|The IUCr is an International Scientific Union. Its objectives are to promote international cooperation in crystallography and to contribute to all aspects of crystallography, to promote international publication of crystallographic research, to facilitate standardization of methods, units, nomenclatures and symbols, and to form a focus for the relations of crystallography to other sciences.|
proteins and large protein complexes are notoriously difficult to study with
X-ray crystallography, not least because they are often very difficult, if not
impossible, to crystallize, but also because their very nature means they are
highly flexible. The result is that when a structure can be obtained it is
often of low resolution, ambiguous and reveals a mosaic-like spread of protein
domains that sometimes create more puzzles than they solve. [Schröder, Levitt
& Brunger. (2014), Acta Cryst. D70, 2241-2255; doi: 10.1107/S1399004714016496 ]
Now, Gunnar Schröder of the Institute of Complex Systems at the Forschungszentrum Jülich and the University of Düsseldorf, Germany and colleagues at Stanford University School of Medicine, USA have reviewed their earlier refinement technique known as Deformable Elastic Network (DEN) and found ways to optimize it successfully for the investigation of several particularly problematic protein structures including soluble proteins and membrane proteins up to a resolution limit ranging from 3 to 7Å.
The team explains that advances in X-ray technology and light sources have in
recent years led to structures for previously intractable proteins such as the
ribosome, transcription complexes and even viruses. The details then lie in a
successful refinement that can provide valuable information about the structure
in question despite lower resolution than would normally be desirable.
"The interpretation of low-resolution diffraction data is generally
difficult," the team says, "owing to the unfavorable ratio of
parameters (variable degrees of freedom, such as flexible torsion angles or
Cartesian atomic coordinates) to observables (observed diffraction
intensities)." Ambiguities and errors of interpretation abound.
The DEN approach begins with a model, a prediction, of the target structure containing as much information as is known ahead of the insertion of the diffraction data, and determines which features of the model ought to be adjusted to fit the diffraction data emerging from the X-ray experiments. In other words, a null hypothesis is applied; those parts of the model not predicted to alter the diffraction data are retained as is. Distances between randomly chosen pairs of atoms within the structure are tested and tweaked accordingly within a distance restraint, customarily referred to as the elastic network.
Professor Ian Robinson of the London Centre of Nanotechnology has been awarded the 2015 Gregori Aminoff Prize in Crystallography.
The prize, conferred since 1979 by the Royal Swedish Academy of Sciences – the body that awards the Nobel prizes – recognises a documented, individual contribution in the field of crystallography, including areas concerned with the dynamics of the formation and dissolution of crystal structures. In its citation, the Academy highlighted Professor Robinson’s development of diffraction techniques for the investigation of surfaces and nanomaterials.
Professor Ian Robinson has made a number of pioneering contributions in the field of X-ray diffraction. He is in the forefront when it comes to utilising the opportunities provided by increasingly advanced synchrotron light sources and free-electron lasers in the study of the electronic and structural properties of solids.
During the 1980s, Robinson further developed x-ray diffraction, allowing the study of surfaces. Until that time the standard technique for studying surface structures had been LEED (Low Energy Electron Diffraction), which uses electrons rather than X-rays to create a diffraction pattern. The use of electrons results in great surface sensitivity, whereas X-ray radiation penetrates much further into a material. When the technique of X-ray diffraction could be made sufficiently surface sensitive, it had many advantages. X-ray diffraction can provide more precise results. The ability of X-rays to penetrate further into a material also makes it possible to look inside a reaction cell and study the chemical processes occurring on a catalyst surface in such a cell. Robinson’s development work has been related to both the experimental techniques and the methods used in interpreting the results, and his method is used at a number of the world’s foremost laboratories.
Ian Robinson is also active in the development of new synchrotron radiation-based techniques, which use the high degree of coherence of these light sources, i.e. the fact that the light waves are in phase with each other. Over the last decades, diffraction based methods have been developed that allow detailed three-dimensional mapping of materials – and Robinson is one of the pioneers in this area. He has demonstrated how it is possible to obtain a three-dimensional representation of deformations and defects in nanomaterials. Using the extremely short X-ray pulses from the LCLS (Linac Coherent Light Source) free-electron laser at Stanford, Robinson and his colleagues have also shown how one can excite motion (phonons) of the atoms in individual nanoparticles and follow how these movements propagate in the particles.
The prize will be presented at the Annual meeting of the Royal Swedish Academy of Sciences, 31 March 2015.
You can view a selection of Professor Ian Robinson’s papers here.
The current Ebola virus outbreak in West Africa, which has claimed more than 2000 lives, has highlighted the need for a deeper understanding of the molecular biology of the virus that could be critical in the development of vaccines or antiviral drugs to treat or prevent Ebola hemorrhagic fever. Now, a team at the University of Virginia (UVA), USA – under the leadership of Dr Dan Engel, a virologist, and Dr Zygmunt Derewenda, a structural biologist – has obtained the crystal structure of a key protein involved in Ebola virus replication, the C-terminal domain of the Zaire Ebola virus nucleoprotein (NP) [Dziubanska et al. (2014). Acta Cryst. D70, 2420-2429; doi:10.1107/S1399004714014710].
The team explains that their structure reveals a novel tertiary fold that is expected to lead to insights into how the viral nucleocapsid is assembled in infected cells. The structure could also provide a basis for the design of drugs to halt infection in humans. "The structure is unique in the RNA virus world," Derewenda explains. "It is not found in viruses that cause influenza, rabies or other diseases." It distantly resembles the β-grasp protein motif found in ubiquitin, most likely the result of convergent evolution.
Like many other related viruses, Ebola virus contains a negative-sense, single-stranded RNA that encodes seven different proteins, one of which is known as the nucleoprotein (NP) for its ability to interact with the viral RNA genome. It is the most abundant viral protein found in infected cells and also inside the viral nucleocapsid. While five of the seven viral proteins have succumbed to structural characterization by X-ray crystallography, NP so far has resisted such attempts, although analogous proteins from other viruses have had their structures analysed.
The UVA team produced the Ebola protein using an engineered form of Escherichia coli bacteria as a protein factory. This allowed them to identify the boundaries of two globular domains and to crystallize the unique C-terminal domain spanning amino-acid residues 641 to 739. The study revealed a molecular architecture unseen so far among known proteins, the team says. There is existing evidence that the newly characterized domain is involved in transcription and the self-assembly of the viral nucleocapsid. As such, the results obtained by the UVA team will be useful in deciphering precisely how these various functions are accomplished by the virus; such a detailed description offers up a potential target for the design of anti-viral drugs.
Cover illustration: Artistic impression of the new MAX IV facility, currently under construction in Lund, Sweden, and one of a new generation of storage-ring-based synchrotron light sources employing a multibend achromat lattice to reach emittances in the few hundred pm rad range in a circumference of a few hundred metres. [Image courtesy of FOJAB arkitekter.]
Progress is being made in improving accelerator technology, enabling a significant increase in brightness and coherent fraction of the X-ray light provided by storage rings. Two facilities will open shortly; MAX IV will open to users in 2016, SIRIUS soon thereafter. Many existing facilities are working on upgrades of their present machines based on these concepts, and entirely new machines are under consideration.
These developments cannot come soon enough, because higher brightness of the source will be of advantage for almost any experiment. This is not only the case for numerous X-ray microscopy applications but also if a small spot of the sample needs to be illuminated like in high-resolution X-ray spectroscopy or in experiments under high pressure in a tiny diamond anvil cell.
While diffraction limited storage rings (DLSRs) provide high average brightness, they cannot compete with Free Electron Lasers (FELs) as regards the peak brightness required for ultra-fast time resolution or single-shot experiments. This complementarity makes it attractive to locate a DLSR and a FEL on the same site. In this case a large number of scientific experiments can be conducted simultaneously on many beamlines at the DLSR, while specialized experiments can be scheduled for the FEL, at which only one or a few experiments can be conducted at a given time.
Exploitation of the full potential of a DLSR requires near-perfect optics, dedicated beamlines and sample environments, and specialized detectors. Together they can produce huge data rates (~10 GB/s) and data volumes (~10 TB/experiment) requiring dedicated infrastructure and specialized software that also allows non-expert synchrotron users to extract the relevant information within a realistic time.
Light sources are a tool to see the world around us and storage rings are nothing but light sources for the X-ray range. The significant improvement provided by the DLSRs under construction and in the design stage will enlighten our view of the world and allow science which is not possible, or not even thinkable, today.
We hope you enjoy this special issue and our glimpse into the science of tomorrow.Mikael Eriksson, J. Friso van der Veen and Christoph Quitmann
After having successfully stored electrons from the 20 MeV Microtron in the Booster in July 2014, on 3 September 2014, the SESAME team succeeded in accelerating the electrons in the Booster to their final energy of 800 MeV.
The SESAME Injector consists of a 20 MeV Microtron and the 800 MeV Booster-Synchrotron. Electrons are produced in the Microtron where they are accelerated to 20 MeV (Million Electron Volt), and these electrons are then transferred to the Booster-Synchrotron.
SESAME’s Microtron became operational in 2012, installation of its Booster was completed in 2013, and storage in the Booster, in July 2014, of the electrons from the Microtron meant that they were then circulating several million of turns in the Booster at their initial energy of 20 MeV.
These electrons have now been accelerated from 20 to 800 MeV, which is the final energy of the Booster. This was done by increasing the magnetic field in the Booster Magnets and feeding microwave power into an accelerating unit (cavity), steering the beam in the center of the vacuum chamber and keeping all this synchronized.
Successfully having brought SESAME’s Booster to full operation is of particular significance since this is the first high-energy accelerator in the Middle East, and this achievement with the Booster is to be attributed to a team of young scientists and technicians from the region for whom accelerator technology is a new field. They were led in this work by Erhard Huttel, the Technical Director of SESAME.
When the facility starts operations (probably in early 2016), scientists from the Middle East and neighbouring countries, in collaboration with the international synchrotron light community, will have the possibility of performing world-class scientific studies: for example, to determine the structure of a virus to improve medical remedies, to get insight into the interior and the three-dimensional micro-structure of objects such as materials of interest to cultural heritage and archaeology, and to investigate magnetisation processes which are highly relevant for magnetic data storage, to cite just a few examples of the vast scientific potential of SESAME.
Direct chemical editing of DNA might be useful in synthetic biology for modifying genes in an organism for a specific task or in medicine for repairing damaged genes involved in diseases, such as cancer and certain inherited disorders. Unfortunately, as Guillermo Montoya of the Spanish National Cancer Research Centre in Madrid, Spain, and colleagues point out sloppy editing can lead to damage to the genome as a whole.
Writing in the journal Acta Crystallographica Section D Biological Crystallography [Stella et al. (2014), Acta Cryst. D70, 2042-2052; doi:10.1107/S1399004714011183] Montoya and colleagues, explain how a new protein-DNA binding domain can allow the necessary modifications to be made due to its high specificity. Their strategy involves allowing specific gene modifications to be made through the addition; removal or exchange of DNA sequences using customized proteins and endogenous DNA-repair machinery.
Key to such successful editing will be the engineering of protein-DNA interactions, the team says and this in turn will rely on finding DNA-binding domains. The researchers recently identified one such protein, BurrH, from the bacterium Burkholderia rhizoxinica which contains highly polymorphic DNA-targeting modules. They have demonstrated that BurrH recognizes a 19 base-pair DNA target. Now, they have determined the crystal structure of the apo, unattached, form of the protein and its structure bound to DNA to reveal insights into how form affects function. The team explains that structural data were obtained using a single-wavelength anomalous diffraction (SAD) technique on a selenium derivative (all methionines were substituted for selenomethionines) of the protein crystals chilled to 100 Kelvin to enhance the signals.
The crystal structure uncovered a central region containing 19 repeats of a helix-loop-helix modular domain (BurrH domain; BuD) within the protein's complete chain of 794 amino acids. This molecular recognition unit identifies its DNA target through a single residue-to-nucleotide code. As such, the team reports, this could make it relatively easy to change to allow it to target specific genes. The tailored domains can target specific DNA regions and so by fusing BuD to catalytic domains with DNA-modifying activity the team can shuttle enzymes such as nuclease, methylase etc to specific regions of the genome. In their work Montoya and co-workers show that the process allows targeted mutagenesis to be induced specifically by delivery of the nuclease activity and the insertion of exogenous DNA at the binding site without widespread disruption of the genome.
Indeed, while B. rhioxinica is a symbiont of crop fungi that cause devastating blight in rice, onions and other food crops, the team has now exploited its molecular biology to engineer a protein system based on BuD-derived nucleases (BuDNs) that can target genes in human hemoglobin beta (HBB). The targeting can home in on genes close to the mutations that give rise to sickle-cell anemia and so might ultimately be used to carry out genetic repairs in this debilitating and ultimately lethal disorder.
"Our work shows that this novel platform can be engineered to recognize DNA sequences in human cells," the team explains. They add that it is the combination of efficiency and specificity of BuD as well as the fact that only a single residue, the 13th, need be changed to allow it to target other DNA sequences make it a very useful tool. The researchers add that BuD is "well suited to multiple genome- modification approaches for cell or organism redesign, opening new avenues for precise and safe gene editing for biomedical purposes."