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Site-specific recognition of DNA in eukaryotic organisms depends on the arrangement of nucleosomes in chromatin. In the yeast Saccharomyces cerevisiae, ISW1a and related chromatin remodelling factors are implicated in establishing the nucleosome repeat during replication and altering nucleosome position to affect gene activity. Here we have solved the crystal structures of S. cerevisiae ISW1a lacking its ATPase domain both alone and with dNa bound at resolutions of 3.25 Å and 3.60 Å, respectively, and we have visualized two different nucleosome-containing remodelling complexes using cryo-electron microscopy. The composite X-ray and electron microscopy structures combined with site-directed photocrosslinking analyses of these complexes suggest that ISW1a uses a dinucleosome substrate for chromatin remodelling. Results from a remodelling assay corroborate the dinucleosome model. We show how a chromatin remodelling factor could set the spacing between two adjacent nucleosomes acting as a 'protein ruler'., DNA in eukaryotic cells is packaged in nucleosomes to comprise chromatin, serving generally to protect DNA integrity and to repress gene transcription. Chromatin structure depends on regular arrays of nucleosomes [...]

Author(s): Thomas Schalch [1]; Sylwia Duda [1]; David F. Sargent [1]; Timothy J. Richmond (corresponding author) [1] DNA in eukaryotic chromosomes is organized in arrays of nucleosomes compacted into chromatin [...]

Researchers have solved the high resolution X-ray structure of the nucleosome core particle. The crystal structure of the nucleosome core particle of chromatin includes a 146 base pair DNA molecule. The atomic model shows these pairs in a superhelix around the histone protein octamer. Each histone contains a histone fold domain and there are well-ordered structure elements extending from the histone motif. These two aspects determine the histone/histone and histone/DNA interactions. The DNA differs from ideal superhelix geometry due to the lack of uniformity in these interactions.

A study of the crystal structure of a yeast complex shows that the transcription factor IIA (TFIIA) has a boot-like appearance and consists of a 12-strand beta-barrel and a four-helix bundle motif. The beta-barrel helps in extending the TATA-box-binding protein (TBP) beta-sheet. The beta-barrel assists in bridging over the DNA major groove immediately against the TATA box. The four-helix bundle offers its substantial contribution to the surface of the TFIIA/TBP/DNA complex. The complex interacts with additional transcription factors.

A new micro-agent is proposed to assist in automated protein crystal harvesting. The microrobot, named the RodBot, is a wireless mobile device driven by rotating magnetic fields (field strength 5-10 mT). When the RodBot rolls on a substrate in a low Reynolds number liquid environment, it generates flows to lift up and trap crystals in a vortex above itself. The gentle fluidic force acting on the crystals is in the range of a few nanonewtons to tens of nanonewtons and is spread over the whole surface of the crystal. The RodBot is capable of trapping protein crystals ranging from a few micrometres to sub-millimetre size. The trapped crystal can be transported to and deposited onto a loop positioned to accept it, obviating the need for more complicated 'fishing' systems dependent on particular motions of the loop, the presence of further manipulators or the use of mechanical grippers. The RodBot can be driven in 24- and 96-well plates or in a crystal soaking dish, making the system compatible with existing crystallization hardware. [ABSTRACT FROM AUTHOR]

2-Hydroxybiphenyl 3-monooxygenase (HbpA; EC 1.14.13.44) from Pseudomonas azelaica HBP1 was produced in Escherichia coli both as native and SeMet-labelled protein. The two enzymes were purified to homogeneity and crystallized by the hanging-drop vapour diffusion method. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 108.6, b = 196.8, c = 79.3 Å, β = 97.7° for the native protein and a = 108.3, b = 196.8, c = 79.0 Å, β = 97.8° for SeMet HbpA. Crystal-packing considerations led to the assumption of two HbpA subunits per asymmetric unit, which corresponds to a V[sub M] value of 3.3 ų Da[sup -1] and a solvent content of 62%. The crystals were radiation-sensitive and only had a lifespan of about 120 s when exposed to synchrotron radiation on an undulator beamline. To obtain complete data sets, data were collected from 23 native and 26 derivative crystals. The high-resolution limit was 2.0 Å for native and 2.25 Å for SeMet HbpA. [ABSTRACT FROM AUTHOR]

Presents a letter to the editor about flash-cooling of macromolecular crystals to overcome increased mosaicity.