Tag: biology

Bringing cryo-correlative hard X-ray microscopy to life science

Bringing cryo-correlative hard X-ray microscopy to life science

Scientists led by the ESRF, UGA and INSERM have developed cryo-correlative nano-imaging, a new technique that combines lab cryo-fluorescence microscopy, cryo X-ray fluorescence nanoimaging and phase-contrast nano-tomography on ID16A. The results are published in ACS Nano.

Biologists have long wanted to answer a deceptively simple question: what are the structures we see inside cells actually made of? Visible light fluorescence microscopy shows where organelles are, but not their chemical composition. Hard X-rays can map the chemistry but do not necessarily see the organelles. Cryo-correlative nanoprobe work remains rare, particularly for 3D elemental imaging of whole frozen cells.

A new study at ID16A beamline of the ESRF offers a practical solution. An international team has developed an integrated cryogenic workflow that links laboratory cryo-fluorescence microscopy to targeted cryo X-ray fluorescence (XRF) nano-imaging and phase-contrast nano-tomography.

With this new method, they have tracked therapeutic nanoparticles from the European ScanNtreat project as they moved through cancer cells, showing both where the particles went and what happened to them.

The first author of the publication, Dmitry Karpov, former ESRF scientist and now researcher at the Université Grenoble Alpes, explains how this new development can lead to applications: “This is an example of what the ESRF aims to do: to turn cutting-edge instrumentation into discoveries with direct impact on people’s lives, in this case for medicine and life sciences”.

Read more on the ESRF website

Nanoscale under gigapressure

Nanoscale under gigapressure

Research team led by DESY and MAX IV scientists adapts important X-ray analysis method for use with difficult-to-move samples

Sometimes a change of perspective can make a world of difference. A team of scientists from DESY and MAX IV as well as University of Bayreuth has rearranged the method in which one can use an X-ray beam to image a sample without using high-quality lenses. The method, called ptychography, has been widely used at synchrotrons and free-electron lasers to analyse the inner workings of materials quickly enough while avoiding major damage to the sample by the X-rays. The team has turned the standard method of ptychography on its head: Instead of moving the sample around the X-ray beam, they have figured out how to move the X-ray beam itself in a way that does not alter the properties of the X-rays while still accomplishing the effect of ptychographic analysis. Moreover, they have tested the method on a sample that is in and of itself difficult to move – short-lived states of matter under extreme conditions of pressure and temperature. The team has published their findings in the Proceedings of the U.S. National Academy of Sciences (PNAS).

X-ray ptychography has become, in recent years, a standard technique in the toolbox of researchers using X-ray light sources. In a wide variety of fields, including biology and geology, the technique has been critical for imaging the interiors of samples up to atomic-scale detail non-destructively, revealing details on a scale that methods of light and electron microscopy cannot reach. Up to now, ptychography has been accomplished by using extremely precise sample movers that would change the position of the sample relative to the X-ray beam by tiny lengths – sometimes to the nanometre level – creating a grid pattern of sequentially imaged spots that eventually revealed the full image. Called high-resolution phase-contrast imaging, it has provided insights into the nanoscale structures of tiny biological structures, mineral deposits, computer chips and much more.

Read more on the DESY website

Image: Two views of an extreme-states experiment: To the left is an X-ray micrograph of the sample set up, which consisted of a piece of elemental iron surrounded by solid oxygen, itself surrounded by a rhenium gasket within a diamond anvil cell creating intense pressure. To the right is a ptychographic reconstruction of the area of the sample hit by X-rays, shown with a green circle. In that area using their new ptychographic method, the team could reconstruct the oxidation of the iron being melted by the intense pressure. An extreme-states experiment of this kind has not before been imaged in this way.

Credit: Tang Li, DESY

Dynamic measurements in liquids now possible in the laboratory

Dynamic measurements in liquids now possible in the laboratory

A team of researchers in Berlin has developed a laboratory spectrometer for analysing chemical processes in solution – with a time resolution of 500 ps. This is of interest not only for the study of molecular processes in biology, but also for the development of new catalyst materials. Until now, however, this usually required synchrotron radiation, which is only available at large, modern X-ray sources such as BESSY II. The process now works on a laboratory scale using a plasma light source.

“Our laboratory setup now makes this measurement method available to a wider community,” says HZB physicist Dr. Ioanna Mantouvalou, who drove the development together with partners from the Technische Universität Berlin, the Max Born Institute, the Physikalisch-Technische Bundesanstalt and the company Nano Optics Berlin. “In a first step, the laboratory measurements can also more precisely define where further analyses at synchrotron sources are useful and promising. This allows us to make better use of scarce resources,” says Mantouvalou.

Time-resolved soft X-ray spectroscopy provides access to the properties of organic materials and is therefore ideal for studying dynamic changes in the electronic structure of individual elements in disordered systems. However, measurements of liquid solutions in which these molecules or complexes are dissolved are particularly challenging. They require a high photon flux and extremely low noise. Therefore, these experiments require usually large-scale facilities such as modern synchrotron light sources.

Read more on the HZB website

Image: The dashed black lines mark the first thin liquid ‘sheet’ in which the molecules are dissolved. There are two nozzles in the upper part and a collecting vessel in the lower part (left image). Transmission image of the flat jet (centre image). X-ray spectrum of the solution on the CCD detector (right image).

Credit: © HZB

Shedding Light on Sea Creatures’ Secrets

Shedding Light on Sea Creatures’ Secrets

A nanoscale look at how shells and coral form revealed a mineral that, until now, had never been seen in living organisms – and indicates that biomineralization is more complex than we imagined.

Exactly how does coral make its skeleton, a sea urchin grow a spine, or an abalone form the mother-of-pearl in its shell? A new study at the Advanced Light Source at the Department of Energy’s Lawrence Berkeley National Laboratory (Berkeley Lab) revealed that this process of biomineralization, which sea creatures use to lock carbon away in their bodies, is more complex and diverse than previously thought.

Researchers studied the edges of samples from coral, sea urchin, and mollusks, where temporary building blocks known as “mineral precursors” start to form the new shell or skeleton. There, they found a surprise: Corals and mollusks produced a mineral precursor that had never been observed before in living organisms, and had only recently been created synthetically.

They also found variety in the types of building blocks present. Scientists expected to see “amorphous” precursors, minerals that lack a repeating atomic structure. They did – but they also found “crystalline” precursors, minerals that are more structured and orderly. The research is published in the journal Nature Communications.

Read more on the ALS website

Credit: LazingBee/iStock

Gerold Rosenbaum’s #My1stLight – First Synchrotron X-ray Diffraction Pattern

Gerold Rosenbaum’s #My1stLight – First Synchrotron X-ray Diffraction Pattern

August 1970:  First Synchrotron X-ray Diffraction Recorded at DESY

In order to verify that we could get the flux from the 7.5 GeV synchrotron DESY we had calculated actually onto a small specimen, I designed and we had built an in-vacuum, remote-controlled, focusing x-ray monochromator which we were allowed to insert into the vacuum-ultraviolet beamline of the F41 group at DESY. Preparing for the last trip to DESY from our home lab at Heidelberg, my supervisor Ken Holmes told me to pack a muscle fiber and put it into the beam. Being a physicist by education, I asked why, we measure the flux and X-rays are X-rays and do the same whether from a synchrotron or from a tube at home. Ken: “Not for biologists.” Good that I followed his advice. Thus, I recorded the first synchrotron X-ray diffraction pattern (in the universe – as I like to brag and nobody can dispute this).

There it is and it shows the same pattern as with a home source. So, biologists and everybody else could be confident in what synchrotrons promised.

Credit: The results of the flux verifications were published in Nature in April 1971:

Understanding how motor proteins shape our cells

Understanding how motor proteins shape our cells

Understanding the busy networks inside our cells can help researchers develop new cancer treatments and prevent dangerous fungal infections.

With the help of the Canadian Light Source (CLS) at the University of Saskatchewan, a research team led by John Allingham from Queen’s University and Hernando Sosa from the Albert Einstein College of Medicine has shed light on a protein that regulates the intricate microscopic networks that give cells their shape and helps ship important molecules to diverse locations.

Using the CMCF beamline at the CLS and the cryo-EM facility at the Simons Electron Microscopy Center (SEMC) at the New York Structural Biology Center, the team found the missing pieces of an important puzzle.

In their published work, they are the first group to clearly describe the mechanism of action of a tiny motor protein called Kinesin-8 that enables it to control the structures of microtubule fiber networks inside the cell.

Read more on the CLS website

Image: Cells, Canadian Light Source.

Targeting a parasite’s DNA could be more effective way to treat malaria

Targeting a parasite’s DNA could be more effective way to treat malaria

Research from the University of Sheffield using Diamond has explored a new way of killing the Plasmodium parasite that causes malaria. 

According to the World Health Organisation, there were 241 million cases of malaria and 627,000 deaths worldwide in 2020 – making the study and treatment of this disease a high-priority issue for scientists around the world. In a feasibility study, researchers from the University of Sheffield used Diamond to reveal a novel way of fighting the life-threatening disease, malaria. The study discovered molecules that interfered with the parasite’s DNA processing enzyme, but not the equivalent human one. 

A research team from the University of Sheffield’s Department of Infection, Immunity and Cardiovascular Disease examined and targeted an enzyme that maintains the classic double-helical structure of the malaria parasite’s DNA, which contains the blueprint of life, which could be a more effective way to combat malaria.

Read more on the Diamond website

Image: A flap endonuclease cuts DNA (the orange intertwined worms), credit University of Sheffield

New insight into how mammal ancestors became warm-blooded

New insight into how mammal ancestors became warm-blooded

The shapes of the ear canals of mammal ancestors reveal when warm-bloodedness evolved. The study published in Nature demonstrates that mammal ancestors became warm-blooded later than previously thought – nearly 20 million years later-, and that the acquisition of endothermy seems to have occurred very quickly in geological terms, in less than a million years. The international team of scientists, led by London’s Natural History Museum, the University of Lisbon’s Instituto Superior Técnico, the Field Museum in Chicago, and including the University of Witwatersrand, used the ESRF bright X-rays to scan delicate and dense fossils.

Read more on the ESRF website

Image: Comparison of bony labyrinth shape in two examples of warm-blooded (left) and cold-blooded (right) prehistoric mammal ancestors. © Romain David and Ricardo Araújo.

Assembly lines for designer bioactive compounds

Assembly lines for designer bioactive compounds

Researchers successfully bioengineered changes to a molecular “assembly line” for bioactive compounds, based in part on insights gained from small-angle x-ray scattering at the Advanced Light Source (ALS).

The ability to re-engineer these assembly lines could improve their performance and facilitate the synthesis of new medically useful compounds.

Microbes are known to possess molecular “assembly lines” that produce an important class of compounds, many of which have uses as antibiotics, antifungals, and immunosuppressants. The compounds are peptides—chains of amino acids like RNA, but shorter and produced, not by ribosomes, but by cellular machines known as nonribosomal peptide synthetases (NRPSs).

>Read more on the Advanced Light Source website

Image: Top: Comparison of experimental SAXS scattering data (black) with theoretical curves (green) obtained using an ensemble optimization method (EOM) shows excellent agreement. Bottom: LgrA structural models corresponding to the EOM analyses show large differences in conformation, similar to the differences observed using crystallography.

Researchers use CHESS to map protein motion

Researchers use CHESS to map protein motion

Cornell structural biologists took a new approach to using a classic method of X-ray analysis to capture something the conventional method had never accounted for: the collective motion of proteins.

And they did so by creating software to painstakingly stitch together the scraps of data that are usually disregarded in the process.
Cornell structural biologists took a new approach to using a classic method of X-ray analysis to capture something the conventional method had never accounted for: the collective motion of proteins. And they did so by creating software to painstakingly stitch together the scraps of data that are usually disregarded in the process.
Their paper, “Diffuse X-ray Scattering from Correlated Motions in a Protein Crystal,”published March 9 in Nature Communications.
As a structural biologist, Nozomi Ando, M.S. ’04, Ph.D. ’08, assistant professor of chemistry and chemical biology, is interested in charting the motion of proteins, and their internal parts, to better understand protein function. This type of movement is well known but has been difficult to document because the standard technique for imaging proteins is X-ray crystallography, which produces essentially static snapshots.

>Read more on the CHESS website
>Read also: Diffuse X-ray Scattering from Correlated Motions in a Protein Crystal

Image: This slice through the three-dimensional diffuse map shows intense peaks resulting from lattice vibration, as well as cloudy features caused by internal protein motions.

Using European XFEL to shed light on photosynthesis

Using European XFEL to shed light on photosynthesis

First membrane protein studied at European XFEL

In a paper now published in Nature Communications an international group of scientists show that the fast X-ray pulse rate produced by the European XFEL can be used to study the structure of membrane proteins such as those involved in the process of photosynthesis. These results open up eagerly awaited experimental opportunities for scientists studying these types of proteins.

Large proteins and protein complexes are difficult to study with traditional structural biology approaches. Large protein complexes, such as those that sit across cell membranes and regulate traffic in and out of cells, are difficult to crystalize and generally only produce small crystals that are hard to analyse. The extremely fast X-ray pulses generated by European XFEL now enable scientists to collect large amounts of data from a stream of small crystals to develop detailed models of the 3D structure of these proteins.

>Read more on the European XFEL website

Image (extract, full illustration in the article): Graphic shows the basic design of a serial femtosecond crystallography experiment at European XFEL. X-ray bursts strike crystallized samples resulting in diffraction patterns that can be reassembled into detailed images.
Credit: Shireen Dooling for the Biodesign Institute at ASU

New approach for solving protein structures from tiny crystals

New approach for solving protein structures from tiny crystals

Technique opens door for studies of countless hard-to-crystallize proteins involved in health and disease

Using x-rays to reveal the atomic-scale 3-D structures of proteins has led to countless advances in understanding how these molecules work in bacteria, viruses, plants, and humans—and has guided the development of precision drugs to combat diseases such as cancer and AIDS. But many proteins can’t be grown into crystals large enough for their atomic arrangements to be deciphered. To tackle this challenge, scientists at the U.S. Department of Energy’s (DOE) Brookhaven National Laboratory and colleagues at Columbia University have developed a new approach for solving protein structures from tiny crystals.

The method relies on unique sample-handling, signal-extraction, and data-assembly approaches, and a beamline capable of focusing intense x-rays at Brookhaven’s National Synchrotron Light Source II (NSLS-II)—a DOE Office of Science user facility—to a millionth-of-a-meter spot, about one-fiftieth the width of a human hair.

>Read more on the NSLS-II at Brookhaven Lab website

Image: Wuxian Shi, Martin Fuchs, Sean McSweeney, Babak Andi, and Qun Liu at the FMX beamline at Brookhaven Lab’s National Synchrotron Light Source II, which was used to determine a protein structure from thousands of tiny crystals.

Biological material discovered in Jurassic fossil

Biological material discovered in Jurassic fossil

Ichthyosaurs were reptiles that roamed the Jurassic oceans 180 million years ago. They are extremely well studied and the form will probably be instantly recognisable from museums and textbooks. They resemble modern toothed whales such as dolphins and this similarity led researchers to hypothesise that the two creatures had similar strategies for survival in the marine environment. However, until now, there was little evidence to support this hypothesis. The research team led by Lund researcher Johan Lindgren went on the search for biological material within fossils to help solve this puzzle. After a lot of preparation in the lab and traveling around the world to perform experiments, they discovered that the fossil contained remnants of smooth skin and subcutaneous blubber. This is compelling evidence that the Ichthyosaurs were indeed warm-blooded and confirms the previous hypothesis. Lindgren showed visible delight when he described how you could see that the 180-million-year-old blubber was indeed visibly flexible after treatment in his laboratory.

>Read more on the MAX IV Laboratory website

Image: MAX IV’s Anders Engdahl was part of a team that published a landmark study about biological tissue found in a Jurassic fossil. The work published this week in Nature is one of the most comprehensive studies of its kind and sheds new light on the life of a prehistoric sea creature.

New research helps pursuit for malaria vaccine

New research helps pursuit for malaria vaccine

Scientists from The Hospital for Sick Children (SickKids) identify structure of key malaria protein

Using technology available at the Canadian Light Source synchrotron, SickKids scientists have taken an important step forward on the path to finding effective biomedical interventions to halt the spread of malaria, a disease that affected an estimated 216 million people worldwide in 2016 alone.

Jean-Philippe Julien, a scientist in the Molecular Medicine program at SickKids, and his colleagues focused on a molecule known to be essential for the malaria parasite Plasmodium falciparum to go through the sexual stages of its lifecycle. Disrupting that stage of the lifecycle has the potential to reduce infections and deaths from malaria because parasite transmission between humans would be blocked by inhibiting parasite development in the Anopheles mosquito.

“The protein we looked at was identified several years ago as an important target for malaria parasite biology,” says Julien, who is also a Canada Research Chair in Structural Immunology and an Assistant Professor in the Departments of Biochemistry and Immunology at the University of Toronto. “The field has tried for over a decade to clarify its structure in order to guide the development of biomedical interventions that can curb the spread of malaria.”

>Read more on the Canadian Light Source website

Image: One of the structures of the malaria protein (orange) being recognized by the humanized blocking antibody (green and blue).

The ESRF CryoEM excels in its first year

The ESRF CryoEM excels in its first year

In November 2017, a Titan Krios cryo-electron microscope (cryo-EM) was inaugurated at the ESRF, the European Synchrotron, France. Data collected on this cryo-EM features in a Nature publication describing the activation cycle of a serotonin receptor, which is targeted by medication against chemotherapy- and radiotherapy-induced nausea.

“This publication is a true reward for us: the first one in less than a year from inauguration and we hope this kind of rewards will grow in number”, explains Isai Kandiah, ESRF scientist who runs the facility. “It shows the revolution that cryo-EM is leading in structural biology”, she adds. Thanks to cryo-EM, researchers can now freeze biomolecules, including membrane proteins of high medical importance, in several different conformations in action and visualise each of these to atomic resolution. Cryo-EM thus allows researchers to produce snapshots revealing the dynamics of proteins when they interact with other molecules, information that is crucial both for a basic understanding of life’s chemistry and for the development of pharmaceuticals. The user programme of the cryo-electron microscope at the ESRF is run jointly with the European Molecular Biology Laboratory (EMBL), the Institut de Biologie Structurale (IBS) and the Institut Laue-Langevin (ILL).

The research in Nature is a result of an international collaboration of scientists from the Institute of Structural biology (IBS-mixed research unit CEA-CNRS-University Grenoble Alps), CEA, CNRS, the Institut Pasteur, the University of Lorraine (France), the University of Copenhagen (Denmark), the University of Illinois (US) and the biotech company Theranyx. The focus of the paper, featuring data from the ESRF cryo-EM, is the activation cycle of the 5-HT3 receptor, belonging to the family of serotonin receptors. These receptors are well-known because they influence various biological and neurological processes such as anxiety, appetite, mood, nausea, sleep and thermoregulation, among others. Unlike the other serotonin receptors, which are G protein-coupled receptors, 5-HT3 is a neurotransmitter-gated ion channel and changes its conformation during activation. It is present in the brain, as well as in the enteric nervous system, the peripheral nervous system that drives the digestive tract.

>Read more on the European Synchrotron website

Image: A close-up view of the Cryo-EM at the ESRF.
Credit: S. Candé.

SwissFEL makes protein structures visible

SwissFEL makes protein structures visible

Successful pilot experiment on biomolecules at the newest large research facility of PSI

For the development of new medicinal agents, accurate knowledge of biological processes in the body is a prerequisite. Here proteins play a crucial role. At the Paul Scherrer Institute PSI, the X-ray free-electron laser SwissFEL has now, for the first time, directed its strong light onto protein crystals and made their structures visible. The special characteristics of the X-ray laser enable completely novel experiments in which scientists can watch how proteins move and change their shape. The new method, which in Switzerland is only possible at PSI, will in the future aid in the discovery of new drugs.

Less than two years after the X-ray free-electron laser SwissFEL started operations, PSI researchers, together with the Swiss company leadXpro, have successfully completed their first experiment using it to study biological molecules. With that, they have achieved another milestone before this new PSI large research facility becomes available for experiments, at the beginning of 2019, to all users from academia and industry. SwissFEL is one of only five facilities worldwide in which researchers can investigate biological processes in proteins or protein complexes with high-energy X-ray laser light.

>Read more on the SwissFEL website

Image: Michael Hennig (left) and Karol Nass at the experiment station in SwissFEL where their pilot experiment was conducted.
Credit: Paul Scherrer Institute/Mahir Dzambegovic