small-angle x-ray scattering (SAXS)

Taking candid shots of radical proteins

2024/02/092024/02/15

Scientists capture how radical electrons influence protein structure before they have time to react

Some enzymes in the body carry radicals, chemical groups with highly reactive unpaired electrons, to catalyse biochemical reactions, but it has proven challenging to study the structure of these enzymes with the radicals intact. The X-ray crystallography techniques conventionally used to study protein structure introduce ‘X-ray damage’ that would neutralise radicals and alter the protein structure. To study how radicals influence proteins, researchers turned to a ribonucleotide reductase enzyme subunit called R2 that uses a radical to synthesise DNA bases. The team previously used X-ray crystallography at beamline I24 and small angle X-ray scattering at beamline B21 of the Diamond Light Source to solve the structure of this enzyme without safeguarding the radical. In the recent study, they harnessed X-ray free electron laser (XFEL) serial femtosecond crystallography at the Linac Coherent Light Source in collaboration with Diamond’s XFEL Hub to zero in on the radical. With XFEL, they used X-rays to rapidly capture the structure of the protein within femtoseconds — 10¹⁵ times quicker than a second and too quick for X-rays to neutralise the radical or distort protein structure. By comparing the enzyme with and without the radical, they revealed that the presence of the unpaired electron greatly influences the structure of the enzyme’s active site. Their research will allow them to explore the workings of this and related enzymes in finer detail and holds promise for designing drugs that target radical enzymes in cancer cells and infectious microbes.

Read more on the Diamond website

Image credit: Martin Högbom

Protein family shows how life adapted to oxygen

2022/10/042022/10/12

Cornell scientists have created an evolutionary model that connects organisms living in today’s oxygen-rich atmosphere to a time, billions of years ago, when Earth’s atmosphere had little oxygen – by analyzing ribonucleotide reductases (RNRs), a family of proteins used by all free-living organisms and many viruses to repair and replicate DNA.

“By understanding the evolution of these proteins, we can understand how nature adapts to environmental changes at the molecular level. In turn, we also learn about our planet’s past,” said Nozomi Ando, associate professor of chemistry and chemical biology in the College of Arts and Sciences and corresponding author of the study. “Comprehensive phylogenetic analysis of the ribonucleotide reductase family reveals an ancestral clade” published in eLife Digest Oct. 4.

Co-first authors of the study are Audrey Burnim and Da Xu, doctoral students in chemistry and chemical biology, and Matthew Spence, Research School of Chemistry, Australian National University, Canberra. Colin J. Jackson, professor of chemistry, Australian National University, Canberra, is a corresponding author.

This undertaking involved a large dataset of 6,779 RNR sequences; the phylogeny took several high-performance computers a combined seven months (1.4 million CPU hours) to calculate. Made possible by computing advances, the approach opens up a new way to study other diverse protein families that have evolutionary or medical significance.

RNRs have adapted to changes in the environment over billions of years to conserve their catalytic mechanism because of their essential role for all DNA-based life, Ando said. Her lab studies protein allostery – how proteins are able to change activity in response to the environment. The evolutionary information in a phylogeny gives us a way to study the relationship between the primary sequence of a protein and its three-dimensional structure, dynamics and function.

Read more on the CHESS website

Image: Tree inference on a ribonucleotide reductase (RNR) sequence dataset as included in the original report, “Comprehensive phylogenetic analysis of the ribonucleotide reductase family reveals an ancestral clade“.

Creating tastier vegan cheese using synchrotron X-rays

2022/09/302022/10/12

The quest for tastier, more sustainable vegan cheese has led Swedish food company Cassius AB to take a closer look at cheese protein structures. Using synchrotron X-rays at MAX IV, Cassius are searching for the perfect scientific recipe for plant-based cheese.

When regular cheese is produced, the milk proteins react with rennet and form a cheese curd. These specific proteins, formed in a certain structure, are unique to mammalian milk, which makes them difficult to mimic.

Cassius AB:s research project focuses on getting a deeper understanding of how the proteins in regular cheese form structures spontaneously. It also investigates whether this could happen with mammalian milk proteins produced by genetically engineered microorganisms, in a process called precision fermentation.

Since mimicking all proteins in regular cheese is not necessary, Cassius is concentrating on two of the protein types that play a key role in how cheese coagulates.

Cheese gel balls as protein samples

Johan Krakau, founder of Cassius and brands like GoVego, has teamed up with researchers from RISE within the NextBioForm center to perform the experiment at the MAX IV CoSAXS beamline.

Using Small-Angle X-ray Scattering techniques (SAXS), the research team studies different types of protein samples in the form of micelles – spherical protein aggregations that resemble gel balls – and how different conditions affect their shape and size. For example, when mixed with different amounts of salt, or when the pH value is changed. The team also investigates if these proteins coagulate into a curd structure in the same way that mammalian milk proteins do.

Read more on the MAX IV website

Capybara gut holds valuable enzymes for biotechnology

2022/04/132022/06/23

Study elucidates unprecedented processes of herbivore metabolism involved in the efficient degradation of plant fibers

A group of researchers from the Brazilian Biorenewables National Laboratory (LNBR), Brazilian Center for Research in Energy and Materials (CNPEM), an organization supervised by the Brazilian Ministry of Science, Technology and Innovations (MCTI), has published in the journal Nature Communications a study that explores some of the most modern resources of current science to reveal unprecedented and valuable details of the capybara’s digestive process.

The capybara, the largest rodent on the planet, is known for its ability to degrade very efficiently the biomass it consumes, but the details of the animal’s microbiota metabolism that contribute to this characteristic have not yet been elucidated. Researcher Mario Murakami recalls that, in Brazil, this animal is used to eating sugarcane. “Since Brazilian biodiversity is an invaluable source of biotechnological solutions, our hypothesis was that the microorganisms inhabiting capybaras’ intestines have, throughout evolution, developed highly effective molecular strategies for the degradation and use of this biomass of great industrial and economic importance. And that was demonstrated in our study.”

“Population and molecular inventory” of the gut microbiome

The meticulous and unprecedented work started with a complete survey of the bacteria present in the capybara’s intestine, in addition to the expressed genes and metabolites produced from plant fibers. To understand the processes of depolymerization of lignocellulosic fibers and the efficient transformation of sugars into energy, a vast combination of techniques, methodologies and resources, including synchrotron light at the MX2 and SAXS1 beamlines of the Brazilian Synchrotron Light Laboratory (LNLS), was required, from the population scale of microorganisms to the atomic and molecular level of enzymes.

Read more the the LNLS website

Measuring complex fluids under extreme flow conditions

2022/02/042022/02/10

Utilizing the unique focusing optics, flexible sample space, and SAXS capabilities at the FMB-beamline, a group of researchers from the National Institute of Standards and Technology measured the rheology and structure of complex fluids subjected to extreme flow velocities while confined within micrometer-sized capillaries.

What did the scientists do?

A capillary rheometer capable of producing high shear rates at the wall, previously developed for neutron scattering, was modified to expand the accessible shear rates up to 10⁷ s^-1 when using a high-flux x-ray source with small spot sizes, such as the FMB-beamline at CHESS. Using the new setup optimized for x-ray scattering, the structure and rheology of worm-like micelle solutions were measured at high shear rates to better understand the microstructural alignment, breakdown, and shear thinning rheology of these widely utilized surfactants.

Why is this important?

Worm-like micelle surfactant systems have numerous applications ranging from pharmaceutical formulations to enhanced oil recovery. The simultaneous rheology and x-ray scattering measurements will help link the changes in macroscopic rheological properties to the changes in nanoscale fluid structure such as micelle orientation and length distribution. These measurements are also important to improve rheological models, which currently fail to accurately predict the viscosity of complex fluids at high shear rates.

Read more on the CHESS website

Image: SAXS measurements at the FMB-beamline showed distinct changes in the worm-like micelle structure under flow

We all love science!

2021/12/302021/12/30

#LightSourceSelfie from users of the Australian Light Source

Marta Krasowska (Associate Professor), Sarah Otto (PhD Student) and Stephanie MacWilliams (Early Career Researcher) are scientists based at the University of South Australia. They share a passion for soft matter research and conduct experiments at ANSTO’s Australian Synchrotron. Their research questions relate to structural ordering in soft matter and its relevance in applications such as food, personal care products, biomaterials and pharmaceuticals.

In their #LightSourceSelfie, Marta, Sarah and Stephanie discuss what attracted them to this area of research, how they felt the first time they conducted experiments at the Australian Synchrotron, the support they receive from the team based at the facility, their top tips for surviving night shifts and how their research will benefit from the new BioSAX beamline, which is part of the synchrotron’s major upgrade. When it came to single words to describe their research, they agreed on “Challenging, unpredictable and super rewarding!”

Delivering drugs using nanocrystals

2021/09/272021/09/30

Monash University researchers have used advanced techniques at ANSTO to investigate the production of new, elongated polymer nanocapsules with a high payload of drug nanocrystals to potentially increase drug targetability, and also decrease dosage frequency and side effects.

This method had not been investigated previously and represents a pioneering method of investigation in the field of colloidal science applications for drug delivery.

Nanoparticles have been used to increase the delivery efficiency of cancer therapy because of their biocompatibility, versatility and the easiness of functionalisation.

The team engineered novel elongated polymer nanocapsules, which are unlike the more well-known spherical nanocapsules.

The elongated polymer nanocapsules were made with elongated liposomes or surfactant vesicles and used drug nanocrystals as a template.

The results provided strong evidence that the elongated structure could be retained, and also confirmed that the loading method to form rod-like drug nanocrystals inside liposomes was a practical solution.

The combination of the high drug payload, in the form of encapsulated nanocrystals, and the non-spherical feature of liposomes represented a more efficient delivery system.

Spherical hollow nanocapsules have been studied extensively, but the formation of elongated nanocapsules containing active pharmaceuticals as therapeutic agents has been previously largely unsuccessful.

Read more on the ANSTO website

Image: Elongated nanocapsules can be prepared by polymerisation at the surface of elongated liposome templates with drug nanocrystals

Molecular IgG3 structure paves the way for new applications of antibodies

2021/09/032021/10/07

A combination of scattering and analytical techniques has provided the first atomic-level structural model for the IgG3 antibody

In humans, Immunoglobulin (IgG) is the most common type of antibody found in blood circulation. IgG molecules are created by plasma B cells, and there are four subclasses. Of the four, IgG3 is the least understood. It has a uniquely long hinge region separating its Fab antigen-binding and Fc receptor-binding regions. The presence of this elongated hinge makes it challenging to perform structural studies, for example, with X-ray crystallography. Due to this lack of structural information, IgG3 is the only subclass not currently exploited for therapeutic uses. In work recently published in the Journal of Biological Chemistry, researchers from University College London and the University of Birmingham have used a combination of imaging and analytical methods to provide the first experimentally determined molecular structural model for a full-length IgG3 antibody. This new information should enable the use of IgG3 to develop new therapies and antibody tests.

Getting a good look at IgG3

A high-resolution structure for part of the IgG3 molecule, the globular IgG3-Fc fragment, is available. And previous studies of the whole molecule using Small Angle X-ray Scattering (SAXS) and analytical ultracentrifugation (AUC) showed that IgG3 is elongated compared to IgG1, IgG2 and IgG4. SAXS also showed that IgG3 has a more extended central hinge than IgG1 and IgG2 that links its three globular regions together. 

Read more on the Diamond website

Image: The IgG3 structural model is formed from two globular Fab regions, a long hinge in the centre, and one Fc region, as shown from the scattering modelling fits. The structure is reminiscent of a giraffe with an extended and semi-rigid neck.

Credit: Dr Valentina Spiteri, UCL.

Insights into coronavirus proteins using SAXS

2021/07/062021/07/15

A collaboration led by researchers from the European Molecular Biology Laboratory (EMBL) used small angle X-ray scattering (SAXS) at the European XFEL and obtained interesting data on samples containing coronavirus spike proteins including proteins of the isolated receptor biding domain. The results can, for example, help investigate how antibodies bind to the virus. This gives researchers a new tool that may improve understanding of our bodies’ immune response to coronavirus and help to develop medical strategies to overcome COVID-19

SAXS is a powerful technique as it allows researchers to gain insights into protein shape and function at the micro- and nanoscales. The technique has proven to be extremely useful in investigating macromolecular structures such as proteins, especially because it removes the need to crystallize these samples. This means researchers can study the sample in its native form under physiological conditions under which biological reactions occur.

Read more on the European XFEL website

Image: Seen here, the instrument SPB/SFX, where the SAXS experiment was carried out. Using this instrument researchers can study the three-dimensional structures of biological objects. Examples are biological molecules including crystals of macromolecules and macromolecular complexes as well as viruses, organelles, and cells.

Credit: European XFEL / Jan Hosan

Researchers discover foam “Fizzics”

2021/06/212021/06/24

Chemical engineers at the University of Illinois Chicago and UCLA used the U.S. Department of Energy’s Advanced Photon Source (APS) in answering longstanding questions about the underlying processes that determine the life cycle of liquid foams. The breakthrough in understanding how liquid foams dissipate could help improve the commercial production and application of foams in a broad range of industries and could lead to improved products. Findings of the research were featured in the Proceedings of the National Academy of Sciences of the United States of America.

Foams are a familiar phenomenon in everyday lives — mixing soaps and detergents into water when doing dishes, blowing bubbles out of soapy water toys, sipping the foam off a cup of lattes or milk shake. Liquid foams can occur in a variety of natural and artificial settings. While some foams are produced naturally, as in bodies of water creating large ocean blooms on the beaches, others arise in industrial processes. In oil recovery and fermentation, for example, foams are a byproduct.

Whenever soapy water is agitated, foams are formed. They are mostly gas pockets separated by thin liquid films that often contain tiny molecular aggregates called micelles. Oily dirt, for example, is washed away by hiding in the water-phobic cores of micelles. In addition, fat digestion in our bodies relies on the role of micelles formed by bile salts.

Over time, foams dissipate as liquid within the thin films is squeezed out. Soap and detergent molecules that are by very nature amphiphilic (hydrophilic and hydrophobic) aggregate within water to form spherical micelles, with their outward-facing heads being hydrophilic and water-phobic tails forming the core.

Read more on the ANL website

Image: Micellar foam films show grayscale intensity variations that correspond to rich nanoscopic topography mapped using IDIOM protocols.

Credit: Chrystian Ochoa and Vivek Sharma/UIC

Quantifying oriented myelin in mouse and human brain

2021/05/262021/06/08

Myelin “insulates” our neurons enabling fast signal transduction in our brain; myelin levels, integrity, and neuron orientations are important determinants of brain development and disease. However, myelin imaging methods used in clinics or research are non-specific or destructive.

Using small-angle X-ray scattering tensor tomography (SAXS-TT), we exploited myelin’s ~17nm periodicity to non-invasively derive 3D myelin and neuron orientation maps in macroscopic tissue volumes (Figure). We demonstrated the method on a mouse brain (a-d), a mouse spinal cord, a human visual cortex and two human white matter specimens. We validated the readouts with 2D and 3D histology, and correlated the results with MRI contrasts.

How X-rays could make reliable, rapid COVID-19 tests a reality

2021/05/182021/06/04

Vaccines are turning the tide in the pandemic, but the risk of infection is still present in some situations. If you want to visit a friend, get on a plane, or go see a movie, there is no highly accurate, instant test that can tell you right then and there whether or not you have a SARS-CoV-2 infection. But new research from Lawrence Berkeley National Laboratory (Berkeley Lab) could help get reliable instant tests on the market.

A study led by Michal Hammel and Curtis D. Hodge suggests that a highly sensitive lateral flow assay – the same type of device used in home pregnancy tests – could be developed using pairs of rigid antibodies that bind to the SARS-CoV-2 nucleocapsid protein. Such a test would only require a small drop of mucus or saliva, could give results in 15 minutes, and could detect a COVID-19 infection one day before the onset of symptoms. Their work was published in the journal mABs.

The current gold standard tests for COVID-19 use a form of polymerase chain reaction (PCR) to identify the presence of SARS-CoV-2 nucleic acid (RNA) rather than a viral protein. They are quite accurate, with false negative rates ranging less then 5% (depending primarily on the sampling site, sample type, and stage of infection). However, PCR tests must be sent away for analysis at an accredited lab.

Read more on the Berkeley Lab website

Image: Molecular models constructed from the X-ray data show different antibodies bound to the SARS-CoV-2 nucleocapsid protein (pink). The scientists determined that the linear arrangement (right) has higher detection sensitivity than the sandwich arrangement (left).

Credit: Berkeley Lab

Virus recognition skills

2021/04/122021/04/15

A virus recognizes the starting point on the DNA to be packaged inside its protein shell

A bacteriophage – a virus that attacks bacteria – assembles into an infectious species using a powerful nanomachine to stuff its DNA into a protein shell. In several types of phage, this genome packaging motor is composed of several copies of large and small terminase subunits (TerL and TerS, respectively) that attach to a portal into the protein procapsid.

Figure 1. Envelope of NV1 TerS from SAXS data, overlaid with modeled structure with open HTHs. Circle highlights one HTH motif.

The Cingolani group (Thomas Jefferson U) has now determined the structure of TerS from the Pseudomonas phage PaP3. Phage DNA to be packaged contains multiple copies of the genome, but just one copy is needed to fill a procapsid. Terminases attempt to package this one copy by various methods; in PaP3 a termination signal is provided by the interaction of a specific sequence in the DNA (the cos sequence) with TerS.

A crystal structure of PaP3 TerS reveals a nonameric ring of mixed alpha/beta composition, sitting atop a 9-stranded beta-barrel. Projecting out from the ring are spokes tipped with helix-turn-helix (HTH) DNA-binding domains. In the crystal, with no DNA present, the HTH domains are packed tightly against the inner parts of the nonamer (a “closed” form). Crystals of TerS from the related NV1 phage were also studied; their quality was not as good but the same conformation was found. BioSAXS coupled to size-exclusion chromatography, at CHESS, was then used to examine the PaP3 TerS structure, and that of the related NV1 protein, in solution. Both turned out to be ~25% larger than predicted from the crystal structure. The molecular envelope determined from SAXS data for NV1 clearly showed protuberances on the outside of the nonameric ring that did not match the crystal structure. However, by rotating the HTH domain of each monomer about an obvious hinge region, an “open” model could be built that fit the SAXS envelope well (Figure 1).

Unravelling the molecular structure, self-assembly, and properties of a cephalopod protein variant

2021/04/062021/05/06

Cephalopods, such as the loliginid in Figure 1A, are known for their remarkable ability to rapidly change the color and appearance of their skin. These capabilities are enabled in part by unique structural proteins called reflectins, which play essential roles in optical behavior of cephalopod skin cells. Moreover, reflectins have demonstrated exciting potential as functional materials within the context of biophotonic and bioelectronic systems. Given reflectins’ demonstrated significance from both fundamental biology and applications perspectives, some research effort has been devoted to resolving their three-dimensional (3D) structures. However, the peculiar sequence composition of reflectins has made them extremely sensitive to subtle changes in environmental conditions and prone to aggregation, thus significantly complicating the study of their structure-function relationships and precluding their definitive molecular-level structural characterization. In this work, we have elucidated the structure of a reflectin variant at the molecular level, demonstrated a robust methodology for controlling its assembly and optical properties.

We began our studies by rationally selecting a prototypical reflectin variant (RfA1TV) by using a bioinformatics-guided approach (Figure 1B). Next, we not only produced the variant in high yield and purity but also optimized conditions for maintaining this protein in a monomeric state (Figure 1C). We then probed the protein with small angle X-ray scattering (SAXS) using the Austrian SAXS beamline at the Elettra Synchrotron Laboratory in Trieste, Italy. For this purpose, a well-dispersed solution of RfA1TV was prepared in a low-pH buffer and transferred into a glass capillary, which was positioned in the path of an incident X-ray beam. The X-rays scattered by the solution-borne RfA1TV molecules formed a 2-D pattern on a Pilatus3 1M detector (Figure 1D). Subsequently, radial averaging and image calibration of the two-dimensional data furnished corresponding one-dimensional curves, which were further processed, analyzed, and correlated with other experiments to obtain insight into the protein’s geometry (Figure 1E).

Read more on the Elettra website

Image: (A) A camera image of a Doryteuthis pealeii squid. (B) An illustration of the selection of the prototypical truncated reflectin variant (RfA1TV) from full-length Doryteuthis pealeii reflectin A1. (C) A digital camera image of a solution of primarily monomeric RfA1TV (Upper) and a corresponding cartoon of RfA1TV monomers (Lower Inset). (D) An illustration of the SAXS analysis of the reflectin variant, wherein incident X-rays are scattered by the solution-borne proteins to furnish a corresponding scattering pattern. (E)The 3D structure of RfA1TV (random coils – gray, helices – orange, β-strands – purple).

Credit: This figure has been adapted from M. J. Umerani*, P. Pratakshya* et al.Proc. Natl. Acad. Sci. U.S.A 117, 32891-32901 (2020).

Blood disorder mechanism discovered

2021/01/192021/03/18

G6PD deficiency affects about 400M people worldwide and can pose serious health risks. Uncovering the causes of the most severe cases could finally lead to treatments.

With a name like glucose-6-phosphate dehydrogenase deficiency, one would think it is a rare and obscure medical condition, but that’s far from the truth. Roughly 400 million people worldwide live with potential of blood disorders due to the enzyme deficiency. While some people are asymptomatic, others suffer from jaundice, ruptured red blood cells and, in the worst cases, kidney failure.

Now, a team led by researchers at the Department of Energy’s SLAC National Accelerator Laboratory has uncovered the elusive mechanism behind the most severe cases of the disease: a broken chain of amino acids that warps the shape of the condition’s namesake protein, G6PD. The team, led by SLAC Professor Soichi Wakatsuki, report their findings January 18th in Proceedings of the National Academy of Sciences.

Read more on the SLAC website

Image: The G6PD enzyme plays a crucial role in red blood cells, removing molecules such as hydrogen peroxide from the body. In some cases, mutations can bend the molecule awkwardly, interfering with G6PD’s function. In the worst cases, the mutations lead red blood cells to rupture.

Credit: Mio Wakatsuki, from protein images by Naoki Horikoshi/SLAC National Accelerator Laboratory

How a very “sociable” protein can hold clues about Alzheimer’s origin

2020/12/162020/12/17

The origin of the most prevalent form of Alzheimer’s disease, which accounts for 95% of cases, is still not clear despite decades of scientific studies. “Before understanding the pathology, we need to understand the biology”, explains Montse Soler López, scientist leading research on Alzheimer’s disease at the ESRF. “The only thing we are sure about is that the most common form of Alzheimer’s is linked with ageing”, she asserts.

So researchers have been focusing on parts of the body that degrade dramatically with age. Neurons, for example, are long-lived cells, meaning that they don’t renew themselves like other cells do. Neurons lodge mitochondria, which are so-called the “powerhouse of cell” because of their active role generating energy in the body. With time, mitochondria suffer oxidative stress and this leads to their malfunction. It has been recently discovered that people with Alzheimer’s may have an accumulation of amyloids inside mitochondria (previously it was thought amyloids were only outside the neurons). Montse Soler López is trying to find whether there is a link between mitochondrial dysfunction, presence of amyloids and early disease symptoms. “We believe that malfunctioning of the mitochondria can take place 20 years before the person shows symptoms of the disease”.

Read more on the ESRF website