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Microscopy & Imaging JoVE (Open Access) Citable · DOI

Structural Information from Single-molecule FRET Experiments Using the Fast Nano-positioning System

DOI: 10.3791/54782-v
What you'll learn
  • ✓ Set up and execute smFRET measurements using TIRF microscopy for large donor-acceptor networks
  • ✓ Apply Bayesian inference with Fast-NPS software to analyze FRET efficiency data
  • ✓ Interpret credible volumes and dye models to extract high-resolution structural information
  • ✓ Model protein-nucleic acid complexes using smFRET data and nano-positioning analysis
Protocol

We present the setup and experimental procedure to obtain smFRET data from large donor-acceptor networks with a TIRF microscope. The step-by-step analysis of these measurements with the Bayesian inference software Fast-NPS yields high-resolved structural information via the application of adapted dye models.

Difficulty
advanced
Total time
~3–5 days (including sample preparation, image acquisition, and computational analysis per dataset)

Steps

1
Perform smFRET measurements with TIRF microscopy

Set up total internal reflection fluorescence (TIRF) microscopy to acquire single-molecule FRET data from samples containing large donor-acceptor dye networks. Configure optical path, laser lines, and detection parameters for simultaneous donor and acceptor signal acquisition.

▶ 00:57
2
Calculate mean FRET efficiency from raw data

Process acquired smFRET trajectories to determine mean FRET efficiency values for individual molecules or molecular complexes. Apply standard FRET calculation methods to establish baseline structural information.

▶ 05:19
3
Model credible volumes using Fast-NPS software

Apply the Fast Nano-positioning System (Fast-NPS) Bayesian inference software to FRET efficiency measurements. Generate credible volumes that define spatial restraints for dye positions within the macromolecular complex.

▶ 06:45
4
Interpret structural results for archaeal RNA polymerase

Analyze Fast-NPS modeling output to extract high-resolution structural information from smFRET data of an archaeal RNA polymerase open promoter complex. Validate dye models and interpret credible volume geometry.

▶ 10:09
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