Powders show their strength

Oct 09, 2007
Powders show their strength
After purification, the SH3.2 domain spontaneously formed a microcrystalline material suitable only for powder diffraction measurements. Credits: Dr. Nikos Pinotsis (EMBL - Hamburg).

[PIC=:left]Growing a single crystal of a protein can be very difficult. Thanks to recent developments, a powder sample may be enough to solve a structure.

Researchers from the European Molecular Biology Laboratory and the ESRF have successfully used the powder diffraction technique to determine the structure of the second SH3 domain of ponsin, which plays an important role in muscle differentiation. The results are comparable to those obtained by single crystal techniques and demonstrate the power and the potential applicability of the powder technique in structural biology.

The knowledge of the 3D structure of proteins and protein assemblies provides valuable information in health science research and biotechnological applications. X-ray crystallography is the method researchers use to visualise the structures of macromolecules. However, it can be challenging, even impossible, to grow crystals that are suitable for standard single crystal measurements.

Often, macromolecules such as amyloid fibrils, supramolecular assemblies or even proteins are particularly hard to crystallise.

On the other hand, the preparation of a powder sample – comprising a large number of very tiny randomly oriented crystallites– is much less demanding. Powders can be prepared for many samples simply by batch precipitation. Nevertheless, analysing data from powders is more complicated, and generally limited to comparably simple crystal structures.

The two teams from the ESRF and the EMBL, focused on the study of an SH3 domain of ponsin, a protein involved in muscle differentiation. Furthermore, the SH3 motif is one of the most abundant found in a large variety of signalling processes thus linked to several diseases and is therefore an important therapeutic target for drug design.

Upon sample purification the specific SH3-domain protein sample spontaneously formed a suspension of microcrystals. This material was suitable only for powder diffraction and therefore it was brought to the European Synchrotron Radiation Facility in Grenoble where scientists collected very high quality data. The molecular arrangement of the domain was gradually unravelled from the powder diffraction patterns using the joint expertise of the two research teams.

They created novel data processing strategies drawing on both traditional single crystal and powder methods. For example, radiation damage or modest changes in sample preparation conditions lead to small shifts of the diffraction peak positions in a powder pattern. Overlapped diffraction peaks can then be better resolved by combining multiple patterns and exploiting these differences. The final result was a powder dataset which approaches single crystal quality (dmax ~ 2.4 Å).

In order to start the analysis, scientists used a similar SH3 domain as a structural model. They determined the orientation and position of the protein into the unit cell via molecular replacement. The structure was refined initially to produce electron density maps which were then employed to trace the main amino acid chain alterations and find the missing atoms. The structure was completed via a multiple-data-set Rietveld fit.

To the team’s surprise, they could even detect water molecules in electron density maps; a substantial observation from powder diffraction data. These results were later verified by single crystal measurements, as the conditions for growing a single crystal of the protein, suitable for structural analysis, were subsequently discovered.

The study on this SH3 domain represents a step forward in the development of modern X-ray diffraction methods for micro- and nano-crystalline samples. “We believe that powder methods may have a significant impact in the fields of structural biology and biomaterial sciences – just as it has had in solid-state and structural chemistry over the last twenty years”, explains Irene Margiolaki, first author of the paper. “The structure of this important protein confirms that powder diffraction is now a technique ready to tackle genuine biological problems”, she concludes.

Reference: I. Margiolaki, J. P. Wright, M. Wilmanns, A. N. Fitch & N. Pinotsis, "Second SH3 Domain of Ponsin Solved from Powder Diffraction", J. Am. Chem. Soc., 129 (38), 11865 -11871, 2007.

Source: ESRF

Explore further: Researchers bring clean energy a step closer

add to favorites email to friend print save as pdf

Related Stories

How magnetic dimers interact to create long-range order

Oct 13, 2014

Researchers carrying out experiments at the U.S. Department of Energy's Advanced Photon Source (APS) and Spallation Neutron Source have shed new light on how magnetic long-range order forms and remains stable ...

Chemist develops X-ray vision for quality assurance

Jul 24, 2014

It is seldom sufficient to read the declaration of contents if you need to know precisely what substances a product contains. In fact, to do this you need to be a highly skilled chemist or to have genuine ...

Recommended for you

Researchers bring clean energy a step closer

Feb 27, 2015

For nearly half a century, scientists have been trying to replace precious metal catalysts in fuel cells. Now, for the first time, researchers at Case Western Reserve University have shown that an inexpensive metal-free catalyst ...

The construction of ordered nanostructures from benzene

Feb 27, 2015

A way to link benzene rings together in a highly ordered three-dimensional helical structure using a straightforward polymerization procedure has been discovered by researchers from RIKEN Center for Sustainable ...

Superatomic nickel core and unusual molecular reactivity

Feb 27, 2015

A superatom is a combination of two or more atoms that form a stable structural fragment and possess unique physical and chemical properties. Systems, that contain superatoms, open a number of amazing possibilities ...

Oat breakfast cereals may contain a common mold-related toxin

Feb 25, 2015

Oats are often touted for boosting heart health, but scientists warn that the grain and its products might need closer monitoring for potential mold contamination. They report in ACS' Journal of Agricultural and Food Chemistry that s ...

User comments : 0

Please sign in to add a comment. Registration is free, and takes less than a minute. Read more

Click here to reset your password.
Sign in to get notified via email when new comments are made.