Molecular self-assembly with scaffolded DNA origami enables building custom-shaped nanometer-scale objects with molecular weights in the megadalton regime. Here we provide a practical guide for design and assembly of scaffolded DNA origami objects. We also introduce a computational tool for predicting the structure of DNA origami objects and provide information on the conditions under which DNA origami objects can be expected to maintain their structure.

We present an approach that allows us to simultaneously access structure and dynamics of a multi-domain protein in solution. Dynamic domain arrangements are experimentally determined by combining self-consistent networks of distance distributions with known domain structures. Local structural dynamics are correlated with the global arrangements by analyzing networks of time-resolved single-molecule fluorescence parameters. The strength of this hybrid approach is shown by an application to the flexible multi-domain Hsp90. The average solution structure of Hsp90’s closed state resembles the known x-ray crystal structure with Angstrom precision. The open state is represented by an ensemble of conformations with inter-domain fluctuations of up to 25 Å. The data reveal a state-specific suppression of the sub-millisecond fluctuations by dynamic protein-protein interaction. Finally, the method enables localization and functional characterization of dynamic elements and domain interfaces.

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