Continuous and Discontinuous Changes in the Unit Cell of HIV-1 Reverse Transcriptase Crystals on Dehydration

A signi®cant improvement in the X-ray resolution of crystals of Escherichia coli inorganic pyrophosphatase at cryotemperature was obtained as a result of studying the relationship between the crystal order and cryosolution component concentrations. To perform the experiments, the ability to reverse the¯ash-cooling process and to return a crystal to ambient temperature was used. In each cycle, the crystal was transferred from a cold nitrogen-gas stream to a cryosolution with modi®ed concentrations of the components. The crystal was then¯ash-cooled again and the diffraction quality checked. Such a technique allows the screening of a wide concentration range rather quickly without using a large number of crystals and allows the determination of optimal cryosolution component concentrations. The resolution limit for crystals of pyrophosphatase increased by almost 0.7 A Ê , from 1.8 to 1.15 A Ê .

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Improving the X-ray resolution by reversible flash-cooling combined with concentration screening, as exemplified with PPase

Samygina¹,

Antonyuk²,

Lamzin³

et al. 2000

“…Below 86% r.h. the diffraction quality deteriorated and the process was not reversible. This is reminiscent of the complex dehydration behaviour observed in HIV-1 reverse transcriptase crystals (Esnouf et al, 1998). WbdD556 crystals often cracked during dehydration with the FMS, degrading the diffraction quality.…”

Section: Figurementioning

confidence: 99%

“…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.…”

Section: Effect Of Dehydration On the Structurementioning

confidence: 99%

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

Hagelueken

Huang

Harlos

et al. 2012

WbdD is a bifunctional kinase/methyltransferase that is responsible for regulation of lipopolysaccharide O antigen polysaccharide chain length in Escherichia coli serotype O9a. Solving the crystal structure of this protein proved to be a challenge because the available crystals belonging to space group I23 only diffracted to low resolution (>95% of the crystals diffracted to resolution lower than 4 Å and most only to 8 Å ) and were non-isomorphous, with changes in unit-cell dimensions of greater than 10%. Data from a serendipitously found single native crystal that diffracted to 3.0 Å resolution were non-isomorphous with a lower (3.5 Å ) resolution selenomethionine data set. Here, a strategy for improving poor (3.5 Å resolution) initial phases by density modification and cross-crystal averaging with an additional 4.2 Å resolution data set to build a crude model of WbdD is desribed. Using this crude model as a mask to cut out the 3.5 Å resolution electron density yielded a successful molecular-replacement solution of the 3.0 Å resolution data set. The resulting map was used to build a complete model of WbdD. The hydration status of individual crystals appears to underpin the variable diffraction quality of WbdD crystals. After the initial structure had been solved, methods to control the hydration status of WbdD were developed and it was thus possible to routinely obtain high-resolution diffraction (to better than 2.5 Å resolution). This novel and facile crystal-dehydration protocol may be useful for similar challenging situations.

“…In addition, it is fairly common that crystals of large macromolecular complexes such as the ribosome suffer from a lack of isomorphism on cryo-cooling. This apparent problem could be exploited to provide a vehicle for phasing such complexes, because of the differential sampling of the molecular transform which occurs on changes in the unit cell (Esnouf et al, 1998;Ren et al, 1995). Thus, EM reconstructions could provide starting models for each of the crystal forms and would establish initial values for the rotation matrices and translation vectors which relate the molecules in the different forms prior to non-crystallographic averaging between them.…”

Section: Prospects For the Futurementioning

confidence: 99%

Complementing crystallography: the role of cryo-electron microscopy in structural biology

Grimes

Fuller

Stuart

1999

Dramatic improvements in experimental methods and computational techniques have revolutionized three-dimensional image reconstruction from electron micrographs (EM) of vitri®ed samples. Recent results include the ®rst determination of a protein fold (for the core protein of the hepatitis B virus) by non-crystalline imaging techniques. These developments have generated interest within the crystallographic community and have led to a re-evaluation of the technique, particularly amongst those working in the ®eld of virus structure or struggling with the phasing of large macromolecular assemblies. A simple discussion of the techniques of EM image reconstruction and its advantages and problems in terms familiar to crystallographers will hopefully allow an appreciation of the essential complementarity of the two techniques and the practical potentials for phasing applications.