Structural basis of the Methanothermobacter thermautotrophicus MCM helicase activity

The MCM complex from the archaeon Methanother-mobacter thermautotrophicus is a model for the eukaryotic MCM2-7 helicase. We present electron-microscopy single-particle reconstructions of a DNA treated M.thermautotrophicus MCM sample and a ADP·AlF(x) treated sample, respectively assembling as double hexamers and double heptamers. The electron-density maps display an unexpected asymmetry between the two rings, suggesting that large conformational changes can occur within the complex. The structure of the MCM N-terminal domain, as well as the AAA+ and the C-terminal HTH dom-ains of ZraR can be fitted into the reconstructions. Distinct configurations can be modelled for the AAA+ and the HTH domains, suggesting the nature of the conformational change within the complex. The pre-sensor 1 and the helix 2 insertions, important for the activity, can be located pointing towards the centre of the channel in the presence of DNA. We propose a mechanistic model for the helicase activity, based on a ligand-controlled rotation of the AAA+ subunits

Image formation and image analysis in electron microscopy

Dit proefschrift, behandelt diverse onderwerpen die betrekking hebben op de beeldvorming en beeldanalyse in de elektronenonenmicroscopie. ... Zie: Samenvatting

Molecular shape of Lumbricus terrestris erythrocruorin studied by electron microscopy and image analysis

The molecular structure of erythrocruorin (hemoglobin) from Lumbricus terrestris has been studied by electron microscopy of negatively stained particles. Over 1000 molecular projections were selected from a number of electron micrographs and were then classified by multivariate statistical image-processing techniques. The two main groups of top and side views were each subdivided into smaller classes with significantly different features. About half of the top-view projections exhibit perfect hexagonal symmetry at the current resolution of about 2.0 nm, while the other top views lack this symmetry, probably as a result of tilting of the molecules relative to the carbon support film. The side views were separated into two 'families', each associated with the two different stable side-view positions the molecules can take. From these narrow stable side-views, the two families of projections are, again, generated by tilting. The symmetry properties of the three non-tilted projections show that Lumbricus erythrocruorin has a pointgroup D6 (622) symmetry rather than D3 (32).