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Niels Volkmann

Publications and source records attributed to Niels Volkmann.

6 recordsLinked to original sources
PubMed2003

Full-length archaeal Rad51 structure and mutants: mechanisms for RAD51 assembly and control by BRCA2.

To clarify RAD51 interactions controlling homologous recombination, we report here the crystal structure of the full-length RAD51 homolog from Pyrococcus furiosus. The structure reveals how RAD51 proteins assemble into inactive heptameric rings and active DNA-bound filaments matching three-dimensional electron microscopy reconstructions. A polymerization motif (RAD51-PM) tethers individual subunits together to form assemblies. Subunit interactions support an allosteric 'switch' promoting ATPase activity and DNA binding roles for the N-terminal domain helix-hairpin-helix (HhH) motif. Structural and mutational results characterize RAD51 interactions with the breast cancer susceptibility protein BRCA2 in higher eukaryotes. A designed P.furiosus RAD51 mutant binds BRC repeats and forms BRCA2-dependent nuclear foci in human cells in response to gamma-irradiation-induced DNA damage, similar to human RAD51. These results show that BRCA2 repeats mimic the RAD51-PM and imply analogous RAD51 interactions with RAD52 and RAD54. Both BRCA2 and RAD54 may act as antagonists and chaperones for RAD51 filament assembly by coupling RAD51 interface exchanges with DNA binding. Together, these structural and mutational results support an interface exchange hypothesis for coordinated protein interactions in homologous recombination.

Adenosine Triphosphatases↗
PubMed2003

Myosin isoforms show unique conformations in the actin-bound state.

Crystallographic data for several myosin isoforms have provided evidence for at least two conformations in the absence of actin: a prehydrolysis state that is similar to the original nucleotide-free chicken skeletal subfragment-1 (S1) structure, and a transition-state structure that favors hydrolysis. These weak-binding states differ in the extent of closure of the cleft that divides the actin-binding region of the myosin and the position of the light chain binding domain or lever arm that is believed to be associated with force generation. Previously, we provided insights into the interaction of smooth-muscle S1 with actin by computer-based fitting of crystal structures into three-dimensional reconstructions obtained by electron cryomicroscopy. Here, we analyze the conformations of actin-bound chicken skeletal muscle S1. We conclude that both myosin isoforms in the nucleotide-free, actin-bound state can achieve a more tightly closed cleft, a more downward position of the lever arm, and more stable surface loops than those seen in the available crystal structures, indicating the existence of unique actin-bound conformations.

Actins↗
PubMed2003

Electron microscopy.

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Computational Biology↗
PubMed2002

Microscopic evidence for a minus-end-directed power stroke in the kinesin motor ncd.

We used cryo-electron microscopy and image reconstruction to investigate the structure and microtubule-binding configurations of dimeric non-claret disjunctional (ncd) motor domains under various nucleotide conditions, and applied molecular docking using ncd's dimeric X-ray structure to generate a mechanistic model for force transduction. To visualize the alpha-helical coiled-coil neck better, we engineered an SH3 domain to the N-terminal end of our ncd construct (296-700). Ncd exhibits strikingly different nucleotide-dependent three-dimensional conformations and microtubule-binding patterns from those of conventional kinesin. In the absence of nucleotide, the neck adapts a configuration close to that found in the X-ray structure with stable interactions between the neck and motor core domain. Minus-end-directed movement is based mainly on two key events: (i) the stable neck-core interactions in ncd generate a binding geometry between motor and microtubule which places the motor ahead of its cargo in the minus-end direction; and (ii) after the uptake of ATP, the two heads rearrange their position relative to each other in a way that promotes a swing of the neck in the minus-end direction.

Adenosine Diphosphate↗
PubMedDate not supplied

A novel three-dimensional variant of the watershed transform for segmentation of electron density maps.

Electron density maps at moderate resolution are often difficult to interpret due to the lack of recognizable features. This is especially true for electron tomograms that suffer in addition to the resolution limitation from low signal-to-noise ratios. Reliable segmentation of such maps into smaller, manageable units can greatly facilitate interpretation. Here, we present a segmentation approach targeting three-dimensional electron density maps derived by electron microscopy. The approach consists of a novel three-dimensional variant of the immersion-based watershed algorithm. We tested the algorithm on calculated data and applied it to a wide variety of electron density maps ranging from reconstructions of single macromolecules to tomograms of subcellular structures. The results indicate that the algorithm is reliable, efficient, accurate, and applicable to a wide variety of biological problems.

Actin Cytoskeleton↗