Monthly Archives: March 2017

A large-solid-angle X-ray Raman scattering spectrometer

The large-solid-angle X-ray Raman scattering spectrometer at ID20. Photo credit: ESRF/McBride

S. Huotari and co-workers [J. Synchrotron. Rad. (2017), 24, 521-530] describe an end-station for X-ray Raman spectroscopy at beamline ID20 of the European Synchrotron Radiation Facility. The end-station is dedicated to the study of shallow core electronic excitations using non-resonant inelastic X-ray scattering.
X-ray Raman scattering (XRS) spectroscopy is a versatile tool for studying shallow X-ray absorption edges using hard X-rays. It has proven to be an invaluable technique for the study of electronic excitations in a variety of sample systems such as crystals, liquids and gases. Over the past decades, XRS has been applied to solve geoscientific questions by studying shallow core edges under extreme pressure and temperature conditions, follow chemical reactions in situ, and study liquid samples under well defined thermodynamic conditions.
A drawback of XRS is the orders-of-magnitude weaker scattering cross section in comparison with the probability for photoelectric absorption. This can be compensated for by using light sources with a very high brilliance and efficient signal collection; this has been the guiding motive for the design of the spectrometer presented in this paper.
The new end-station provides an unprecedented instrument for X-ray Raman scattering, and will open the door to renewed studies of low-energy X-ray absorption spectra in materials under in situ conditions, such as operando batteries and fuel cells, in situ catalytic reactions, and extreme pressure and temperature conditions.

P14.EH2: Getting ready to turn the light on

Dr Rosemary Wilson @rawilson80 Scientific training and outreach officer, EMBL Hamburg

Long after the lights went on in the PETRA III Max von Laue experimental hall on the DESY campus, and the 14 beamlines became hives of activity, one corner has remained dark and seemingly forgotten. But there have always been plans for this corner of the Max von Laue Hall, and now those plans are taking shape. This hutch, situated directly behind the EMBL crystallography beamline P14, will house P14.EH2, a new experimental endstation which will cater for time-resolved crystallography experiments. In the autumn of 2016, EMBL group leader Thomas Schneider and Professor Arwen Pearson from the Centre of Ultrafast Imaging (CUI) at the University of Hamburg, were successful in securing a grant from the German federal government which in part provides funds for designing and building the endstation. Since then basic designs for the instruments have been decided, the first bits of equipment ordered and positions advertised for postdocs to help construct and run the endstation. The group hope to be able to welcome friendly users by the end of 2018. So how about some details?

Why a new endstation?

PETRA III produces some of the most brilliant and intense X-rays on the planet. The EMBL macromolecular crystallography beamline P14 is optimised to create a tight and parallel beam. With so many photons reaching the sample at the same time, scientists can start to watch the structural changes a protein goes through during a reaction, rather than a time-averaged snapshot as we are familiar with in traditional crystallography. Schneider, Pearson and their teams have already used P14 to do these types of time resolved crystallography experiments, but the set-up is not ideal in the long run.

Briony Yorke in the hutch that will house the new endstation. The X-ray beam will enter the hutch through the hatch in the back wall. Photo: Rosemary Wilson

The hutch for P14.EH2 sits directly behind P14 and the new endstation will be fed by the same X-ray beam. Each time-resolved experiment will need a different instrumental set-up, optimised to the type of results the scientists want to achieve. Having a separate hutch dedicated to the complicated time-resolved set-ups means that the regular operation on P14 doesn’t need to be interrupted and the scientists can take time to plan, design and build their set-up before pressing the button to allow the X-ray beam into the hutch. “The idea is that the set-up will be very flexible and modular” explains Wellcome Postdoctoral Fellow Briony Yorke (@BrionyYorke), who is working with Pearson on the endstation. Due to the tight angle that the X-ray beam leaves the PETRA III storage ring, the other beamline hutches and endstations are narrow, with access to equipment only possible by climbing over and under beamlines and detectors. The nature of the time-resolved experimental set-up means that access has to be easy and comfortable, and this was a major consideration during the design process. (See the project webpage for more details on the hutch layout design and specifications.)

What methods and hardware will be available?

Time-resolved crystallography works by taking a series of snapshots of a molecule such as a protein in quick succession to build up a movie of the protein’s structural changes during a reaction (see also: Taking crystallography to the fourth dimension). In practical terms, the X-ray beam has to be interrupted to create many successive images. “The trick is to turn the X-ray beam on and off faster than the process we are interested in” explains Briony. “The current shutter on P14 is a milli-second shutter, so the only possibility to look at shorter time slices is to get the detector to count for a shorter time period by repeatedly turning it on and off, since we physically cannot open and shut the shutter fast enough.” On the new endstation, a circular metal disc with pattern of holes stamped in it called a chopper will repeatedly ‘chop’ the X-ray beam, breaking up the signal to provide the sought after shorter time slices.

The prototype Hadamard chopper designed by Briony’s master student Anke Puckert and made on the DESY site. Of the three outer ring sequences, the central pattern encodes the Hadamard sequence, the other two are control patterns. Credit: Anke Puckert

Briony’s particular interest is Hadamard time-resolved crystallography, a method she developed during her PhD, which will be an available option in the new set-up. While a chopper for standard time-resolved crystallography experiments may have a single hole in it, creating an interruption every, say second as it rotates through the beam, Hadamard choppers require a much more intricate pattern of holes. This will create many more interruptions, resulting in more data per crystal, and a more detailed ‘movie’ of the protein dynamics.

“The choppers pose quite a challenge” says Briony. “During experiments they will be spinning at very high speeds (several 1000 rpm), so they have to be cooled with water and placed under a vacuum, and shielded in a bullet-proof case something goes wrong!” Another key bit of equipment that will be found in the new hutch will be a laser for initiation of the (bio)chemical process the experiment aims to study. “The laser impulse will be delivered with fibre optics” explains Briony. “This will enable a really precise initiation of the experiments” she adds.

What science will it be used for?

“We are interested in looking at structural changes within proteins that happen during a chemical reaction, for example in an enzyme. The rate determining steps of these reactions happen on the microsecond to millisecond time-scale which P14.EH2 is designed to handle. We want to provide a regular service for people interested in exploring protein dynamics” says Briony. She will be using the time-resolved crystallography endstation, for example, to investigate how cataracts form in our eyes. “Cataracts form when proteins in our eyes start to stick together. But we don’t know what triggers this process. If we can get a picture of how and why this happens, we might be able to design medication to prevent them forming or even reverse the process” she says. P14.EH2 is likely to be interesting for a whole range of applications – from biotechnology to medicine. “For example, we could use the endstation to understand how some bacteria make crude oil, or eat plastic” explains Briony, “or gain a better understanding of how enzymes interact with medicines in our bodies. And all this evidence can be used to help build better computer simulations of molecular motions” she concludes.

What’s next?

Any day now the first instruments should arrive and the instrumentation group at EMBL can start constructing the endstation. Visit the project webpage for regular updates on their progress!

You can see papers published by Dr Thomas R. Schneider in IUCr Journals here

You can see papers published by Professor Arwen R. Pearson in IUCr Journals here


In-cell NMR: a topical review

The structure of biological macromolecules is critical to understanding their function, mode of interaction and relationship with their neighbours, and how physiological processes are altered by mutations or changes in the molecular environment.

Ideally, classical structural biology research should interface more with cellular biology, as it is crucial for the structural data obtained in vitro to be validated within the cellular or tissue context. A true cellular structural biology approach should allow macromolecules to be characterised directly in their native environment. Such an approach would guarantee the high significance of data obtained in vivo or in the cell with the high resolution of a structural technique.

In the Past decade, NMR spectroscopy has been applied to obtain structural and functional information on biological macromolecules inside intact, living cells. The approach, termed “in-cell NMR”, utilises the improved resolution and sensitivity of modern high-field NMR spectrometers and exploits selective enrichment of the molecule(s) of interest with NMR-active isotopes.

Since its inception, in-cell NMR has gradually emerged as a possible link between structural and cellular approaches. Being especially suited to investigate the structure and dynamics of macromolecules at atomic resolution, in-cell NMR can fill a critical gap between in vitro-oriented structural techniques such as NMR spectroscopy, X-ray crystallography and single-particle cryo-EM techniques and ultrahigh-resolution cellular imaging techniques, such as cryo-electron tomography.

In a topical review IUCrJ (2017), 4, 108-118 Lucia Banci and her co-worker Enrico Luchinat, both based at the University of Florence, summarise the major advances of in-cell NMR and report the recent developments in the field, with particular focus on its application for studying proteins in eukaryotic and mammalian cells and on the development of cellular solid-state NMR.


NMR Crystallography

Crystallography is the study of the crystalline state of matter. The meaning is contained in the etymology of the word: κρυσταλλογραφ[{\acute \iota}]α (krustallograpia) arising from κρ[{\acute \upsilon}]σταλλος (krustallos, `clear ice’) and γραφο (grapo, `I write’). The modern inter­pretation of this term is widely construed as `the study of crystals.’ Crystallography since the `Traité de Crystallographie‘ of René-Just Haüy (Paris, 1822) has been based on a direct space description of crystals. There are many ways to study crystals, for example, by optical, thermal, mechanical or diffraction techniques. The relationship between the crystalline state of matter and its ability to diffract waves of properly matched wavelength has played and will always play a special role in this area of research. Nuclear magnetic resonance (NMR) spectroscopy has, however, from its earliest days, provided structural information on both periodic and amorphous compounds, ranging from specific inter­nuclear distances to complete structural models of complex materials and biomolecules. The term `NMR Crystallography’ presents a broad polysemy. To some, it represents a stand-alone structure elucidation method for single crystal, polycrystalline or amorphous compounds. For others, it is a source of additional structural information when compounds fail to yield crystals of sufficient quality or size suitable for single-crystal diffraction-based structure determination, or when powder diffraction patterns exhibit a too high degree of complexity for structure model elaboration. Some consider NMR, diffraction and modelling as a synergistic complementary set of methods. Others consider that the multiplicity of specific NMR experiments allows for the progressive build-up of topological sub-graphs of the crystal graph, and thus drives the structure model search. These are all established uses of magnetic resonance toward the investigation of the crystalline state.

The Commission on NMR Crystallography and Related Methods of the Inter­national Union of Crystallography was established at the Montreal General Assembly in August 2014. The commission envisions `NMR Crystallography’ in an even more expansive light, as being: the use of the spin degrees of freedom of the magnetic resonance phenomenon to study the crystalline state of matter. As NMR may also be applied to the liquid state, it may also be used to study crystal genesis. The increasing profile of NMR in the crystallographic community is also nicely evidenced by an inspection of the planned lectures and microsymposia scheduled for the 24th Congress & General Assembly of the Inter­national Union of Crystallography, to be held in Hyderabad (August 21–28, 2017).

The contributions to this special issue of Acta Crystallographica Section C serve as an excellent introduction to the power and scope of NMR crystallographic methods and applications. The issue begins with two insightful review articles, one from Stebbins and co-workers on looking at short-range order in paramagnetic samples, and one from Harris and co-workers on in-situ techniques for time-resolved monitoring of crystallization processes.

NMR crystallographic methods have proven to be popular in characterizing organic co-crystals, particularly those with an active pharmaceutical component. Three contributions explore this area. Brown and co-workers describe the application of advanced two-dimensional solid-state NMR experiments to elucidate the role of weak inter­actions in a co-crystal of two fungicides. Kerr et al. have applied related methods to determine the crystal structure of a co-crystal of naproxen with picolinamide. Vigilante and Mehta report their 13C solid-state NMR work on co-crystals of caffeine and theophylline.

It is important to point out that there are many flavours of NMR crystallography, and many applications combine the information available from many techniques to gain structural insights. The information obtained from X-ray diffraction is typically used to its full potential, in combination with the additional information obtained from NMR. Computational chemistry, often in the form of density functional theory (DFT) calculations, is also often incorporated to the refinement protocol to obtain final structural results in best agreement with all available data. For example, Szell et al. report on a combined NMR, X-ray and DFT study of halogen-bonded frameworks featuring nitro­gen-containing heterocycles. DFT also plays a key role, along with 14N NMR, in the report from Alonso and co-workers on inter­molecular inter­actions in AST zeolites. Laurencin and co-workers describe the prospects available via 25Mg and 43Ca NMR spectroscopy in low-coordination-number organo-complexes with the help of DFT methods. Mali reports on the ab initio crystal structure prediction of magnesium (poly)sulfides, and on how the calculated NMR parameters may be used to distinguish between possible structures.

Nishiyama and co-workers describe a beautiful example of the power of combining the information available from electron diffraction with that of NMR spectroscopy to report on crystalline polymorphs of organic microcrystalline samples. Inorganic aluminophos­phates and layered silicates are explored in eloquent and insightful contributions from Ashbrook and co-workers and from Brouwer et al. Finally, an excellent example of the application of NMR crystallography methods to a metal–organic framework is provided by Pourpoint and co-workers, wherein carbon–aluminum distances are measured using advanced techniques.

This collection of papers provides a broad perspective on the diversity of NMR crystallographic methods and applications. It may be useful to keep in mind some of the particular advantages of NMR methods, including, for example, their sensitivity to H-atom positions and weak inter­molecular inter­actions, and their capacity to characterize disordered and noncrystalline samples. The continuing advances in NMR technology, pulse sequence development and robust structure refinement protocols, which include data available from all available sources, all bode well for the future of this exciting field.

David L. Bryce and Francis Taulelle

Guest editors