DOI: 10.5281/zenodo.14990529
title: Data for LRET-derived HADDOCK structural  models describe the conformational  heterogeneity required for DNA cleavage  by the Mre11-Rad50 DNA damage  repair complex

description: Time resolved lanthanide resonance energy transfer and small angle X-ray scattering data using to model structures of the ATP-bound P. furiosus Mre11-Rad50 DNA double strand break repair complex
description: PDBIHM_LRET_structures directory contains additional files corresponding to submissions of structures from the manuscript to PDB-IHM.
description: Labeling Rad50 cysteine mutants with LRET probes  Thiol-reactive Tb3+ chelate DTPA-cs124-EMPH (LanthaScreen, Life Technologies, Inc) was used as the LRET donor for all samples and Bodipy FL maleimide (Invitrogen) or Cyanine3 (Cy3) maleimide (GE Healthcare) were used as acceptor fluorophores. 50 μM purified Rad50NBD containing a single cysteine was combined with a twofold excess of one of the labels in degassed Labeling Buffer (25 mM Tris, pH 8, 100 mM NaCl, 10% glycerol, 1 mM TCEP) in 100 μL reactions. The reaction was incubated in the dark at room temperature for 1.5–2 hr. Labeling of full-length Rad50 cysteine mutants followed the same protocol, except the donor and one acceptor label were added simultaneously to each reaction (twofold molar ratio of each). Unreacted label was removed by running the labeling reaction over a Superdex 200 Increase 10/300 GL column (GE Healthcare) equilibrated with 25 mM HEPES, 200 mM NaCl, 10% glycerol, 1 mM TCEP, pH 7. Eluted protein was concentrated using centrifugal concentrators (VivaSpin, Sartorius) and then assessed for successful labeling by measuring fluorescence using the following settings and the monochromator in the BioTek Synergy Neo2 plate reader: ex337/em490 for DPTA-Tb3+, ex485/em515 for Bodipy FL, and ex535/em570 for Cy3. Concentrated labeled proteins were aliquoted, frozen in liquid nitrogen, and stored at –80°C.  LRET data collection and analysis  For LRET experiments, 40 μL mixtures of 1 μM Mre11 + 0.5 μM Tb 3+-labeled Rad50NBD + 0.5 μM Bodipyor Cy3-labeled Rad50NBD were heated at 60°C for 15 min to form the MRNBD complex. Full-length MR LRET mixtures were 1 μM Mre11 + 1 μM dual-labeled (donor + acceptor) Rad50. Each LRET sample was subsequently diluted to 160 μL (0.25 μM complex) with LRET buffer (50 mM HEPES, 100 mM NaCl, 5 mM MgCl2, 0.1 mM EDTA, 1% glycerol, 1 mM TCEP, pH 7). Next, for samples containing nucleotide, 2 mM of either ATP or ATPγS was added and the samples were heated again for 20 min at 50°C. Finally, for samples containing DNA, either 34 μM hairpin DNA (5'-CACGCACGTAGAAGCT TTTGCTTCTACGTGCGTGAC-3′) or 16.6 μM ssDNA (5'-TGTAGTGCATTGCGTTTTTGCTTCTACG TGCGTGAC-3') was added to ATP-bound LRET samples and heated once more for 15 min at 50°C. These concentrations of DNA should give >95% bound complex in LRET conditions at 50°C. 150 μL were transferred into a 3 mm pathlength cuvette, and LRET data were collected after a final 5 min incubation in the fluorimeter at 50°C. Donor and acceptor lifetimes were calculated from intensity decays measured with an Optical Building Blocks phosphorescence lifetime photometer (EasyLife L). LRET samples were excited through a narrow band 335 nm filter (Semrock FF01-335/7) and after a 200 μs delay (to allow the decay of sample autofluorescence, emission due to direct excitation of the acceptor, and scattering of the excitation pulse), donor emission intensity was collected for Tb3+ at 50 Hz through a 490/10 nm band-pass filter (Omega Optical), and donor-sensitized acceptor emission intensities were collected at 100 Hz through 520/10 nm (Bodipy FL) or 570/10 nm (Cy3) band-pass filters. PTI FeliX32 software was used to fit the Bodipy FL and Cy3 emission decay curves to either a two- or three-exponential function depending on the identity of the LRET pair. For samples where the distance between the LRET pair in the nucleotide-bound crystal structure was ≤42 Å, the data fit well to three exponentials for both Bodipy- and Cy3-labeled samples. For samples where the LRET pair was separated by more than 45 Å, the Bodipy emission decays fit to only two exponentials while the Cy3 decays fit to three. Lifetime distributions shorter than ~100 μs were discarded as these are largely a function of the instrument response time (Cooper and Altenberg, 2013; Zoghbi et al., 2017; Zoghbi et al., 2012; Zoghbi and Altenberg, 2018). Donor Tb3+-chelate fluorescence decays were recorded at each probe position in donor-only-labeled MRNBD complexes. Each Tb3+-chelate fluorescence decay fit well to two exponentials, with the longer lifetime comprising >85% of the signal. This longer lifetime was used in the distance analysis. From the Tb3+-chelate, Bodipy FL, and Cy3 lifetimes, the distances between donor and acceptor molecules (R) were calculated with  E = 1 − τDA/τD and  R = R0  (  E^−1 − 1  )^1/6  where E is the efficiency of energy transfer, τDA is the donor-sensitized lifetime of the acceptor (Bodipy FL or Cy3), τD is the lifetime of the donor (Tb3+), and R0 is the Förster distance between Tb3+ and Bodipy FL (44.9 Å) or Tb3+ and Cy3 (61.2 Å). Errors are the standard deviations from the mean of at least three measurements.  HADDOCK  Molecular docking of the MRNBD complex was done using the GURU interface on the HADDOCK 2.4 webserver (Dominguez et al., 2003; van Zundert et al., 2016). For the three-body docking protocol, the PDB inputs were 3DSC (Pf Mre11 dimer lacking the helix-loop-helix [HLH] motifs and C-termini, DNAs deleted) (Williams et al., 2008) and two monomers of 3QKU (AMPPNP-bound Pf Rad50NBD in complex with the Mre11 HLH motif) (Williams et al., 2011). Except for increasing the number of structures for rigid body docking (it0) to 3000, all settings used were default. The three linkers attaching the Pf Mre11 capping domain to the nuclease domain were allowed to be fully flexible (Y222-V236, Y249-G254, and V266-F273). To allow HADDOCK to move the two Rad50 monomers relative to each other, we input Rad50 as two identical monomers. C2 symmetry was enforced between the two Rad50NBD monomers and between the two monomers of the Mre11 dimer. HADDOCK Mre11 to Rad50 active ambiguous interaction restraints (AIRs) were based on M. jannaschii (3AV0) (Lim et al., 2011) and E. coli (6S6V) (Käshammer et al., 2019) ATP-γ-S-bound MR structures. Passive AIRs (solvent accessible surface neighbors of active residues) were automatically defined by HADDOCK based on these active AIRs. Two extra passive AIRs were included on the ‘back side’ of the Mre11 capping domain to allow for the extended structure seen in ATP-free T. maritima MR (PDB: 3QG5; Lammens et al., 2011). The same set of active and passive AIRs were used in all of the HADDOCK runs with 50% random exclusion of AIRs in each structure calculation. The measured LRET distances were input as unambiguous restraints and defined as the Cβ-Cβ distance between the LRET-labeled residues, ± 5 Å. For distance restraints greater than 75 Å, the bounds were increased to ±7 Å as the lifetime fits giving these distances were in a more error-prone region (i.e., the flat section) of the Tb3+-Cy3 LRET efficiency curve. The model with the lowest HADDOCK score in each run was considered as the best structure. To ensure that the unambiguous distance restraints obtained from one probe position were not dominating the structure calculations, HADDOCK runs were performed with systematic dropouts of all restraints calculated from a specific cysteine position. For the closed model, all of the dropout structures maintained the proper D-loop/Walker A juxtaposition.