Advances in digital topography for characterizing imperfections in protein crystals

Lovelace, Jeffrey J.; Murphy, Cameron R.; Bellamy, Henry D.; Brister, Keith; Pahl, R.; Borgstahl, Gloria E. O.

doi:10.1107/s0021889805009234

Cited by 7 publications

(11 citation statements)

References 20 publications

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“…Cryocooling in the capillary also worked very well. An increase in mosaicity was expected after cryocooling and the ' R values were near 0.40 , which is comparable with the best mosaicities reported for cryocooled lysozyme crystals [0.47 (Sauter et al, 2001) and 0.33 (Lovelace et al, 2005)]. This result indicates that the cryocooling in a capillary with epoxy to immobilize the crystal works similarly to the loop method with glycerol and oil that we had used previously (Lovelace et al, 2005).…”

Section: Sample Mountingsupporting

confidence: 85%

“…The other was the collection of single-reflection topographs using a high-resolution imaging CCD. The topography system was installed parasitically at the beamline as described previously (Lovelace et al, 2005). This method has the benefit of allowing topographs to be collected with the existing beamline control software and collecting conventional data when the topography camera is stowed.…”

Section: Overall Experimental Setupmentioning

confidence: 99%

“…A multi-step process was used to clean-up and correct the data. The steps included subtraction of the dark current, a genetic-algorithm-based correction of a gain imbalance between odd and even pixels, and a wavelet-based resolution enhancement (Lovelace, Soares et al, 2004;Lovelace et al, 2005). This procedure was automated with Matlab (The Math Works Inc., 1992).…”

Section: Processing Of Digital Topography Fine-u-sliced Datamentioning

confidence: 99%

“…Such highresolution CCDs generally cover only a very small active area ($8 Â 8 mm) as compared with CCD-based area detectors ($200 Â 200 mm) commonly used in protein crystallography. In order to index the crystal as well as collect topographs, we use both the large-area detector and the topography CCD (Lovelace et al, 2005). Fig.…”

Section: Introductionmentioning

confidence: 99%

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Tracking reflections through cryogenic cooling with topography

et al. 2006

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The mosaic structure of a single protein crystal was analyzed by reflection profiling and topography using highly parallel and monochromatic synchrotron radiation. Fine-'-sliced diffraction images (0.002 stills) were collected using a conventional large-area CCD detector in order to calculate reflection profiles. Fine-'-sliced topographic data (0.002 ) stills were collected with a digital topography system for three reflections in a region where the Lorentz effect was minimized. At room temperature, several different mosaic domains were clearly visible within the crystal. Without altering the crystal orientation, the crystal was cryogenically frozen (cryocooled) and the experiment was repeated for the same three reflections. Topographs at cryogenic temperatures reveal a significantly increased mosaicity, while the original domain structure is maintained. A model for the observed changes during cryocooling is presented.research papers

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Section: Sample Mountingsupporting

confidence: 85%

Section: Overall Experimental Setupmentioning

confidence: 99%

Section: Processing Of Digital Topography Fine-u-sliced Datamentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Tracking reflections through cryogenic cooling with topography

et al. 2006

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“…In order to avoid this problem, a differential evolution algorithm was used to fit anisotropic mosaicities from the data (Wormington et al, 1999). The differential evolution algorithm allows constrained fitting and has proven robust for other applications (Wormington et al, 1999;Vahedi-Faridi et al, 2003;Lovelace et al, 2004Lovelace et al, , 2005Lovelace et al, , 2006. Once the anisotropic mosaicities had been calculated, the Pearson's coefficient of regression (R 2 ) was calulated in the standard fashion.…”

Section: Data Processingmentioning

confidence: 99%

Changes to crystals ofEscherichia coliβ-galactosidase during room-temperature/low-temperature cycling and their relation to cryo-annealing

Juers

Lovelace

Bellamy

et al. 2007

Acta Crystallogr D Biol Crystallogr

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Flash-cooling of macromolecular crystals often compromises diffraction quality by increasing the mosaicity. In some cases, cycling the crystal between low temperature (LT) and room temperature (RT) can reverse this increase in mosaicity. Previous studies of RT/LT cycling have focused on the quality of the crystal as it was repeatedly returned to the LT state. Here, crystal quality is explored not only at LT but also when the crystal is returned to RT. The domain model is used to extract information about crystal order from reflection profiles measured from crystals of Escherichia coli beta-galactosidase at both temperatures. Despite optimization of the cryocooling protocol, the mosaicity increases by about sixfold with cooling and is anisotropic at both temperatures. The mosaicity increase is the consequence of a decrease in domain volume, an increase in the variation of domain cell dimensions and an increase in the angular spread between domains. Upon rewarming, the mosaicity recovers substantially, including the somewhat surprising recovery of domain volume, but incompletely. Over multiple RT/LT cycles disorder in both states increases, which appears to mainly arise from radiation damage, although a contribution from cool-thaw processes cannot be ruled out. The analysis further suggests that LT disorder is governed by variability inherent in the cooling process combined with the overall history of the crystal. In contrast, RT disorder appears to be governed principally by the overall history of the crystal. This suggests that with these particular crystals under the experimental conditions used, particularly at high-intensity synchrotron X-ray sources, RT/LT cycling annealing protocols should involve few cycles so as to limit the hysteresis in both temperature states while taking advantage of the inherent variability in the cooling process that may result in improved crystal order at LT.

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Molecular structure from X-ray diffraction

Powell

2006

Annu. Rep. Prog. Chem., Sect. C: Phys. Chem.

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While there are some developments in structure solution and refinement, these represent gradual improvements rather than fundamental leaps in methodology. An exception is the assessment of crystal quality by X-ray diffraction topography using a dedicated solid-state electronic detector; this is an elegant example where the precise physical nature of the crystal has been studied and analyzed. Similarly, using medial axis analysis of calculated electron density potentially extends automatic structure building to substantially lower resolution. Phase analysis by crystallography of powdered lysozyme shows that this is likely to be of real use, rather than an intellectual curiosity.

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Advances in digital topography for characterizing imperfections in protein crystals

Cited by 7 publications

References 20 publications

Tracking reflections through cryogenic cooling with topography

Tracking reflections through cryogenic cooling with topography

Changes to crystals ofEscherichia coliβ-galactosidase during room-temperature/low-temperature cycling and their relation to cryo-annealing

Molecular structure from X-ray diffraction

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