Abstract
We studied laser-microdissection of standard and Langmuir-Blodgett (LB) nanotemplate protein crystals in glycerol solution. The time required for microdissection was significantly longer for LB-crystals as compared to standardcrystals which also more rapidly dissolve. Microfragmentation of lysozyme crystals was observed after extended solvent exposure. Synchrotron radiation nanobeam mapping allowed localizing and aligning cryofrozen lysozyme microfragments. 3D data-sets obtained from two microfragments were refined to atomic resolution. The well-defined electron density maps showed no evidence for damage of radiation of sensitive side-groups. Our results suggest applications of laser-microdissection techniques in structural studies on crystals with a high mosaicity. They also provide a new window for the characterization of protein crystal organization down to the submicron scale, pointing to a new emerging biophysical technique.
Highlights
While, a lot of efforts are being invested at Synchrotron Radiation (SR) sources on high-throughput protein-to-structure pipe-lines in the context of structural genomics [1,2,3,4], methods allowing manipulating and tailoring of protein micro-crystals in a laboratory or SR-beamline environment have been less explored
We note the idealized model of a protein crystal composed of coherently scattering mosaic blocks [5] which can be experimentally studied by X-ray line-profile analysis or X-ray topography [6,7]
We are aware that the selection of mosaic blocks for X-ray nanodiffraction techniques would represent a methodological progress and could in particular improve the quality of structural refinements for high mosaicity crystals
Summary
A lot of efforts are being invested at Synchrotron Radiation (SR) sources on high-throughput protein-to-structure pipe-lines in the context of structural genomics [1,2,3,4], methods allowing manipulating and tailoring of protein micro-crystals in a laboratory or SR-beamline environment have been less explored Such techniques could, be useful for separating and sorting crystals from a batch, for dissection of crystals, polymorphs or crystalline domains from an aggregate or a cellular environment. We are aware that the selection of mosaic blocks for X-ray nanodiffraction techniques would represent a methodological progress and could in particular improve the quality of structural refinements for high mosaicity crystals For all of these reasons it is of interest exploring techniques allowing dissecting protein crystals, down to the level of individual mosaic blocks
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