Interface refinement of low- to medium-resolution Cryo-EM complexes using HADDOCK2.4

ATG9A and ATG2A form a heteromeric complex essential for autophagosome formation

“…Another full-length model was generated similarly using the experimental structure of ATG9A (PDB ID 6WQZ) instead of the AF model. The interfaces were refined using HADDOCK 2.4's coarse grain refinement procedure via the web server (Neijenhuis et al, 2022). Model quality was assessed at various steps during the modelling and in the final models by determining how well the models satisfied the CXL-MS derived distance constraints and binding site data from S2; Video S1).…”

The Ku complex promotes DNA end-bridging and this function is antagonized by Tel1/ATM kinase

“…In order to obtain a wild-type Ku–DNA complex as well, we built a homology modeling structure of the wild-type heterodimer bound to DNA with the mutant heterodimer structure as a model. The models obtained were refined by the HADDOCK2.4 refinement protocol ( 71 ), which exploits molecular dynamics simulation in water solvent allowing freedom of movement first to the residues side chains all over the protein and then to the region of the protein taking contact with the interactor. The simulations generated structures with minimized energy and HADDOCK scores of -118.5 for the wild-type and -124.3 for the mutant heterodimer, mainly due to a higher number of electrostatic interactions in the mutant complex (the specific scores for electrostatic interactions being -548.7 versus -516.8 for Ku70 C85Y –Ku80 versus Ku70–Ku80).…”

“…The best position for DNA within the Ku–DNA complex was obtained with the Ku70 C85Y –Ku80 complex, and adopted for the wild-type complex as well. The HADDOCK2.4 refinement protocol ( 71 ) was used to refine the models of wild-type Ku70–Ku80 and Ku70 C85Y –Ku80 heterodimers with the DNA molecule described above, with the minimized energy protocol with standard parameters ( https://wenmr.science.uu.nl/haddock2.4/settings#refinement ). Briefly, this minimization protocol performs a series of short molecular dynamics (MD) simulations with explicit solvent after a solvent shell is built around the complex with position restraint on the α backbone of the protein, thus allowing the amino acids chains to move.…”

Integrating model simulation tools and cryo‐electron microscopy

“…gmfit uses a simplified gaussian mixture representation, and optimizes their position and rotation to maximize an energy term maximizing the fit between the sum of the Gaussians and the map while minimizing subunits overlap. 122 Later methods such as Multifit 124 used simultaneous rather than sequential fitting, as initial errors in the fitting procedure Mod-EM 118 Correlation maximization HADDOCK 119 Restraints ADP_EM 120 Spherical harmonics PRISM-EM 121 Template matching gmfit 122 Gaussian mixture Vesper 123 Density vector…”

“…123 In addition to fitting structures using cryo-EM data, methods have been developed that take advantage of multiple types of data, coming from native mass spectrometry, crosslinking mass spectrometry or small-angle x-ray scattering (SAXS), and other experiments that may provide spatial restraints for the internal organization of a biomolecular complex. Methods that combine multiple experimental information include Haddock, 119,129 IMP, 113 ROSETTA, 130 and power. 131 This type of integrative approach can be used to resolve assemblies that would remain too ambiguous or difficult to solve by direct fitting in the density, the extra information from other methods providing the necessary information to either find the correct solution, or separate it from others fitting similarly well in the density.…”