkit is Nuclear Magnetic Resonance (NMR), a non-invasive and non-destructive method that utilizes the magnetic properties of atomic nuclei to determine the three-dimensional structure of molecules. NMR has come a long way since its inception in the early 20th century. It was in the 1970s that the technique was first applied to the study of biological molecules. Over the years, advancements in hardware, software, and methodology have made NMR an increasingly powerful tool for structural biologists. Today, NMR is widely used to study a variety of molecules, including proteins, nucleic acids, and carbohydrates, among others.

One of the unique features of NMR is that it can provide information on the dynamic behavior of molecules in solution. This makes NMR particularly useful for studying the conformational changes and interactions of biomolecules in physiological conditions. Additionally, NMR is able to detect weak interactions between molecules which are critical for the stability and function of macromolecular complexes. 

NMR is particularly useful for studying small to medium-sized molecules, such as proteins up to about 50 kDa. Even though NMR is limited in its ability to study large and dynamic molecules and tends to require complex sample preparation, in recent years its application in drug discovery have shifted from a target-centric approach to a compound-centric one. It has become an essential tool for fragment based drug discovery and has played a big part in the development of protein-protein interaction modulations.

Why is Cryo Electron Microscopy (Cryo-EM) the best choice for Structural Biology?

Over the past few decades, cryo-EM has come a long way from its humble beginnings as a niche technique. Advances in hardware, software, and sample preparation have led to remarkable improvements in resolution and throughput, making cryo-EM a formidable tool for structural biologists worldwide.

Cryo-EM is a technique that allows us to visualize the intricate details of molecules providing unprecedented insights into their function and mechanisms. It is mainly used for the structural analysis of targets that are too large or dynamic to be studied by X-ray crystallography or NMR. Examples of such biomolecules are membrane proteins, large macromolecular complexes, and viruses. Additionally, cryo-EM can capture structural snapshots of molecules in different conformations or bound to ligands, providing valuable information about their functional states.

One of the major advantages of cryo-EM is that it can provide high-resolution structures without the prior need to form crystals. Instead, the samples are flash-frozen in a thin layer of vitreous ice, which helps to preserve the native structure of a target. 

3D protein strucutre determination by high resolution cryo-EM structural biology services nano imaging services cryo-EM CRO
3D Reconstruction of Beta Amylase done at NanoImaging Services with cryo-EM.

Despite its advantages, there are some limitations to cryo-EM, such as the need for specialized equipment and expertise. Cryo-EM requires a high-end electron microscope, specialized sample preparation equipment, and skilled operators. Additionally, the data collection and processing can be time-consuming and computationally intensive. 

Nevertheless, cryo-EM has revolutionized the field of structural biology, providing an invaluable tool for visualizing macromolecular structures that were not accessible by x-ray or NMR. While X-ray crystallography and NMR continue to be valuable techniques for solving atomic structures, cryo-EM is rapidly becoming the method of choice for structural biologists who seek to study complex molecular machines or challenging targets such as membrane proteins.

The availability of advanced software and computational tools, combined with the increasing accessibility of cryo-EM CROs, like NanoImaging Services, has made it possible for more researchers to incorporate this technique into their workflow. Cryo-EM enables high resolution structures of challenging targets, and allows the pharmaceutical industry to gain a deeper understanding of biological processes at the molecular level. 

Cryo-EM X-ray Crystallography Nuclear Magnetic Resonance (NMR)
  • Near atomic visualization of proteins in their native state
  • Well developed & widely adopted
  • High resolution
  • Non-invasive, non-destructive and well suited to small to medium-sized molecules
  • Highly specialized equipment and expert sample preparation required
  • Not all proteins crystallize well
  • Not suited for large, dynamic, or membrane-associated molecules
  • Requires complex sample prep
  • Limited ability to study large or dynamic molecules

The potential for cryo-EM to reveal new insights into fundamental biological mechanisms, as well as to facilitate the development of new therapeutics, is enormous.

Each analytical technique has its strengths and limitations, however, cryo-EM stands out as a technique with immense potential for advancing our understanding of life's molecular basis. The ability for cryo-EM to reveal hidden details of biological molecules has already led to groundbreaking discoveries, and as cryo-EM technology and methodology evolve, we can only imagine what new insights and discoveries await us.

Learn about cryo-EM for structure based drug design (SBDD), or request a cryo-EM consultation.

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  6. Shi L, Zhang N. Applications of Solution NMR in Drug Discovery. Molecules. 2021; 26(3):576. DOI: /10.3390/molecules26030576
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Understand the pros and cons of cryo-EM, x-ray crystallography, and nuclear magnetic resonance (NMR) for protein structure determination | Nano Imaging cryo-EM CRO

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