How legionella manipulates the host cell by means of molecular mimics

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

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.

>Read more on the MAX IV Laboratory website

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

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.

>Read more on the SwissFEL at Paul Scherrer Institute website

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

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.

>Read more on the Canadian Light Source website

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

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.

Respiratory virus study points to likely vaccine target

The proteins that bind

Researchers reveal the structure of a protein that helps bacteria aggregate

Serine-rich repeat proteins (SRRPs), which help bacteria attach to surfaces, have been structurally characterised in pathogenic bacteria but not in beneficial bacteria such as those present in the gut. Dr Nathalie Juge’s team at the Quadram Institute Bioscience has previously identified SRRP as a main adhesin in Lactobacillus reuteri strains from pigs and mice. Now, together with colleagues at the University of East Anglia, they have described the structure and activity of the binding region of L. reuteri SRRPs in a paper published in PNAS. Using the Macromolecular Crystallography beamlines (I03 and I04) at Diamond Light Source, they discovered that the structure of these proteins is unique among characterised SRRPs and is surprisingly similar to pectin degrading enzymes. Molecular simulations and binding experiments revealed a pH-dependent binding to pectin and to proteins from the epithelium known as mucins. Altogether, these findings shed light on the activity of a key protein in these bacteria and may help guide the development of more targeted probiotic interventions.

>Read more on the Diamond Light Source website

Figure: (Left) Cartoon representation of crystal structures of the binding region of SRRP53608. (Right) Cartoon representation of crystal structures of the binding region of SRRP100-23. The N-terminus is shown with blue balls and the C-terminus is shown with red balls.

Fighting malaria with X-rays

Today 25 April, is World Malaria Day.

Considered as one of humanity’s oldest life-threatening diseases, nearly half the world population is at risk, with 216 million people affected in 91 countries worldwide in 2016. Malaria causes 445 000 deaths every year, mainly among children. The ESRF has been involved in research into Malaria since 2005, with different techniques being used in the quest to find ways to prevent or cure the disease.

Malaria in humans is caused by Plasmodium parasites, the greatest threat coming from two species: P. falciparum and P. vivax. The parasites are introduced through the bites of infected female Anopheles mosquitoes. They travel to the liver where they multiply, producing thousands of new parasites. These enter the blood stream and invade red blood cells, where they feed on hemoglobin (Hgb) in order to grow and multiply. After creating up to 20 new parasites, the red blood cells burst, releasing daughter parasites ready for new invasions. This life cycle leads to an exponential growth of infected red blood cells that may cause the death of the human host.

The research carried out over the years at the ESRF has aimed to identify mechanisms critical for the parasite’s survival in the hope of providing an intelligent basis for the development of drugs to stop the parasite’s multiplication and spread.

>Read more on the European Synchrotron website

Image: Inside the experimental hutch of the ESRF’s ID16A nano-analysis beamlin.
Credit: Pierre Jayet

Solution to plastic pollution on the horizon

Engineering a unique plastic-degrading enzyme

The inner workings of a recently discovered bacterium with a fascinating ability to use plastic as an energy source have been recently revealed in PNAS. The world’s unique Long-Wavelength Macromolecular Crystallography (MX) beamline here at Diamond Light Source was used to successfully solve the structure of the bacterial enzyme responsible for chopping up the plastic. This newly evolved enzyme could be the key to tackling the worldwide problem of plastic waste.

Plastic pollution is a global threat that desperately needs addressing. Plastics are rarely biodegradable and they can remain in the environment for centuries. One of the most abundant plastics that contributes hugely to this dire situation is poly(ethylene terephthalate) (PET).

PET is used largely in textiles, where it is commonly referred to as polyester, but it is also used as packaging for liquids and foodstuffs. In fact, PET’s excellent water-repellent properties led to it being the plastic of choice for soft drink bottles. However, once plastic bottles are discarded in the environment the water resistance of PET means that they are highly resistant to natural biodegradation. PET bottles can linger for hundreds of years and plastic waste like this will accumulate over time unless a solution is found to degrade them.

A recent breakthrough came in the discovery of a unique bacterium, Ideonella sakaiensis 201-F6, which was found feeding on waste from an industrial PET recycling facility. PET has only been widely used since the 1970s, so the bacterium had evolved at breakneck speed to be able to take advantage of the new food source.

The bacterium had the amazing ability to degrade PET and use it to provide carbon for energy. Central to this ability was the production of a PET-digesting enzyme, known as PETase.

>Read more on the Diamond Light Source website


Serial crystallography develops by leaps and bounds at the ESRF

Serial crystallography is a new way of studying macromolecular structures using synchrotron and X-FEL sources around the world.

The Structural Biology group at the ESRF is continuously developing new methods to advance the field. Two articles describing advances made are published today in Acta Crystallographica Section D.

“On the Structural Biology Group beamlines one of the ultimate aims is that users can define protocols for experiments, click ‘go’ and let the experiments run by themselves”, explains Gordon Leonard, head of the Structural Biology group at the ESRF. With this idea in mind and to get as much information as possible from the samples available, the team has already adopted serial crystallography, a technique which involves taking diffraction data from many, sometimes hundreds or thousands, of crystals in order to assemble a complete dataset, piece by piece. Indeed, the members of the group are constantly developing new ways to improve the method through collaboration involving scientists from the ESRF, DESY, the Hamburg Centre for Ultrafast Imaging, the European X-FEL and the University of Hamburg.

>Read more on the European Synchrotron website

Image: Daniele de Sanctis on the ID29 beamline.
Credit: S. Candé.

Discovery of the novel green fluorescent protein by NSRRC

Scientist Dr. Chun-Jung Chen and Research Assistant Mr. Yen-Chieh Huang of the National Synchrotron Radiation Research Center collaborated with the researchers at the University of the Philippines – Diliman to analyze the three-dimensional structure and functional characteristics of the novel green fluorescent protein asFP504 isolated from a soft coral species, Alcyonium sp. found at the Taklong Island, Guimaras, Philippines. The results of the study were published and selected as the cover story on the Philippine Journal of Science in March, which is considered as one of the representative research results of the Southward Policy of NSRRC.

>Read more on the National Synchrotron Radiation Research Center (NSSRC) website

Image: Extract of the cover on the Philippine Journal of Science (2018.03)

Supporting World Cancer Day 2018

Diamond is proud to be supporting World Cancer Day and highlighting our role, working with our user community, in pioneering synchrotron research in every area of cancer – from developing a better understanding of how cancer cells work to delivering new cancer therapies.
Despite major advances in diagnosis and treatment, cancer still claims the lives of 8.8 million people every year around the world. About 4 million of these die prematurely (under the age of 70). World Cancer Day aims to raise the awareness of cancer and its treatment around the world. With the tagline ‘We can. I can.’, World Cancer Day is exploring how everyone can play their part in reducing the global burden of cancer.

Diamond has published over 900 publications in the last 12 months, with around 10% of these focusing on cancer. The wide-ranging research currently covers at least 12 cancer types, with many more general studies on the structure of cancer cells and pathways, potential drug targets and possible drug candidates. Building on last year’s review of some of the key studies in cancer that have taken place at Diamond, here is an update on studies that have been published in the last 12 months.

>Read more on the Diamond Light Source website


ID23-EH2: Gearing up for serial crystallography

ID23-EH2 is up and running, catering to small samples and serial crystallography experiments. Its small beam and unique diffractometer are the trademarks of this new MX beamline.

“This is amazing”, says David Drew, a user from Stockholm University, on the new ID23-EH2. “There is a perfect beam line to be screening LCP crystals. After 5 years working on this… it is amazing to be able to speed up finding the best spot to collect”, he adds. Drew and his team are on ID23-EH2. They are the first users since ID23-EH2 opened for business this month and have just started the experiment. He works with his team in transport proteins, which carry nutrients across membrane proteins and are important drug targets. 

>Read more on the ESRF website

Picture: Max Nanao with the users from the University of Stockholm (Sweden).


The search for an Ebola vaccine

Researchers expertly solved the crystal structures of drugs bound to the outer coating of the Ebola virus to pinpoint the regions that are essential for inhibitory activity.

Ebola is a viral disease that is highly infectious and associated with a high risk of death. It first arose in 1976, from which point it was associated with dozens of small-scale outbreaks; however, in 2013 Ebola was responsible for a huge epidemic in West Africa. Emergency was declared and over 11,000 people lost their lives to the virus. Despite this horrific state of affairs, Ebola still remains an untreatable disease and there is no vaccine to prevent infection.

>Read more on the Diamond Light Source website


Serial microcrystallography at CHESS

What if large crystals are not available?

The standard X-ray protein crystallography experiment requires a single protein crystal specimen that is large enough to collect a “complete” data set, that is, to collect all the available diffraction peaks to a given resolution.

But what if large crystals are not available? A team of scientists at MacCHESS and the University of Toronto is pushing what is possible for small protein crystals at storage ring sources.

While structural biologists have expanded their purview to increasingly large and complex biological systems, the necessity for reliable, atomic resolution structural data for those systems has not changed. However, it is simply not possible to grow sufficiently large crystals for many systems. The necessity of large crystals in protein crystallography stems primarily from two factors. First, all other things being equal, microcrystals diffract more weakly than large ones, because the crystal volume, and thus number of protein molecules diffracting the X-rays, is lessened. Second, and more insidiously, protein microcrystals succumb more quickly to radiation damage – a loss of diffraction intensity resulting from X-ray induced, stochastic ionization and bond cleavage. These factors result in apparently contradictory solutions: increase the beam intensity to induce more diffraction, but at the expense of crystal lifetime; or lower the beam intensity, but collect weak data.

>Read more on the CHESS website

Image caption: The sample chip loaded and placed on the piezo stage.

Crystallographers identify 1,000 protein structures

The Canadian Light Source is celebrating two milestones reached by scientists who have conducted research at the national facility at the University of Saskatchewan.

Scientists have solved 1,000 protein structures using data collected at CLS’s CMCF beamlines. These have been added to the Protein Data Bank – a collection of structures solved by researchers globally. Researchers have also published 500 scientific papers based on their work using the crystallography beamlines.

Proteins are the building blocks of life and are described as the body’s workhorses. The body is made of trillions of cells. Cells produce proteins, which do the work of breaking down food, sending messages to other cells, and fighting bacteria, viruses and parasites. The discoveries at the CLS range from how the malaria parasite invades red blood cells to why superbugs are resistant to certain antibiotics and how parkin protein mutations result in some types of Parkinson’s disease. Understanding how these and other such proteins work can potentially save millions of lives.

>Read more on the Canadian Light Source website

Image: PDB ID: 6B0S