Computational protocol: Using stable MutS dimers and tetramers to quantitatively analyze DNA mismatch recognition and sliding clamp formation

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Protocol publication

[…] For crystallization, 100 µM MutS D835R (monomer concentration) was combined with 50 µM 21-bp DNA containing a mismatch (same 21-bp sequence as in the SAXS DNA-binding studies), and 100 µm ADP. Crystals were grown using hanging drop vapor diffusion from a well solution of 25 mM Tris (pH 8), 750 mM NaCl, 12% PEG 6000, 10 mM MgCl2. Microseeding was used to improve crystal quality. Before data collection, crystals were transferred to a cryobuffer consisting of the mother liquor supplemented with 30% glycerol and flash cooled in liquid nitrogen.Diffraction data were collected at beamline ID14-4 at the ESRF in Grenoble, France. Data reduction was performed using XDS () and Scala () in the CCP4 suite (). The structure of the C-terminally truncated MutS:DNA complex (PDB entry 1E3M) () was used as a search model for structure solution using Phaser (). Refinement jobs were carried out using REFMAC5 (). PDB_REDO () was used to optimize refinement parameters for REFMAC5. During the refinement process, the structure of the dimer of the C-terminal 33 residues of MutS () was used as an initial model to fit the density for the C-terminal domain using the program Coot (). Most of the structure could be modeled confidently, but electron density for the mismatch binding domain in subunit B and the C-terminal domains (823–853) is relatively weak and residues 658–669 and 749–757 of the ATPase domain of subunit A (the mismatch-contacting subunit), residues 1–25, 55–74 and 95–106 of the mismatch-binding domain of subunit B, residues 801–822 in both subunits and six bases are missing. We did not observe clear density for nucleotides in either of the two subunits of the crystallized protein, and the positions of the P-loops resembled empty nucleotide binding sites. Nevertheless, some residual difference density is present in the nucleotide-binding site of the mismatch-contacting subunit, suggesting a small fraction of ADP-bound protein in the crystal. Coordinates of the refined model of MutS D835R have been deposited in the Protein Data Bank with entry code 3ZLJ. For crystallographic statistics, see . Figures were generated using PyMOL ( [...] SAXS measurements were performed at beamlines P12 and X33 () at EMBL Hamburg. Samples of MutS D835R were prepared in buffer containing 25 mM Hepes (pH 7.5), 250 mM KCl, 5% glycerol. MutS tetramer samples were prepared in buffer A as described earlier in the text. The samples were thawed and centrifuged at high speed for 1 min just before measurement. Samples were exposed to X-rays in a measuring cell cooled to 10°C. Data were analyzed using the ATSAS software package (): data processing was performed using PRIMUS () where the Guinier plots were used to assess Rg values and data quality at low-angles (Supplementary Figure S2D), after which GNOM () was used to generate distance distribution plots. GNOM results were used as input for DAMMIF () to generate 10 independent ab initio models for both the dimer and the DNA-free tetramer, which were subsequently averaged using DAMAVER (). For SAXS statistics, see . Figures were generated using PyMOL ( […]

Pipeline specifications

Software tools XDS, CCP4, REFMAC5, Coot, PyMOL, ATSAS, DAMMIF
Databases PDB_REDO
Applications Small-angle scattering, Protein structure analysis
Organisms Escherichia coli, Dipturus trachyderma
Chemicals Adenosine Triphosphate