Improvements in microscopy technology have focused on elusive details in biology

Posted by Jerry Carter on November 24th, 2021

In the late 17th century, Dutch businessman Anthoni van Leeuwenhoek began to use the first microscope to study a very small world and discovered a colorful world composed of protists, bacteria, and other organisms that had never been seen before. Later generations of scientists have developed more sophisticated means to explore microcosms, bringing many of the mysteries of the biological field into an alarming situation.

Now, researchers at the Center for Applied Structure Discovery in Biological Design (CASD) and Arizona State University’s School of Molecular Sciences (SMS) has taken the field of microscopy one step forward as part of a multi-institutional research collaboration and improved a technique called cryo-electron microscopy, or low temperature em.

This technology involves the rapid freezing of biological samples of interest, and then using electron beam imaging and recording thousands of two-dimensional images, which are then assembled into the atomic outline of the sample structure by a computer. These known density maps can be converted into detailed 3D images.

This method is particularly useful for finding out the subtleties of protein structure, which are often overlooked in traditional modeling strategies. This information is essential for understanding health and disease. Since protein is the main target of most drugs, a comprehensive understanding of its structure and function is essential for designing more effective treatments with fewer side effects.

This new research describes a way to produce more precise structures through a complex statistical method called maximum entropy. This method has been effectively used in many fields, from protein research and neuroscience to ecology and animal population behavior. It is very suitable for the refinement of frozen em data to produce the most unbiased structural model of biological samples.

Molecules like proteins have complex three-dimensional shapes and can also change shape during their function. Abhishek Singharoy, the corresponding author of the new study, said: \"Complex biomolecules actually exist in a series of states. You can take a snapshot of the different conformations of these molecules.\" Some of these structures may persist over time. But others are extremely short-lived, coming and going on a time scale of one-billionth of a second.

The described new technology allows researchers to simulate these transient structures, which play a vital role in biological processes, but they are often ignored by traditional cryo-electromagnetic techniques.

Researchers from the University of Illinois joined the team at Arizona State University; Purdue University; the Department of Mathematics and Computer Science in Grenoble, France; the University of Florida; and Stony Brook University.

Alberto Perez of the University of Florida said: \"This work highlights how laboratory-developed integration and streamlining tools are used in combination with experimental data to promote our understanding of structural biology.\"

A series of modern imaging technologies enable researchers to study the key molecules of life, including proteins, nucleic acids, and even individual molecules. The cryo-electron microscope is a variant of the electron microscope, which was first developed in the 1970s. This technology later won the 2017 Nobel Prize in Chemistry, awarded to pioneers in the field Jacques Dubochet, Joachim Frank and Richard Henderson), because they developed a cryo-electron microscope, which is used to determine the high-resolution structure of biomolecules in solution.

Like other forms of electron microscopes, cryo-electron microscopes use electron beams to replace the photons used to illuminate samples in traditional optical microscopes. Since the resolution of objects under the microscope is limited to about half the wavelength of the light illuminating the sample, the wavelength of electrons is even shorter, depending on their momentum, allowing scientists to see tiny structures with amazing clarity.

Cryo-EM is one of the three methods used in structural biology research, which is combined with x-ray crystallography and nuclear magnetic resonance spectroscopy (NMR). Each method has its own advantages. Although X-ray crystallography can produce amazingly detailed structures with very high resolution, especially using X-ray free electron lasers or XFEL technology, this method is difficult to image large or complex structures, including membrane proteins, because they are very hard to crystallize. The same is true for nuclear magnetic resonance.

Because samples do not require crystallization and can be studied in their natural surroundings, cryo-electron microscopy has made great progress in large and complex biomolecular imaging. In recent years, a new generation of high-speed cameras has been developed to help capture the dynamic activity of various biomolecules. When researchers try to generate three-dimensional structural models from the raw data provided in the initial two-dimensional density maps of molecules, intractable parts emerge. Previously, this involved making a grounded guess about what the structure might look like and fitting the information provided in the density map into this model.

Overfitting the raw data into the structural model may produce inaccuracies. In contrast, the new method does not have any assumptions about the final molecular structure, except for the known constraints. By producing the most unbiased structure, this maximum entropy method can help investigators fill gaps in the structure determination process and better explain the contribution of various conformations that may exist at very low frequencies. To fully understand biomolecules like proteins, the structure of all relevant states that can be assumed by these molecules needs to be determined simultaneously.

Imagine simply trying to make a model of the behavior of a boy standing still for an hour, except for occasional brief movements of the arms and legs. On average, without any change, the resulting boy model will consist of a static, stationary image. On the other hand, the maximum entropy method will allow all different gestures, no matter how brief, to contribute to the final image, producing a more accurate representation.

This new study provides six examples of carefully folded proteins of different sizes, including large membranes and multi-domain systems. This result emphasizes the ability of the maximum entropy statistical software package (called CryoFold) to discover molecular integration, including rare low-probability structures that have been experimentally verified and identified as functionally related.

The maximum entropy technology can be combined with existing data fitting methods to convert low-resolution data into a high-resolution, high-confidence three-dimensional structure in the iterative process. These advances are helping cryo-electron microscopy to achieve its full potential by characterizing the entire conformational landscape of proteins and other important biomolecules.

\"This work integrates multiple physics-based methods to refine protein structure data from cryo-electron microscopy, providing not a static picture of a protein, but a collection of structures, which represent the correct, dynamic properties of the protein,\" Chitrak Gupta said, Co-author CASD researcher and SMS.