Capturing the dynamic 3D atomic-scale structure of a macromolecular machine while it performs its biological function remains an outstanding goal of biology. Here we provide an update on our project to combine (prior) information from multiple existing static structures of stable states with dynamic datasets of inter-atomic distances obtained by high-throughput non-equilibrium single-molecule FRET (smFRET) measurements in a microfluidic mixer using novel time-resolved multi-pixel single-photon avalanche diode detector. These measurements, performed on libraries of molecular constructs, will sample multiple inter-atomic distances as function of reaction time. The measured distance distributions, together with additional information provided by cross-linking experiments analyzed by mass spectrometry, will then serve as multiple intra- and inter-domain distance constraints which, together with prior information from available structures, will enable large-scale computational energy optimization-based refinement of time- resolved ‘snap shots’ of complex structures with improved accuracy. These time-resolved computational structures, together with intermediary molecular dynamics simulations, will allow solving the 3D atomic-level structure of the macromolecule for each sampled reaction time point, eventually producing a 3D structural dynamic movie of the macromolecule in action. To demonstrate the utility of the proposed method, we study on one hand the dynamic structure of RNA polymerase during transcription initiation (promoter binding, bubble opening, abortive initiation, promoter clearance) and, on the other hand, a pair of intrinsically disordered proteins (IDPs) involved in transcription regulation (ACTR and NCBD) that adopt a fully folded structure during a coupled folding and binding reaction. In addition to elucidating outstanding questions in transcription by combining detector developments, high-throughput and time- resolved out-of-equilibrium single-molecule FRET measurements with new experimentally-constrained molecular structure computational approaches, this multidisciplinary project aims to provide a new generic toolkit applicable to a large array of enzymes, proteins and molecular machines.

3D atomic-scale movies of molecular machines in action

Andrea Bonzi;Serena Farina;Ivan Rech;Angelo Gulinatti;
2021

Abstract

Capturing the dynamic 3D atomic-scale structure of a macromolecular machine while it performs its biological function remains an outstanding goal of biology. Here we provide an update on our project to combine (prior) information from multiple existing static structures of stable states with dynamic datasets of inter-atomic distances obtained by high-throughput non-equilibrium single-molecule FRET (smFRET) measurements in a microfluidic mixer using novel time-resolved multi-pixel single-photon avalanche diode detector. These measurements, performed on libraries of molecular constructs, will sample multiple inter-atomic distances as function of reaction time. The measured distance distributions, together with additional information provided by cross-linking experiments analyzed by mass spectrometry, will then serve as multiple intra- and inter-domain distance constraints which, together with prior information from available structures, will enable large-scale computational energy optimization-based refinement of time- resolved ‘snap shots’ of complex structures with improved accuracy. These time-resolved computational structures, together with intermediary molecular dynamics simulations, will allow solving the 3D atomic-level structure of the macromolecule for each sampled reaction time point, eventually producing a 3D structural dynamic movie of the macromolecule in action. To demonstrate the utility of the proposed method, we study on one hand the dynamic structure of RNA polymerase during transcription initiation (promoter binding, bubble opening, abortive initiation, promoter clearance) and, on the other hand, a pair of intrinsically disordered proteins (IDPs) involved in transcription regulation (ACTR and NCBD) that adopt a fully folded structure during a coupled folding and binding reaction. In addition to elucidating outstanding questions in transcription by combining detector developments, high-throughput and time- resolved out-of-equilibrium single-molecule FRET measurements with new experimentally-constrained molecular structure computational approaches, this multidisciplinary project aims to provide a new generic toolkit applicable to a large array of enzymes, proteins and molecular machines.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11311/1208761
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