|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.|
A group of scientists from Europe and the USA have successfully used high-resolution X-ray crystallography alongside resonance Raman Spectroscopy to “fingerprint” and validate different redox and ligand states in crystal structures of proteins in order to gain maximum functional information and to avoid, for example, the misinterpretation of reaction mechanisms. This breakthrough may prevent future inaccurate predictions in the structure and role of proteins. [Kekilli et al. (2014), Acta Cryst. D70, 1289-1296; doi: doi:10.1107/S1399004714004039]
The combined resonance Raman (RR) and crystallographic data described in the paper demonstrate that online RR spectroscopy is a powerful tool for fingerprinting both redox and ligand states in single crystals of haemproteins.This approach allows information on vibrational states to be gained and, if performed on-axis, is particularly useful for large crystals, where UV-visible absorption micro-spectrophotometry is not feasible owing to the opacity of the haemprotein crystals.
Single-crystal spectroscopies particularly when applied in situ at macromolecular crystallography beamlines, allow spectroscopic investigations of redox and ligand states and the identification of reaction intermediates in protein crystals during the collection of structural data. This complementary combined approach to structure determination has proved to be a powerful tool to obtain useful data and correctly assign the true oxidation and ligand state(s) in redox-protein crystals.
In this study the scientists successfully present a comprehensive, correlated single-crystal resonance Raman and structural study of Alcaligenes xylosoxidans cytochrome c prime (AxCYTcp) in its ferric, ferrous and gas ligand-bound forms as well as characterizing X-ray induced changes to these states. The researchers went on to demonstrate that redox and ligand states in crystal structures can quite effectively be spectroscopically fingerprinted in situ on a macromolecular crystallography beamline, in this case at Swiss Light Source beamline X10SA.
This methodology, if applied routinely to structural studies of haemproteins, has the potential to radically increase and complement the biological significance of the results gained from crystallographic experiments, such as redox-dependent biological mechanisms.
Lead researcher Dr Mike Hough at the School of Biological Sciences, University of Essex, commented, "The resonance Raman technique is applicable to many protein crystals that contain chromophores. UV-visible spectroscopy has been used quite successfully for similar purposes however as a surface technique, resonance is very useful for larger crystals which are often opaque to light and also gives information on vibrational properties".
The technique is robust and could be rolled-out across many X-ray crystallography beamlines or more importantly as an integral part of future beamlines.
To accompany the exhibition Illuminating Atoms, the Royal Albert Hall has teamed up with the Science & Technology Facilities Council to present a very special Q&A with Professor Elspeth Garman.
Professor Garman will describe how X-ray crystallography is used to determine molecular structures, helping us to understand diseases and develop drugs to control them. Beginning with the methods pioneered over a century ago by father and son William and Lawrence Bragg, Elspeth will discuss how their legacy lives on at the forefront of modern drug discovery. Thanks to their contributions, the 3-dimensional shapes of complex biological molecules can be found, helping us to understand and counter many diseases and viruses affecting people all over the globe.
Elspeth Garman is Professor of Molecular Biophysics and Director of the University of Oxford's Doctoral Training Centre Systems Biology Programme. As well as determining several key protein structures (e.g. from TB and bird flu), her group works to improve X-ray diffraction experiments on protein crystals. As an expert on the effects of cryocooling and radiation damage on delicate crystals, Elspeth has appeared on BBC Radio and at the Cheltenham Science Festival.
Elspeth Garman is also Co-editor of the IUCr journal Acta Crystallographica Section D: Biological Crystallography and Guest Editor of a series of special issues in the Journal of Synchrotron Radiation on X-ray damage to biological crystalline samples; you can see the latest special issue here.
Tickets include a glass of wine on arrival and access to the exhibition before the event from 6pm.
To find out more follow this link to the Royal Albert Hall.
The research team of Annette Rompel from the Institute for Biophysical Chemistry, University of Vienna, Austria explore the mechanisms behind the "browning reaction" during the spoilage of mushrooms. The researchers were able to demonstrate that the enzyme responsible is already formed before fungal spoiling [Mauracher et al. (2014). Acta Cryst. D70, 2301-2315; doi:10.1107/S1399004714013777] and [Mauracher et al. (2014). Acta Cryst. F70, 263-266; doi:10.1107/S2053230X14000582]
Understanding the mechanism of the enzyme tyrosinase pigmentation is of both medical and technological interest. The copper-containing enzyme is present in animals and humans and is essential for the protective pathway against UV radiation. It also simultaneously provides the elucidation and potential means with which to prevent the spoilage of food. Mushrooms were selected for the study due to their low cost and ready availability. In addition, they are also a valuable target for researchers, largely because of their high enzyme content, fungi are ideal sources for potential studies of tyrosinase. Mushrooms therefore serve as a model organism for the study of the pathway involved in food spoilage.
Since 2012, it has been known that six different tyrosinases (PPO1 to 6) exist within our common edible mushroom, two of which (PPO3 and PPO4) occur in larger quantities. The enzyme responsible for the mechanism of food spoilage is formed within eukaryotes (organisms that have a nucleus) as an inactive precursor during the developmental phase of an organism. This precursor is then activated via specific chemical cleavage. At this cleavage site, the protein segment covering part of the enzyme active site is removed and the substrates (tyrosine and other monophenols) can be accessed and can take part in key chemical reactions.
Until now, none of the previously established methods of isolation present in the literature could be successfully applied to PPO4. For the first time, in the Institute for Biophysical Chemistry at the University of Vienna, a method has been developed that allows for the one stage isolation of latent tyrosinase from its natural source. The enzyme characterization was undertaken at the Department of Chemistry in close cooperation with the Institute of Mass Spectrometry led by Andreas Rizzi, both within the University of Vienna. After sufficiently large quantities were extracted from pure PPO4, the researchers were able to identify and optimize appropriate crystallization conditions that produced well-formed protein crystals. This was only possible with the use of a relatively unusual co-crystallization reagent, a polyoxometalate of the Anderson type.
Following this research, it is now possible to purify the enzyme in sufficient quantities for characterization. The research has allowed the crystallization and three-dimensional structure of PPO4 to be resolved.
This story is reprinted from material from the University of Wien, with editorial changes made by IUCr. The views expressed in this article do not necessarily represent those of IUCr. Link to original source.
Membrane 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.