Download Student project proposal

15.10.15
Student project proposal
Submitted by: Nadia Opara
PhD student at C-CINA (group of Prof. Henning Stahlberg, Dr. Thomas Braun)
e-mail: [email protected]
Topic: High quality sample of protein nanocrystals from
nanoliter volumes for electron diffraction data collection.
Main aim of the project – establish reproducible conditions for
protein nanocrystal delivery using minimal sample volumes.
Fast and lossless sample delivery system to the electron microscope (EM)
grid is a prerequisite for optimal exploitation of biological material - usually
available in limited amounts - in structural analysis. Additionally, advent of the
new generation of high-speed cameras allows collection diffraction data of high
resolution. In the view of recent advances in structural analysis well-established
path for nanocrystal protein sample preparation is of the high importance.
Trehalose embedding technique was reported as a successful method to
preserve 2D protein crystals especially for high-resolution transmission electron
microscopy (TEM) studies [1, 2]. Recently it was proven that, this method can be
also adapted for adequately small (sufficiently thin) 3D protein crystals for
electron diffraction data collection (Fig. 1) [3]. Nanoliter volumes of protein
crystals solution initially sugar embedded were deposited on EM grid.
A)
B)
Fig. 1 A) Nanocrystals deposited on TEM grid, B) Electron diffraction pattern of lysozyme
The lysozyme protein was used as model system since is readily forming
3D crystals in broad spectrum of chemical conditions. Final dimensions of the
grown protein crystal depend on physical and biochemical system parameters
like: protein concentration, pH, temperature or concentration of precipitat [4].
However, unlike lysozyme, many biologically important proteins are
difficult to obtain in large quantities and forms very small crystals (nanocrystals)
useless for other methods exploited in structural biology.
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Protein crystals in size (thickness) optimal for diffraction and
tomography studies (100-200 nm) can be obtained by crystallization experiment
using vapor diffusion method on either standard sitting drop plate with 96 wells
or silicon chip with anisotropically etched cavities (Fig. 2). Both systems are
compatible with nanovolume dispensing unit together with dew point stage
developed at C-CINA [3], which allows deposition at low temperatures limiting
evaporation of the low-volume sample.
Fig. 2 4x4 array of wells on silicon chip for protein crystallization.
Main advantage of pristine silicon single crystal material is its very good
heat conductivity. Protein crystallization chambers made of silicon can be used
for tests, which require conditions of different temperatures. Application of the
thermal cyclic changes can lead to melting and regrowth of the crystalline
material and improve crystal quality [5]. Nonetheless, in this method protein
material is prone to clustering in the edges of the cavities (Fig. 2).
During realization of the project evaluation of different geometries of
protein growth – sitting drop plate geometry vs silicon chip with cavities using
selected physico-chemical conditions will lead to determination parameters for
growth the well-defined protein crystal size diffracting to the high resolution.
Available at C-CINA electron microscopy equipment will be used to examine of
the quality of the delivered nanocrystalline material.
References:
[1] The use of trehalose in the preparation of specimens for molecular electron
microscopy, Chiu et al., Micron 42, 762-772 (2011)
[2] Low-dose electron diffraction of catalase crystals dried with a matrix of the
disaccharide, trehalose: Is a “dry protein crystallography” feasible?, W. H.
Massover, Microsc. Microanal. 8 (Suppl.2) (2002)
[3] S. Arnold et al., Electron microscopy sample preparation from nanoliter
volumes – poster presented at SNI annual meeting, (September 2015)
[4] Generation of Size-Controlled, Submicrometer Protein Crystals, Falkner et al,
Chem. Mater., 17, 2679-2686 (2005)
[5] Growth of crystals in optical tweezers, U. Gibson et al, Proc. SPIE 5930,
Optical Trapping and Optical Micromanipulation II, 593014 (2005)
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