Abstract:The benefits of protein crystal growth in microgravity are well documented. The crystallization vessels currently employed for microgravity crystallization are far from optimal with regards to cost, sample volume, size, and ease of use. The use of microbatch experiments is a favorable alternative in each respect: 96 experiments of 0.5-2 microL volumes can be performed in a single microtiter tray measuring 5 x 8 cm and costing 1 pound sterling each. To date, the use of microbatch has not been pursued on account… Show more
“… Fig. 1 Crystallisation phase diagram and set ups using the modified hanging drop and microbatch methods ( a ) Schematic illustration of a protein crystallisation phase diagram showing that the adjustable parameters are precipitant or additive concentration, pH and temperature (Chayen 2004 ) ( b ) 24-well crystallisation tool with screwcaps incorporating the coverslips where crystallisation drops are set up, ( c ) the close-up of a single screw cap on its well, ( d ) standard microbatch under oil experiments, ( e ) inverted experiments (Chayen 2005 ; Khurshid and Chayen 2006 ) …”
Section: Introductionmentioning
confidence: 99%
“… ( b ) 24-well crystallisation tool with screwcaps incorporating the coverslips where crystallisation drops are set up, ( c ) the close-up of a single screw cap on its well, ( d ) standard microbatch under oil experiments, ( e ) inverted experiments (Chayen 2005 ; Khurshid and Chayen 2006 ) …”
The techniques of X-ray protein crystallography, NMR and high-resolution cryo-electron microscopy have all been used to determine the high-resolution structure of proteins. The most-commonly used method, however, remains X-ray crystallography but it does rely heavily on the production of suitable crystals. Indeed, the production of diffraction quality crystals remains the rate-limiting step for most protein systems. This mini-review highlights the crystallisation trials that used existing and newly developed crystallisation methods on two muscle protein targets - the actin binding domain (ABD) of α-actinin and the C0-C1 domain of human cardiac myosin binding protein C (cMyBP-C). Furthermore, using heterogenous nucleating agents the crystallisation of the C1 domain of cMyBP-C was successfully achieved in house along with preliminary actin binding studies using electron microscopy and co-sedimentation assays .
“… Fig. 1 Crystallisation phase diagram and set ups using the modified hanging drop and microbatch methods ( a ) Schematic illustration of a protein crystallisation phase diagram showing that the adjustable parameters are precipitant or additive concentration, pH and temperature (Chayen 2004 ) ( b ) 24-well crystallisation tool with screwcaps incorporating the coverslips where crystallisation drops are set up, ( c ) the close-up of a single screw cap on its well, ( d ) standard microbatch under oil experiments, ( e ) inverted experiments (Chayen 2005 ; Khurshid and Chayen 2006 ) …”
Section: Introductionmentioning
confidence: 99%
“… ( b ) 24-well crystallisation tool with screwcaps incorporating the coverslips where crystallisation drops are set up, ( c ) the close-up of a single screw cap on its well, ( d ) standard microbatch under oil experiments, ( e ) inverted experiments (Chayen 2005 ; Khurshid and Chayen 2006 ) …”
The techniques of X-ray protein crystallography, NMR and high-resolution cryo-electron microscopy have all been used to determine the high-resolution structure of proteins. The most-commonly used method, however, remains X-ray crystallography but it does rely heavily on the production of suitable crystals. Indeed, the production of diffraction quality crystals remains the rate-limiting step for most protein systems. This mini-review highlights the crystallisation trials that used existing and newly developed crystallisation methods on two muscle protein targets - the actin binding domain (ABD) of α-actinin and the C0-C1 domain of human cardiac myosin binding protein C (cMyBP-C). Furthermore, using heterogenous nucleating agents the crystallisation of the C1 domain of cMyBP-C was successfully achieved in house along with preliminary actin binding studies using electron microscopy and co-sedimentation assays .
“…New crystallization methods and techniques are very often tested on readily available and easily crystallized proteins, such as lysozyme, glucose isomerase, or thaumatin (for recent examples, see Dunlop & Hazes, 2005;Hansen et al, 2006;Khurshid & Chayen, 2006, among many others). However, this approach is not too informative as these model proteins usually crystallize very readily; it is relatively difficult to find conditions in which lysozyme does not crystallize (Chayen & Saridakis, 2001).…”
Growing crystals is a critical step in the process of determining the 3-D structure of macromolecules by X-ray crystallography. While significant progress has been made in analyzing quantitatively crystallization experiments, in general, crystallization of biological macromolecules remains more of an art than a science. This work presents Xtaldb, an expert system for the quantitative analysis of crystallization experiments.Xtaldb provides tools for efficiently designing crystallization screens, tracks in a semiautomatic manner most of the parameters of the experiments, and provides sophisticated search, analysis, and data graphing tools. The algorithm used to produce balanced random screens is the fastest and most robust available. Xtaldb was tested on a set of six novel proteins that had failed to produce diffraction-quality crystals in a high-throughput structure determination pipeline. Of them, five yielded crystals diffracting to 3.5 Å or better and three 3-D structures were elucidated. One of the three proteins was YfbR, a member of the HD-domain phosphohydrolase superfamily. Structural analysis of the 3-D structure and biochemical work confirmed phosphohydrolase activity. Further studies by a collaborator demonstrated that YfbR is a 5'-deoxynucleotidase. Xtaldb was used to produce two crystals of catalytically inactive YfbR mutants in the presence of metal cofactor and the substrates TMP or dAMP. The structures of the complexes explained the mechanism of the unique pattern of substrate selectivity, further supported by computational docking studies. The complex structures suggested a plausible atomic mechanism of catalysis for the enzyme, the first proposed mechanism for an HD-domain phosphohydrolase based directly from enzyme-substrate complex structures.
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