Improvements in microscopy home in on biology's elusive details


October 14, 2021

In the late 1600s, the Dutch tradesman Antonie van Leeuwenhoek began investigating the world of the very small using the first microscope, discovering a riotous world of protists, bacteria and other previously unseen organisms. Subsequent generations of scientists have developed ever more sophisticated means of probing the microscopic world, bringing many mysteries of the biological realm into stunning relief.

Now, researchers at Arizona State University's Biodesign Center for Applied Structural Discovery and School of Molecular Sciences, as part of a multi-institutional research collaboration, are carrying the field of microscopy a step further, refining a technique known as cryogenic electron microscopy, or cryo-EM. The ASU research group (from left): Abhishek Singharoy, John Vant, Jonathan Nguyen, Petra Fromme, Chitrak Gupta and Wade Van Horn. Download Full Image

The technology involves flash-freezing a biological sample of interest, then using a beam of electrons to image and record thousands of 2D images, assembled by means of computer into an atomic profile of the sample’s structure. Such density maps, as they are known, can then be converted into a detailed 3D image.

The method is particularly useful for ferreting out subtleties of protein structure, which are often missed through conventional modelling strategies. Such information is critical for the understanding of health and disease. Since proteins are the primary targets of most pharmaceutical drugs, a fuller picture of their structure and function is essential for designing more effective treatments, with fewer side effects.

The new research describes a method for producing more accurate structures, through a sophisticated statistical method known as maximum entropy. This approach — which has been effectively applied in many domains ranging from protein research and neuroscience to ecology and the behavior of animal populations — is ideally suited to the refinement of cryo-EM data, producing the most unbiased structural model of a biological sample.

Molecules like proteins assume complex, three-dimensional forms and can also change shape during their functioning.

“Complex biomolecules actually exist in an ensemble of states, and you can take a kind of snapshot of these molecules in different conformations,” said Abhishek Singharoy, corresponding author of the new study and an assistant professor at ASU.

Some of these conformations may persist in time, but others are extremely ephemeral, coming and going on time scales measured in billionths of a second.

The new technique described allows researchers to model these transitory structures, which can play a vital role in biological processes but are often missed using traditional cryo-EM techniques.

The ASU team is joined by researchers from the University of Illinois; Purdue University; Department of Mathematics and Computer Sciences, Grenoble, France; the University of Florida; and Stony Brook University. 

“This work highlights how the integration and streamlining tools developed by labs with complementary expertise can be used in combination with experimental data to advance our understanding of structural biology,” said Alberto Perez of the University of Florida.

The group’s findings appear in the current Cell Press journal Matter, appearing on the journal's cover.

One of a triad of methods

A range of modern imaging techniques allows researchers to investigate crucial molecules of life, including proteins, nucleic acids and even single molecules. Cryo-EM, a variant of electron microscopy, was first developed in the 1970s. The technique was later recognized with the the 2017 Nobel Prize in chemistry, awarded to Jacques Dubochet, Joachim Frank and Richard Henderson, pioneers in the field, for developing cryo-electron microscopy for the high-resolution structure determination of biomolecules in solution. 

Like other forms of electron microscopy, cryo-EM replaces photons of light used for illuminating samples in conventional light microscopy with a beam of electrons. Because resolution of an object by microscopy is limited to roughly one-half the wavelength of light used to illuminate the sample, the shorter wavelengths of electrons, which are dependent on their momentum, enable scientists to visualize tiny structures with astonishing clarity.

Cryo-EM is one of a triad of methods used for research in structural biology, joining X-ray crystallography and nuclear magnetic resonance spectroscopy (NMR). Each method has its own strengths. While X-ray crystallography can produce stunningly detailed structures in very high resolution, especially using X-ray free electron lasers or XFEL technology, the method struggles to image large or complex structures, including membrane proteins, as they are challenging to crystalize. The same is true of NMR.

Cryo-EM comes of age

Imaging large, complex biomolecules is where cryo-EM hits its stride, because samples do not require crystallization and can be studied in their native surrounding environment. In recent years, a new generation of high-speed cameras have been developed to help capture the dynamic activity of various biomolecules. The tricky part arises when researchers try to produce a 3D structural model from the raw data provided in the initial 2D density map of the molecule. Previously, this has involved making an educated guess about what the structure may look like and fitting the information provided in the density map into this model.

Inaccuracies can arise from the overfitting of raw data into the structural model. The new approach instead makes no assumptions about the final molecular structure apart from constraints that are known with certainty. By producing the most unbiased structure, this maximum entropy approach can help researchers fill in the blanks during structural determination, better accounting for the contribution of various conformations that may exist at very low frequency. A full understanding of biomolecules like proteins requires the simultaneous determination of the structures of all the relevant states that these molecules can assume.

To visualize this in simple terms, imagine trying to produce a model that illustrates the behavior of a boy who is standing still for an hour, except for occasional, fleeting movements of arms and legs. On average, nothing changes, and a resulting model of the boy would consist of a static, motionless image. A maximum entropy approach on the other hand would allow all various gestures, however brief, to contribute to the final image, yielding a much more accurate representation.

The new study presents six examples of elaborately folded proteins of various sizes, including large membrane and multi-domain systems. The results emphasize the ability of a maximum entropy statistical package known as CryoFold to discover molecular ensembles, including rare low-probability structures that have been experimentally validated and recognized as functionally relevant.

The maximum entropy technique can be used in conjunction with existing methods of data fitting in an iterative process capable of turning low-resolution data into high-resolution, 3D structures with a high degree of confidence. Such advances are helping cryo-EM reach its full potential by characterizing the entire conformational landscape of proteins and other important biomolecules.

“This work integrates multiple physics-based approaches to refine protein structures from cryo-EM data, providing not a single, static image of the protein, but rather a collection of structures, which is more representative of the true, dynamic nature of proteins,” said Chitrak Gupta, co-author and a researcher in the Center for Applied Structural Discovery and School of Molecular Sciences.

Richard Harth

Science writer, Biodesign Institute at ASU

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Evolving engineering for a better world


October 14, 2021

Investigating how engineers can define a new engineering paradigm that values social and environmental justice is what motivates Darshan Karwat.

As an assistant professor at both The Polytechnic School, one of the seven Ira A. Fulton Schools of Engineering at Arizona State University, and ASU’s School for the Future of Innovation in Society, Karwat is dedicated to shifting the focus of engineering design and practice through research, policy impact and advocacy. re-Engineered Lab From left: Darshan Karwat, assistant professor in the Ira A. Fulton Schools of Engineering and the School for the Future of Innovation in Society; Madison Macias, urban and environmental planning master’s student in ASU’s School of Geographical Sciences and Urban Planning; Eddie Schmitt, postdoctoral fellow in the School for the Future of Innovation in Society; Jorge Morales Guerrero and Mokshda Kaul, sustainable energy doctoral students in ASU’s School of Sustainability; and Eric Stribling, innovation in global development doctoral student in the School for the Future of Innovation in Society. Not pictured: David Oonk, postdoctoral fellow in the School for the Future of Innovation in Society. Photo by Erika Gronek/ASU Download Full Image

Karwat’s path started in Mumbai, India, where he grew up and frequently confronted a range of environmental and social issues. Breathing the city’s polluted air and witnessing poverty shaped his outlook into adulthood and led him to become an environmental activist as an undergraduate studying aerospace engineering at the University of Michigan.

After earning his doctoral degree in aerospace engineering and environmental ethics, Karwat designed programs for low-cost air pollution sensors and climate resilience as an American Association for the Advancement of Science fellow at the Environmental Protection Agency in Washington, D.C. He also helped design and run the Wave Energy Prize, a design-build-test competition, and advanced desalination research efforts at the U.S. Department of Energy.

Merging his educational background with his concerns for social justice, peace and environmental protection has given Karwat a platform to develop his own unique research and impact niche dedicated to these interests.

re-Engineered

In Karwat’s interdisciplinary lab, re-Engineered, students and researchers have experience in anthropology, urban planning, science and technology policy, mechanical and chemical engineering, and community development.

Some of the questions at the heart of re-Engineered are: Why are we engineers? For whose benefit do we work? What is the full measure of our moral and social responsibility?

“Without clear answers to these questions, many engineering solutions end up doing just as much harm as they do good because we are engrossed with making something efficient and affordable rather than making something equitable or sustainable,” says re-Engineered lab student Madison Macias, an ASU mechanical engineering alumna earning her master’s degree in urban and environmental planning in ASU’s School of Geographical Sciences and Urban Planning.

Prior to joining re-Engineered, Macias says she likely would’ve gone through her entire academic career feeling like her work wasn’t true to her initial intent — “to better the world through engineering.”

Exploring the engineering mindset

Karwat believes “the mindsets of engineers and the political economy of the engineering industry can unknowingly hinder progress in helping build a more peaceful world, further contributing to a lack of resources available in communities that are underserved.”

The re-Engineered lab recently surveyed engineers to gain a better understanding of why this is the case. They found that along with less time and money, many engineers don’t feel equipped to take on work that directly addresses issues of environmental or social concern and “the ‘undone’ technical work in sectors that are not currently economically valued,” Karwat says.

Engineers also may not be connected to the communities they serve, making it difficult to fully understand the challenges they face and to collaborate to address those challenges.

“Many engineering students choose to study engineering because they feel like engineering can help make a positive contribution in the world, but when they join the workforce and job opportunities are limited or their income becomes a priority, they might lose their passion, become distracted and stray from their original goals and intentions,” Karwat says. “This is in line with growing research that shows current modes of engineering education and professionalization strip concerns for public welfare from engineers.”

His research team recently completed a pilot project funded by the Engineering Change Lab-USA to understand environmental protections, social justice and diversity, equity and inclusion values among practicing engineers.

The project uncovers a generational divide — that younger engineers are coming into the workforce with stronger environmental and social values and there are political factors at play that affect the kind of engineering people want to pursue.

“While we need to continue to study and affect change among engineering students, most engineers are not in school. They are out in the working world, living their lives, earning money to pay bills and supporting their families,” Karwat says. “To see the change we want in the world, we need to focus on understanding practicing engineers and changing their practice. Unfortunately, there is very little to no research on them, and this project is an attempt to begin to address that gap.”

Fusing engineering with social science

Karwat’s latest research project, funded by the National Science Foundation, explores collaborations between academic and community groups that are addressing engineering and scientific questions at the heart of environmental, climate and energy challenges — a project he describes as “an expression of the ASU Charter.”

Four teams of nonprofit leaders and ASU-affiliated faculty and staff are combining their skills to develop engineering and scientific road maps to address issues like water resiliency, urban heat, tree health monitoring and injustices on the lands of Indigenous communities in the U.S. and Canada.

“Along with developing plans and strategies to tackle these important issues, we are studying the dynamics of the collaboration process and trying to understand how that changes perceptions of what constitutes meaningful engineering and science,” Karwat says. “One of the goals is to better understand the currencies of collaboration such that we might create a new field of collaboration for engineers that is economically, socially, culturally and symbolically valued.”

Influencing existing policies

Along with research, Karwat is committed to bringing change to the institutions that shape engineering. He was recently elected from a group of nearly 300 applicants to the New Voices program, an initiative launched in 2018 by the National Academies of Sciences, Engineering and Medicine.

The cohort of early career leaders from academia, industry, government and nonprofit organizations will engage in relevant dialogue about how science, engineering and medicine are shaping the global future.

“The New Voices cohort will help inform the efforts of the National Academies of Sciences, Engineering and Medicine as they consider the role of engineering and science in addressing major global challenges,” Karwat says.

Karwat and his lab team are also working with program managers at the National Renewable Energy Laboratory and the U.S. Department of Energy to offer insights and practical recommendations on processes, metrics and outcomes that can shape their technology research and development portfolios to promote environmental and energy justice.

“With questions of environmental and energy justice becoming more salient, it behooves the research and development enterprise to think about the ways in which technological design is informed by values of justice,” Karwat says. “Are we sure the technologies being researched are of public value and address injustices in society? And if not, in what ways might we change what is researched and what is developed?”

A constellation of changemakers

In 2019, Karwat came up with an idea to recognize people and collaborative efforts that are redesigning engineering to elevate the values of environmental protection, social justice, human rights and peace — the Constellation Prize. A committee of educators, students and peace-builders who feel that there is important and boundary-pushing engineering work that should be celebrated to inspire others are moving the prize forward.

Last year’s inaugural cohort of Constellation Prize winners were awarded for their achievements in advancing Indigenous rights, biodiversity, engineering education, community collaboration and policy impact.

Many other winners are doing equally impactful work, and Karwat hopes to expand the reach of the Constellation Prize as the endeavor grows and becomes more widely recognized.

The final frontier

Along with several projects in the works, Karwat also wants to merge his background in aerospace engineering, space systems and environmental protection to craft new kinds of engineering questions and explore projects aimed at better protecting the sanctity and beauty of space as we continue to explore it.

“The momentum to envision the idea of humans as an interplanetary species continues to build,” Karwat says. “I wonder if we can create this future through engineered systems in a way that not only avoids the environmental and justice challenges we’ve been left to deal with on Earth but also inspires engineering design that reflects a new ethos of care for the places we go.”

Sona Patel Srinarayana

Communications specialist, Ira A. Fulton Schools of Engineering

480-727-1590