Operator-assisted harvesting of protein crystals using a universal micromanipulation robot

Viola, R.; Carman, Peter; Walsh, Jace; Miller, Echo; Benning, Matthew M.; Frankel, Daniel; McPherson, Alexander; Cudney, Bob; Rupp, Bernhard

doi:10.1107/s0021889807012149

Cited by 26 publications

(32 citation statements)

References 34 publications

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“…In a subsequent paper [2] we described analyses of nine crystalline proteins grown in the original experiments, each obtained in the presence of a ''cocktail'' of low molecular weight compounds. Another, independent analysis was carried out on bovine trypsin crystals grown in our experiments in the presence of banzamidine and protamine [6].…”

Section: Resultsmentioning

confidence: 99%

Development of an alternative approach to protein crystallization

McPherson

Nguyen²,

Larson

et al. 2007

J Struct Funct Genomics

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We are developing an alternate strategy for the crystallization of macromolecules that does not, like current methods, depend on the optimization of traditional variables such as pH and precipitant concentration, but is based on the hypothesis that many conventional small molecules might establish stabilizing, intermolecular, non covalent crosslinks in crystals, and thereby promote lattice formation. To test the hypothesis, we carried out preliminary experiments encompassing 18,240 crystallization trials using 81 different proteins, and 200 chemical compounds. Statistical analysis of the results demonstrated the validity of the idea. In addition, we conducted X-ray diffraction analyses of some of the crystals grown in the experiments. These clearly showed incorporation of conventional molecules into the protein crystal lattices, and further validated the underlying hypothesis. We are currently extending the investigations to include a broader and more diverse set of proteins, an expanded search of conventional and biologically active small molecules, and a wider range of precipitants. The strategy proposed here is essentially orthogonal to current approaches and has an objective of doubling the success rate of today.

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Section: Resultsmentioning

confidence: 99%

Development of an alternative approach to protein crystallization

McPherson

Nguyen²,

Larson

et al. 2007

J Struct Funct Genomics

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“…There are many accepted and published cases where such ligands have been observed in Fo‐Fc electron density maps below 3 σ cut‐off (Table ). For example, the F o‐ F c electron density maps for 2J9N and 3LK0, two structures from the challengers themselves, do not show clear density for the modeled ligands at 3 σ cut‐off as shown in Figure . Additionally, it was emphasized that the overall B factors of neighboring components in a crystal structure cannot differ drastically .…”

Section: Challenging Publications Involving Structural Datamentioning

confidence: 97%

“… Delete‐refine F o‐ F c maps for representative structures. (A) Maps for one of the two ligands in the asymmetric unit in PDB: 3LK0 , contoured at 3.0 σ and 1.8 σ and (B) maps for two peptides in the asymmetric unit in PDB: 2J9N contoured at 3.0 σ and 2.0 σ . Positive and negative electron densities are shown in green and red colors, respectively.…”

Section: Parameters For Quality Analysismentioning

confidence: 99%

Macromolecular structures: Quality assessment and biological interpretation

Salunke

Nair

2017

IUBMB Life

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Structure determination using X-ray crystallography involves collection of diffraction data, determination of initial phases followed by iterative rounds of model building and crystallographic refinement to improve the phases and minimize the differences between calculated and observed structure factors. At each of these stages, a variety of statistical filters exist to ensure appropriate validation. Biologically important observations often come from interpretations of signals that need to be carefully deciphered from noise, and therefore human intervention is as important as the automated methods and filters. Currently, all structural data are deposited in the Protein Data Bank (PDB), and this repository is continuously evolving to incorporate new developments in macromolecular crystallography. The journals that publish data arising from structural studies modulate their policies to take cognizance of new improved methodologies. Together, the PDB and journals have evolved an accepted protocol to ensure the integrity of crystallographic results. As a result, the quality of available data and associated interpretations have improved over the years. Typically, if there are differences regarding the mechanism of action of a protein revealed by crystallography then new experiments are carried out to provide further evidence for or against a particular hypothesis. Hence, the scientific systems in structural biology are robust and to a large extent capable of correcting any errors in interpretation of structural results. However, this process is hindered by spurious challenges to published research, based on misuse of validation mechanisms. Such activities are counterproductive and will cause damage to the field of structural biology. © 2017 IUBMB Life, 69(8):563-571, 2017.

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“…Initial analysis predominantly relies on a trained observer characterizing crystals visually. Robotic crystal mounting which is being actively developed98, 99, promises to apply reproducible mounting protocols such that initial leads can be examined with an X-ray beam. Similarly in situ diffraction can enhance the process considerably with less sample manipulation.…”

Section: Expert Opinionmentioning

confidence: 99%

Lessons from high-throughput protein crystallization screening: 10 years of practical experience

Luft

Snell

DeTitta

2011

Expert Opinion on Drug Discovery

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Introduction X-ray crystallography provides the majority of our structural biological knowledge at a molecular level and in terms of pharmaceutical design is a valuable tool to accelerate discovery. It is the premier technique in the field, but its usefulness is significantly limited by the need to grow well-diffracting crystals. It is for this reason that high-throughput crystallization has become a key technology that has matured over the past 10 years through the field of structural genomics. Areas covered The authors describe their experiences in high-throughput crystallization screening in the context of structural genomics and the general biomedical community. They focus on the lessons learnt from the operation of a high-throughput crystallization screening laboratory, which to date has screened over 12,500 biological macromolecules. They also describe the approaches taken to maximize the success while minimizing the effort. Through this, the authors hope that the reader will gain an insight into the efficient design of a laboratory and protocols to accomplish high-throughput crystallization on a single-, multiuser-laboratory or industrial scale. Expert Opinion High-throughput crystallization screening is readily available but, despite the power of the crystallographic technique, getting crystals is still not a solved problem. High-throughput approaches can help when used skillfully; however, they still require human input in the detailed analysis and interpretation of results to be more successful.

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Operator-assisted harvesting of protein crystals using a universal micromanipulation robot

Cited by 26 publications

References 34 publications

Development of an alternative approach to protein crystallization

Development of an alternative approach to protein crystallization

Macromolecular structures: Quality assessment and biological interpretation

Lessons from high-throughput protein crystallization screening: 10 years of practical experience

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