crystallography – Page 2 – Lightsources.org

Natural defense against red tide toxin found in bullfrogs

2019/06/262019/06/27

A team led by Berkeley Lab faculty biochemist Daniel Minor has discovered how a protein produced by bullfrogs binds to and inhibits the action of saxitoxin, the deadly neurotoxin made by cyanobacteria and dinoflagellates that causes paralytic shellfish poisoning.
The findings, published this week in Science Advances, could lead to the first-ever antidote for the compound, which blocks nerve signaling in animal muscles, causing death by asphyxiation when consumed in sufficient quantities.
“Saxitoxin is among the most lethal natural poisons and is the only marine toxin that has been declared a chemical weapon,” said Minor, who is also a professor at the UCSF Cardiovascular Research Institute. About one thousand times more potent than cyanide, saxitoxin accumulates in tissues and can therefore work its way up the food chain – from the shellfish that eat the microbes to fish, turtles, marine mammals, and us.

>Read more on the ALS website

Image: A photo illustration showing the atomic structures of saxiphilin and saxitoxin, a red tide algal bloom, and an American bullfrog (R. catesbeiana).
Credit: Daniel L. Minor, Jr., and Deborah Stalford/Berkeley Lab.

Study offers new target for antibiotic resistant bacteria

2019/06/182019/06/24

As antibiotic resistance rises, the search for new antibiotic strategies has become imperative. In 2013, the Centers for Disease Control estimated that antibiotic resistant bacteria cause at least 2 million infections and 23,000 deaths a year in the U.S.; a recent report raised the likely mortality rate to 162,044.
New Cornell research on an enzyme in bacteria essential to making DNA offers a new pathway for targeting pathogens. In “Convergent Allostery in Ribonucleotide Reductase,” published June 14 in Nature Communications, researchers used the MacCHESS research stations at the Cornell High Energy Synchrotron Source (CHESS) to reveal an unexpected mechanism of activation and inactivation in the protein ribonucleotide reductase (RNR).

Understanding the “switch” that turns RNR off provides a possible means to shut off the reproduction of harmful bacteria.
RNRs take ribonucleotides, the building blocks of RNA, and convert them to deoxyribonucleotides, the building blocks of DNA. In all organisms, the regulation of RNRs involves complex mechanisms, and for good reason: These mechanisms prevent errors and dangerous mutations.

>Read more on the CHESS website

Image: William Thomas, a graduate student in the field of chemistry and chemical biology, collects data on ribonucleotide reductase.

Scienstists make breakthrough in creating universal blood type

2019/06/102019/06/13

Enzymes in the human gut can convert A blood type into O.

Half of all Canadians will either need blood or know someone who needs it in their lifetime. Researchers from the University of British Columbia have made a breakthrough in their technique for converting A and B type blood into universal O, the type that is most needed by blood services and hospitals because anyone can receive it.
In a paper published in Nature Microbiology, Stephen Withers and a multidisciplinary team of researchers from the University of British Columbia show how they successfully converted a whole unit of A type blood to O type using their system. They were able to remove the sugars from the surface of the red blood cells with help from a pair of enzymes that were isolated from the gut microbiome of an AB+ donor.
The Canadian Light Source (CLS) at the University of Saskatchewan (UofS) played a critical role in understanding the structure of a previously unknown enzyme that was part of this pair. The researchers were unable to identify what this unique enzyme looked like from the gene sequence they had. Crystallography, done at the CLS, was crucial for the researchers to understand how this enzyme works and why it had a particular affinity for the A type blood.

Potassium hunting on protein factories

2019/06/072019/06/17

Amazing insights into the location of elusive potassium ions on bacterial ribosomes

Groundbreaking research at the new long-wavelength macromolecular crystallography beamline (I23) at Diamond Light Source has for the first time demonstrated the location of potassium ions in bacterial ribosomes. Ribosomes are the protein factories of cells and although they are vital for life, little was known of the sites of metal ions that are crucial for their structure and function. The work recently published in Nature Communications showcases the fantastic applications of the I23 beamline and sheds light on the important role of potassium ions.

Image: (extract, full image here) 70S ribosome elongation complex (potassium atoms rendered as green spheres).

Understanding the viruses that kill cancer cells

2019/04/122019/04/16

Taking inspiration from virology to find better treatments for cancer

There are some viruses, called oncolytic viruses, that can be trained to target and kill cancer cells. Scientists in the field of oncolytics want to engineer these viruses to make them safer and more effective so they can be used to treat more people and different types of cancers. To achieve this, they first have to fully understand at the molecular level all the different ways that the virus has evolved to infect healthy cells and cause disease. A research team from Cardiff University set out to better understand how a protein on the surface of a virus often used to kill cancer, called an adenovirus, binds to human cells to cause an infection. Using X-ray crystallography, the team was able to determine the structure of one the key adenovirus proteins. Using this information and after extensive computational analysis, the research team realised the virus was not binding the receptor on the cells that was originally thought. This has important implications for the development of new virotherapies and engineering of viruses to treat cancer. The more thoroughly the researchers can understand how the adenoviruses interact with cancer cells at the molecular level, the more safe and effective treatments can be brought to clinical trial in the future.

Doubling the DNA alphabet

2019/03/292019/04/16

Implications for life in the universe and DNA storage

Life on Earth is dictated by the DNA alphabet comprised of only four DNA bases or letters: A, T, G and C. It has long been of interest to understand whether there is something very special about the four letters that comprise DNA and whether this is the only code that could support life. At a basic level, this question can be addressed by examining an expanded alphabet and determining the properties of DNA including additional synthetic letters. This study impacts our current understanding of terrestrial DNA and suggests that extraterrestrial life forms could have evolved using a different genetic code than found here on Earth. The work has immediate applications in synthetic biology for the creation of new molecules and greatly expands the ability to store information in DNA.

Now, in breakthrough work, funded by NASA, NSF and NIGMS, Dr. Steven Benner at the Foundation for Applied Molecular Evolution, in collaboration with Dr. Millie Georgiadis at the Indiana University School of Medicine, and colleagues at biotechnology companies and other universities, have provided evidence that the standard DNA code can be expanded to include eight letters forming “hachimoji DNA” (“hachi” eight and “moji” letter in Japanese) using four novel synthetic nucleobases (B, S, P and Z) in addition to A, T, C and G and still retain critical features of natural DNA^1,2. Structurally, hachimoji DNA can adopt a standard double helical form of DNA and retain Watson-Crick complementary base pairing, which allows the expanded DNA to be faithfully replicated and transcribed by polymerases to produce hachimoji DNA copies and hachimoji RNA. These properties are essential for a genetic system that can support life.

Image: Crystal structure of a double helix built from eight hachimoji building blocks, G (green), A (red), C (dark blue), T (yellow), B (cyan), S (pink), P (purple), and Z (orange). The first four building blocks are found in human DNA; the last four are synthetic, and possibly present in alien life. Each strand of the double helix has the sequence CTTAPCBTASGZTAAG. Notable is the geometric regularity of the pairs, a regularity that is needed for evolution.

Low background noise crucial for single particle imaging experiments

2019/03/192019/03/26

Model experiment brings scientists a step closer to SPI at European XFEL

Taking snapshots of single molecules with X-rays has long been a dream for many scientists. Such experiments have successfully been computationally modelled, but have never been practically demonstrated before.
In a model experiment carried out at the European Synchrotron Radiation Facility (ESRF), European XFEL scientists, together with international collaborators, have now come one step closer to successfully carrying out so-called single particle imaging experiments (SPI) at X-ray laser facilities such as European XFEL. In a paper published today in the journal from the International Union of Crystallography (IUCrJ), scientists demonstrate experimentally that, in principle, a 3D structure can indeed be obtained from many tens of thousands of very weak images, using X-rays with similar properties as produced at X-ray free-electron lasers such as European XFEL.

>Read more on the European XFEL website

Image: Reconstruction of the 3D electron density. (a) Reconstruction from the result derived by EMC. The electron density projected along an axis perpendicular to the drawing plane is shown here. (b) Reconstruction from the reference Fourier volume. Again, the projected electron density is shown. (c) 3D iso-surface rendering of the reconstructed electron density shown in panel (a). The threshold of the iso-surface has been set to 0.2, given a normalized density with values between 0 and 1. (d) Scanning electron micrograph from the original sample.
Image source

Structural insights into tiny bacterial harpoons

2019/02/282019/03/07

Bacteria produce complex nano-harpoons on their cell surface. One of their functions is to harpoon and inject toxins into cells that are close by. Producing such a complex weapon requires lots of different moving components that scientists are still trying to understand. Researchers from the University of Sheffield have been using some of Diamond’s crystallography beamlines to understand a particularly enigmatic piece of this tiny puzzle. The team led by David Rice and Mark Thomas worked on a protein component of the harpoon called TssA which they already knew was an integral piece of the machinery. However, unlike the other components of the harpoon, there are distinct variants of the TssA protein that contain radically different amino acid sequences at one end of the protein. The team showed that the structures of the variable region of two different TssA subunits were completely unrelated and they could assemble into distinctly different multisubunit complexes in terms of their size and geometry. This begged the question as to how different bacteria could use this protein with different structures to produce a harpoon with the same function across all species. They found that despite these differences, there was a very specific conserved region at the other end of the protein. They hypothesise that the conserved region is the part that does the work and helps the harpoon to function whereas the variable region acts as a scaffold. They used I02, I03 and I24 in their study and plan to do follow up work using X-ray crystallography and Cryo-EM such as those at the eBIC centre at Diamond. The research was published in Nature Communications.

Image: Macromolecular Crystallography (MX) at Diamond reveals the shape and arrangement of biological molecules at atomic resolution, knowledge of which provides a highly accurate insight into function.

First users on VMXm

2018/11/202018/11/22

First users from the University of Southampton investigated proteins involved in nutrient uptake of photosynthetic or cyanobacteria to understand how these phytoplankton thrive under scarce nutrient conditions.

The work has immense global significance for biofuels production and biotechnology. This beamline marks the completion of Diamond’s original Phase III funding on time and within budget.

First users have now been welcomed by Diamond Light Source, the UK’s national synchrotron light source on its new VMXm beamline. The Versatile Macromolecular Crystallography micro/nanofocus (VMXm) beamline becomes the 32nd operational beamline to open its doors to users, completing the portfolio of seven beamlines dedicated to macromolecular crystallography.
The unique VMXm beamline represents a significant landmark for Diamond. It is a specialist tuneable micro/nanofocus macromolecular crystallography (MX) beamline, with an X-ray beam size of less than 0.5 microns, allowing even the tiniest of samples to be analysed. Integrated into the ‘in vacuum’ sample environment is a scanning electron microscope, making VMXm a hybrid X-ray/cryoEM instrument for detecting and measuring data from nanocrystals. VMXm is aimed at research applications where the production of significant quantities of protein and crystals is difficult.

Image: Principal Beamline Scientist Dr Gwyndaf Evans with his team Dr Jose Trincao, Dr Anna Warren, Dr Emma Beale and Dr Adam Crawshaw. First users – Dr Ivo Tews from Biological Sciences at the University of Southampton and joint Diamond-Southampton PhD student Rachel Bolton investigating proteins involved in nutrient uptake of photosynthetic or cyanobacteria.

Snaphot of molecular mechanism at work in lethal virus

2018/09/13

X-ray crystallography at the Australian Synchrotron contributed to major research findings.

Data collected on the macromolecular crystallography beamlines at the Australian Synchrotron has contributed to major research findings on two deadly viruses, Hendra and Nipah, found in Australia, Asia and Africa. The viruses can be transmitted to humans not directly by the bat which is the natural carrier but by an infected animal like horses or pigs.

Beamline scientist, Dr David Aragao (pictured above), a co-author on the paper in Nature Communications, said that obtaining a clear motion picture of key biological process at the molecular level of viruses is often not available with current biomedical techniques.
“However, using X-ray crystallography from data collected on both MX1 and MX2 beamlines at the Australian Synchrotron, we were able to obtain 8 ‘photograph-like’ snapshots of the molecular process that allows the Hendra and Nipah virus to replicate.“

Two authors of the paper, PhD students Kate Smith and Sofiya Tsimbalyuk, who are co-supervised by Aragao and his collaborator Professor of Biochemistry Jade Forwood of the Graham Centre for Agricultural Innovation Charles Sturt University, used the Synchrotron extensively collecting multiple data sets that required extensive refinements over two years to isolate the mechanism of interest.

Image: Beamline scientist, Dr David Aragao.

Structure reveals mechanism behind periodic paralysis

2018/08/272018/08/30

The results suggest possible drug designs that could provide relief to patients with a genetic disorder that causes them to be overcome suddenly with profound muscle weakness.

A rare genetic disorder called hypokalemic periodic paralysis (hypoPP) causes sudden, profound muscle weakness in people who occasionally exhibit low levels of potassium in their blood, or hypokalemia. When a patient is hypokalemic, hypoPP affects the function of the muscles responsible for skeletal movement. The disease has been known to stem from mutations in certain membrane proteins that channel and regulate the flow of sodium into cells. Exactly how the mutation affects the proteins’ function, however, was not known.

In earlier work, researchers from the Catterall Lab at the University of Washington had solved the structure of a sodium channel called Na_vAb from a prokaryote (single-celled organism). As a next step, the group decided to see if Na_vAb could serve as a model for studying the mutations that cause hypoPP in humans (eukaryotes), with the goal of finding a way to prevent or treat this disorder.

A leak in the pipe?

In a resting state, muscle-cell membranes keep potassium ions and sodium ions separated, inside and outside the cell, respectively, creating a voltage across the membrane. A chemical signal from a nerve cell sets off a cascade of events that results in sodium ions flowing into the cell, changing the membrane potential and and ultimately triggering muscle contraction.

Image: Three states of the voltage-sensing domain (VSD) of a membrane-channel protein. In the normal state, the water-accessible space (magenta) does not extend through the channel, preventing sodium (gray spheres) from passing through. In the disease state, a clear passage allows sodium to leak through, resulting in muscle paralysis. In the “rescued” state, the binding of guanidinium (blue and yellow spheres) effectively closes the channel and blocks sodium leakage. The red sphere represents the location of the disease-causing mutation. The side-chain sticks represent the voltage sensors of the sodium channel.

How legionella manipulates the host cell by means of molecular mimics

2018/07/062018/07/11

Using synchrotron light, researchers from CIC bioGUNE have solved the structure of RavN, a protein that Legionella pneumophila uses for stealing functions and resources of the host cell.

Mimicry is the ability of some animals to resemble others in their environment to ensure their survival. A classic example is the stick bug whose shape and colour make him unnoticed to possible predators. Many intracellular pathogens also use molecular mimicry to ensure their survival. A part of a protein of the pathogen resembles another protein totally different from the host and many intracellular microorganisms use this capability to interfere in cellular processes that enable their survival and replication.

The Membrane Trafficking laboratory of the CIC bioGUNE in the Basque Country, led by Aitor Hierro, in collaboration with other groups from the National Institutes of Health in the United States, have been working for several years in understanding how the infectious bacterium Legionella pneumhopila interacts with human cells. During this research, experiments have been carried out at the XALOC beamline of the ALBA Synchrotron and I04 beamline of Diamond Light Source (UK). The results enabled scientists to solve the structure of RavN, a protein of L. pneumophila that uses this molecular mimicry to trick the infected cell.

>Read more on the ALBA website

Figure: (extract) Schematic representation of the structure of RavN1-123 as ribbon diagram displayed in two orientations (rotated by 90° along the x axis). Secondary elements are indicated as spirals (helices) or arrows (beta strands), with the RING/U-box motif colored in orange and the C-terminal structure colored in slate. (Full image here)

First serial crystallography experiments performed at BioMAX

2018/07/032018/07/11

BioMAX has successfully performed the first serial crystallography experiments at the beamline. This new method is performed at room temperature which allows structural biologists to study their molecules at more biologically relevant conditions. The technique can also be used on smaller crystals which will alleviate some of the restrictions for molecules such as membrane proteins, that do not typically form large crystals. Eventually, it is hoped that this technique will allow users at the BioMAX and MicroMAX beamlines to take snapshots of the dynamic states of proteins in rapid succession giving a dynamic view of protein movement and activity.

The serial crystallography technique promises to be very useful to users of both synchrotrons and XFELs. Over the course of one experiment, users were able to measure between 20 and 50 crystals every second, resulting in 20 TB of data from just 3 proteins. BioMAX hopes to quickly master this complex technique in order to offer it to users as soon as possible. It also gives us a glimpse of what will be possible at the newly funded MicroMAX beamline.

Image: BioMAX serial crystallography setup using a High Viscosity Extrusion (HVE) injector specially designed for the BioMAX endstation by Bruce Doak of the Max Planck Institute for Medical Research, Heidelberg, and fabricated at that institute.

Biological light sensor filmed in action

2018/06/142018/06/27

Film shows one of the fastest processes in biology

Using X-ray laser technology, a team led by researchers of the Paul Scherrer Institute PSI has recorded one of the fastest processes in biology. In doing so, they produced a molecular movie that reveals how the light sensor retinal is activated in a protein molecule. Such reactions occur in numerous organisms that use the information or energy content of light – they enable certain bacteria to produce energy through photosynthesis, initiate the process of vision in humans and animals, and regulate adaptations to the circadian rhythm. The movie shows for the first time how a protein efficiently controls the reaction of the embedded light sensor. The images, now published in the journal Science, were captured at the free-electron X-ray laser LCLS at Stanford University in California. Further investigations are planned at SwissFEL, the new free-electron X-ray laser at PSI. Besides the scientists from Switzerland, researchers from Japan, the USA, Germany, Israel, and Sweden took part in this study.

Image: Jörg Standfuss at the injector with which protein crystals for the experiments at the Californian X-ray laser LCLS were tested. In the near future, this technology will also be available at PSI’s X-ray laser SwissFEL, for scientists from all over the world.
Credit: Paul Scherrer Institute/Mahir DzaAmbegovic

Canadian researchers unlock how seaweed is digested

2018/06/142018/06/15

Cattle on the Prairies are hundreds of kilometres from the coast and yet it’s possible that seaweed could make its way into their diet as an additive.

“Seaweed is an incredible opportunity. It is a sustainable feedstock. It grows rapidly, it doesn’t require arable land or fresh water to grow,” said Wade Abbott, research scientist at Agriculture and Agi-Food Canada’s Lethbridge Research and Development Centre.

It may seem like a leap to go from the human gut to that of cattle, but Abbott explained that by understanding the human gut microbiome, or microorganisms, and the microbiome’s ability to use the sugars found in seaweed in its symbiotic relationship with the host, he sees potential to expand what is now a limited use of algae products.

Image: Culturing gut bacteria in the lab (shown in these test tubes) allows researchers ‎to determine which genes in the genomes of bacteria are activated and discover new enzymes that digest rare substrates like agarose.
Credit: Wade Abbott

New high-precision instrument enables rapid measurements of protein crystals

2018/05/292018/05/31

A team of scientists and engineers at the U.S. Department of Energy’s (DOE) Brookhaven National Laboratory have developed a new scientific instrument that enables ultra-precise and high-speed characterization of protein crystals at the National Synchrotron Light Source II (NSLS-II)—a DOE Office of Science User Facility at Brookhaven, which generates high energy x-rays that can be harnessed to probe the protein crystals. Called the FastForward MX goniometer, this advanced instrument will significantly increase the efficiency of protein crystallography by reducing the run time of experiments from hours to minutes.

Protein crystallography is an essential research technique that uses x-ray diffraction for uncovering the 3D structures of proteins and other complex biological molecules, and understanding their function within our cells. Using this knowledge about the basic structure of life, scientists can advance drug design, improve medical treatments, and unravel other environmental and biochemical processes governing our everyday lives.

>Read more on the NSLS-II website

Image: Yuan Gao, Wuxian Shi, Evgeny Nazaretski, Stuart Myers, Weihe Xu and, Martin Fuchs designed and implemented the new goniometer scanner system for ultra-fast and efficient serial protein crystallography at the Frontier Microfocusing Macromolecular Crystallography (FMX) beamline at the National Synchrotron Light Source II.