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.

ALS passes the 7000-protein milestone

The eight structural biology beamlines at the ALS have now collectively deposited over 7000 proteins into the Protein Data Bank (PDB), a worldwide, open-access repository of protein structures. The 7000th ALS protein structure (entry no. 6C7C) is an enzyme from Mycobacterium ulcerans (strain Agy99), solved with data from Beamline 5.0.2. This bacterium produces a toxin that eats away at skin tissue, causing what’s known as Buruli ulcers (Google at your own risk!). The bacterium is antibiotic-resistant, and treatment involves the surgical removal of infected tissues, including amputation.

The enzyme structure was solved by a group from the Seattle Structural Genomics Center for Infectious Disease (SSGID), whose mission is to obtain crystal structures of potential drug targets on the priority pathogen list of the National Institute of Allergy and Infectious Diseases (NIAID). As of May 2018, SSGCID has deposited 1090 structures in the PDB, with data for more than a quarter of those collected at ALS beamlines.

>Read more on the Advanced Light Source website

Image: PDB 6C7C: Enoyl-CoA hydratase, an enzyme from M. ulcerans (strain Agy99).

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

X-ray laser opens new view on Alzheimer proteins

Graphene enables structural analysis of naturally occurring amyloids

A new experimental method permits the X-ray analysis of amyloids, a class of large, filamentous biomolecules which are an important hallmark of diseases such as Alzheimer’s and Parkinson’s. An international team of researchers headed by DESY scientists has used a powerful X-ray laser to gain insights into the structure of different amyloid samples. The X-ray scattering from amyloid fibrils give patterns somewhat similar to those obtained by Rosalind Franklin from DNA in 1952, which led to the discovery of the well-known structure, the double helix. The X-ray laser, trillions of times more intense than Franklin’s X-ray tube, opens up the ability to examine individual amyloid fibrils, the constituents of amyloid filaments. With such powerful X-ray beams any extraneous material can overwhelm the signal from the invisibly small fibril sample. Ultrathin carbon film – graphene – solved this problem to allow extremely sensitive patterns to be recorded. This marks an important step towards studying individual molecules using X-ray lasers, a goal that structural biologists have long been pursuing. The scientists present their new technique in the journal Nature Communications.

Amyloids are long, ordered strands of proteins which consist of thousands of identical subunits. While amyloids are believed to play a major role in the development of neurodegenerative diseases, recently more and more functional amyloid forms have been identified. “The ‘feel-good hormone’ endorphin, for example, can form amyloid fibrils in the pituitary gland. They dissolve into individual molecules when the acidity of their surroundings changes, after which these molecules can fulfil their purpose in the body,” explains DESY’s Carolin Seuring, a scientist at the Center for Free-Electron Laser Science (CFEL) and the principal author of the paper. “Other amyloid proteins, such as those found in post-mortem brains of patients suffering from Alzheimer’s, accumulate as amyloid fibrils in the brain, and cannot be broken down and therefore impair brain function in the long term.”

Image: On the ultra-thin, extremely regular layer of graphene, the fibrils align themselves in parallel in large domains. The intense X-ray light from the X-rax free-electron laser LCLS at the SLAC National Accelerator Center enabled the researchers to gain partial information about the fibril structure from ensembles of just a few fibrils.
Credit: Greg Stewart/SLAC National Accelerator Laboratory

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.

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)

Structural Mechanisms of Histone Recognition by Histone Chaperones

Chromatin is the complex of DNA and proteins that comprises the physiological form of the genome. Non-covalent interactions between DNA and histone proteins are necessary to compact large eukaryotic genomes into relatively small cell nuclei. The nucleosome is the fundamental repeating unit of chromatin, and is composed of 147bp of DNA wrapped around an octamer of histone proteins: 2 copies of each H2A, H2B, H3 and H4.

Assembly of nucleosomes in the cell requires the coordinated effort of many proteins including ATP-dependent chromatin remodeling enzymes and ATP-independent histone chaperone proteins. Histone chaperones are a large class of proteins responsible for binding the highly basic histone proteins, shielding them from non-specific interactions, facilitating nuclear import of histones, and finally depositing histones onto DNA to form nucleosomes. Despite performing many overlapping functions, histone chaperone proteins are highly structurally divergent. However, nearly all histone chaperones contain highly charged intrinsically disordered regions (IDRs)1. In many cases truncation of these conserved regions results in loss of histone affinity and deposition functions.

>Read more on the Stanford Synchrotron Radiation Lightsource

Image: (extract) SAXS analysis of Npm Core+A2 truncation (1-145) bound to five H2A/H2B dimers. Left: small angle x-ray scattering curve of the complex (purple dots). Simulated SAXS curve from the best scoring structural model shown as a black line. Right: SAXS envelope of the complex (pink) with the best scoring structural model inside. Positioning of H2A/H2B dimers by NMR and SAXS structural restraints. Full image here.