Cryo soft X-ray tomography, a new tool for structural biology

Dr. Eva Pereiro & Dr. Peter Guttmann ALBA & HZB-TXM BESSY II

Structural Cell Biology is a wide area where the need for a detailed structural and functional description of the different cell components must be correlated with a topological map of these components at the whole cellular level. Cryo-electron tomography has proven to be a suitable tool in this context with resolutions in the order of a few nanometers [1]. However, its intrinsic sample thickness limitation obliges to perform cell sectioning which makes it nearly impossible to get 3-D information of the whole cell. In addition, the correlation of information with optical microscopy has become an important issue to study biological events at different levels. But the gap between these two microscopy methods in terms of magnification, sample thickness and spatial resolution has complicated the microscopy information gateway. Cryo soft X-ray tomography (cryo-SXT) is a new complementary approach in this field that, in combination with fluorescence microscopy, can provide answers at medium resolution (25 – 30 nm feature sizes) [2, 3, 4] on the organelles organization in whole, unstained, un-sectioned cells.

Two transmission X-ray microscopes (TXM) devoted to cryo soft X-ray tomography are available in Europe, both partners of the Biostruct-X project. The HZB-TXM at the BESSY II electron storage ring (Berlin, Germany) started operation in spring 2009 and MISTRAL TXM at ALBA (Barcelona, Spain: http://www.cells.es/Beamlines/XM/) came into users operation in February 2013. Access to these beamline facilities for biology research groups of all Europe is available through the specific call for proposals of each facility (please check the Users Office webpages http://useroffice.cells.es/ and http://www.helmholtz-berlin.de/user/index_en.html) as well as funding from Biostruct-X for travelling and subsistence during the experiment at the beamlines. If access to the TXM facilities forms part of an integrated research plan, access can be requested through Instructin the first instance. Instruct will then coordinate with BioStruct-X to steer the user through all or a section of a structural biology technology pipeline, a service that is designed to help researchers that are relatively inexperienced in structural cell biology technologies to achieve new data outcomes.

We encourage new users to contact the scientific staff of both beamlines for helping submitting proposals and further required information such as the use of the available infrastructure at their facilities.

Dr. Eva Pereiro (epereiro@cells.es) for MISTRAL TXM at ALBA

&

Dr. Peter Guttmann (peter.guttmann@helmholtz-berlin.de) for HZB-TXM BESSY II

Ref:

[1] K. Grunewald and M. Cyrklaff, Curr Opin Microbiol, 9, 437-442 (2006).

[2] J.L. Carrascosa et al. J. Struct. Biol. 168, 234-239 (2009).

[3] G. Schneider et al. Nature Mat.7, 985-987 (2010).

[4] F.J. Chichón et al. J. Struct. Biol.177, 202-211 (2012).

[5] G. Schneider et al. J. Struct. Biol. 177, 212-223 (2012).

An example of cutting-edge research can be found in what follows.

Oriented nucleation of hemozoin crystals in Plasmodium-infected red blood cells

Kapishnikov S., Weiner A., Shimoni E., Guttmann P., Schneider G., Dahan-Pasternak N., Dzikowski R., Leiserowitz L., Elbaum M., PNAS, vol. 109, no.28, 11188-11193 (2012)

Malaria is a widespread and severe disease transmitted by mosquitoes carrying parasites of the Plasmodium genus. These invade red blood cells and feed on hemoglobin, releasing free heme that is highly toxic to the parasite itself. In order to detoxify this byproduct, the parasite induces a rapid crystallization of the heme into physiologically insoluble hemozoin.

Two mechanisms have been proposed in recent years to explain the crystal formation in vivo. According to one, crystal growth should occur within small lipid droplets or nanospheres. In the second, nucleation should be induced by a molecular template at the surface of a lipid layer, while crystal growth should occur in the water phase. This model entails a further prediction that crystals nucleated from a common surface should share a common crystallographic orientation.

At HZB-BESSY II we have applied cryo soft X-ray tomography to study hemozoin crystallization in infected red blood cells. Image contrast depends primarily on X-ray absorption by carbon, so lipids are relatively easy to see, indeed, difficult to hide. Crystals were found in the water phase, often adjacent to the inner membrane of the vacuole where hemoglobin digestion takes place. No lipid droplet could be seen surrounding the crystals. Their morphology displayed sharp edges, which indicated mutual orientations with parallel, needle-like c-axes and a-axis orientations facing the membrane, precisely as suggested by complementary diffraction studies. Extensive electron microscopy also supported these conclusions.

These results are of utmost medical importance, as hemozoin growth is the target of common antimalarial drugs. When crystallization is inhibited the parasite effectively poisons itself. With the emergence of drug-resistant strains of Plasmodium, new treatments must be developed to combat the disease. Understanding the crystal growth mechanism of hemozoin is a key step in this direction.

Figure: Cryo X-ray tomography of P. falciparum-infected red blood cells. Top: Slices through tomographic reconstruction and surface rendering of trophozoite-stage parasite with a single nucleus (Nu) and large digestive vacuole (DV) enclosing numerous hemozoin (Hz) crystals. Bottom: theoretical hemozoin crystal morphology compared to biogenic hemozoin appearance on a cut parallel to the DV membrane indicating common {100} face of nucleation.


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