Structural Biology

Biography

Biography: Nadia L. Opara

Abstract

Classical crystallography methods based on synchrotrons usually require crystals of relatively large dimensions, i.e. above about 5 micrometers. The recent availability of X-ray free electron laser sources (XFELs) providing femtosecond X-ray pulses of ultrahigh brightness facilitate the investigation of nanocrystals. However, in this case data collection has to be performed in the mode of the serial crystallography in so-called diffraction-before-destruction regime because the probed area of the sample is completely destroyed after the interaction with ultraintense radiation.  As thousands of crystals have to be provided sequentially to the XFEL beam, selection of an efficient sample delivery system is crucial to minimize protein consumption during data collection.

Delivery methods applied so far include steady streaming liquid jets [1,2] of the crystal suspension. The application of more viscous media like lipidic cubic phase (LCP) [3], agarose [4] or hyaluronic acid [5] matrices has also been demonstrated. However all these methods use significant amounts of the precious protein, which cannot be recovered even if not directly probed.

Recent developments of drop of demand methods [6] or fixed targets [1,7] allow overcoming this problem. But still, handling of the fragile crystals should be gentle or, at best, avoided.

Microfabricated silicon chips with ultrathin Si₃N₄ membranes provide the possibility to regularly position crystals on precisely defined spots by direct crystallization using classical vapor diffusion method [8]. The sample consumption is minimal since crystal growth takes place in nanoliter volume cavities. No additional sample transfer is needed, because X-rays are probing the crystals at the spot where they grew on the X-ray-transparent ultrathin amorphous silicon nitride membranes. Assembly with a second chip to form a hermetically sealed sandwich protects specimens from dehydration and facilitates in situ diffraction data collection at room temperature, as demonstrated in a synchrotron experiment providing high-resolution patterns [Fig. 1].

References:

1. Muniyappan S et al. (2015) Recent advances and future prospects of serial crystallography using XFEL and synchrotron X-ray sources. Bio Design 3:2
2. Steinke I et al. (2016) A liquid jet setup for x-ray scattering experiments on complex liquids at free-electron laser sources. Rev Sci Instrum. 87:063905
3. Weierstall U et al. (2014) Lipidic cubic phase injector facilitates membrane protein serial femtosecond crystallography. Nat Commun. 5:3309
4. Conrad CE et al. (2015) A novel inert crystal delivery medium for serial femtosecond crystallography. IUCrJ 2:421-30
5. Sugahara M et al. (2016) Oil-free hyaluronic acid matrix for serial femtosecond crystallography. Sci Rep. 6:24484