A novel free-mounting system for protein crystals: transformation and improvement of diffraction power by accurately controlled humidity changes

Abstract

A novel device for capillary-free mounting of protein crystals is described. A controlled stream of air allows an accurate adjustment of the humidity at the crystal. The crystal is held on the tip of a micropipette. With a video system (CCD camera), the two-dimensional shadow projections of crystals can be recorded for optical analysis. Instead of the micropipette, a standard loop can also be used. Experiments and results for different crystal systems demonstrate the use of this method, also in combination with shock-freezing, to improve crystal order. Working with oxygen-free gases offers the possibility of crystal measurements under anaerobic conditions. Furthermore, the controlled application of arbitrary volatile substances with the gas stream is practicable.

The crystal structure of dipeptidyl peptidase IV (CD26) reveals its functional regulation and enzymatic mechanism

“…The structure was confirmed by 1 H-NMR, 13 volumes of protein at a concentration of 20 mg͞ml with the reservoir solution (20-22% PEG 2000͞0.1 M ammonium sulfate͞ 0.1 M Tris⅐HCl, pH 8.0) by using the sitting drop vapor diffusion method. We covered the crystallization drop with perfluoropolyether (PFPE) oil and harvested the crystals by using a loop with humidity control (22). The relative humidity was ramped down from 96.5% to 86.5% by using a gradient of 0.5% (150 s Ϫ1 ), leading to a dramatically improved diffraction pattern, typically from below 10 Å to 3 Å.…”

Crystallization, dehydration and experimental phasing of WbdD, a bifunctional kinase and methyltransferase fromEscherichia coliO9a

“…However, a facile and easily implementable protocol may extend the utility of dehydration. The procedure described here combines several previously published ideas (Kiefersauer et al, 2000;Heras & Martin, 2005) into an easy-to-use, low-cost and convenient workflow. It is not anticipated that this will necessarily lead to more success than other approaches, but it is straightforward and amenable to high throughput.…”

“…To investigate the role of crystal dehydration in determining data quality, we mounted crystals in a free-mounting system (FMS; Proteros; Kiefersauer et al, 2000) and lowered the relative humidity (r.h.) of the gas phase surrounding the crystal. During this process, diffraction snapshots were taken and these revealed a clear improvement in the diffraction quality when the r.h. was lowered to $86% (Fig.…”

“…Use of electron density as a molecular-replacement model with, for example, PHASER (McCoy et al, 2007) is now much more automated and this was also critical to our success. Although multiple cases are known in which crystals were successfully dehydrated to improve their diffraction properties (see, for example, Esnouf et al, 1998;Kiefersauer et al, 2000;Chotiyarnwong et al, 2007), it is still a technique that is rarely reported. This perhaps reflects frequent failure or technical difficulties.…”

“…Right to left: dehydration of a WbdD556 crystal using a free-mounting system (Kiefersauer et al, 2000). As indicated, the diffraction images were taken at different relative humidities (r.h.).…”