Abstract
Neutron diffraction experiments are informative for determining the locations of hydrogen atoms in protein molecules; however, much larger crystals are needed than those required for X-ray diffraction. Thus, additional techniques are required to grow larger crystals. Here, a unique crystallization device and strategy for growing large protein crystals are introduced. The device uses two micropumps to control crystal growth by altering the precipitant concentration and regulating the pinpoint injection of dry air flow to the crystallization cell. Furthermore, the crystal growth can be observed in real time. Preliminary microbatch crystallization experiments at various concentration ranges of polyethylene glycol (PEG) 4000 and sodium chloride were first performed to elucidate optimized crystallization conditions. Based on these results, a device to precisely control the sodium chloride and PEG concentrations and the supply of dry air to the crystallization cell was used, and 1.8 mm lysozyme and 1.5 mm alpha-amylase crystals with good reproducibility were obtained. X-ray data sets of both crystals were collected at room temperature at BL2S1 of the Aichi Synchrotron Radiation Center and confirmed that these crystals were of high quality. Therefore, this crystallization device and strategy were effective for growing large, high-quality protein crystals.
Highlights
While X-ray diffraction only reveals the electron density, neutron diffraction can provide complementary data to X-ray diffraction regarding the position of hydrogen atoms and the protonation status to provide a complete biological structure [1]
Only 0.1% of the structural data deposited in the Research Collaboratory for Structural Bioinformatics Protein Data Bank (PDB) used neutron diffraction to determine the macromolecule structure
Because a few single crystals grew in low concentrations of polyethylene glycol (PEG) 4000 and/or sodium chloride, the concentrations were increased to 23% (w/v) PEG 4000 and
Summary
While X-ray diffraction only reveals the electron density, neutron diffraction can provide complementary data to X-ray diffraction regarding the position of hydrogen atoms and the protonation status to provide a complete biological structure [1]. This is mainly because neutron diffraction requires much larger cubic crystals (~1 mm3 ) compared with those needed for X-ray diffraction To grow such large crystals, the control of the nucleation to decrease its probability is one of the necessary processes. The method combines preliminary microbatch experiments with a numerical model, resulting in more efficient and rational crystallization experiments This method still required a prohibitively long time for growing large single crystals. Before attempting protein crystallization using this device, preliminary microbatch crystallization experiments were conducted These used a relatively small amount of protein sample to identify the optimized crystallization solution by defining the sodium chloride and PEG 4000 concentrations where the nucleation probability was low and single crystals would grow [12]. We present the results of using this device to grow large crystals of lysozyme and alphaamylase as example proteins
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