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Open Dataset XSYY8DN2


MutSß (MSH2/MSH3) with Adenosine Nucleotide Analogs and DNA with unpaired loop

Abstract:

His-tagged human MutSß (MSH2/MSH3) aids in the removal of extrahelical small DNA loops and improperly paired DNA bases, which, if unrepaired, lead to cancer and genome instability. Human MutSβ is a heterodimer comprising one subunit each of MSH2 (104 kDa) and MSH3 (127 KDa), both of which undergo an ordered series of nucleotide-dependent steps to initiate repair. Each discrete nucleotide-bound state is a conformational decision point that “sets-up” coupling to the next step along the repair pathway, and must be preserved. Thus, a mechanistic understanding of these steps is key for elucidating how cells avoid mutation. We measured 10 liganded states of MutSβ with and without DNA substrate and in the presence of four distinct nucleotides or ATP analogues. Since DNA lesion recognition depends on ATP hydrolysis, non-hydrolysable analogues and ADP were used to probe conformational states before and after hydrolysis, respectively. Publication of this set can be found at "Hura GL, Budworth H, Dyer KN, Rambo RP, Hammel M, McMurray CT, Tainer JA. Comprehensive macromolecular conformations mapped by quantitative SAXS analyses. Nat Methods. 2013 Jun;10(6):453-4. doi: 10.1038/nmeth.2453. PMID: 23624664; PMCID: PMC3728378."

Experimental description:

The working buffer for all SAXS samples was 25 mM HEPES pH 8, 100 mM NaCl, 5 mM MgCl2, 5 % glycerol. For SAXS measurements with ADP, ATP and ATP analogs the nucleotides were also added to the working buffer at 200 μM. SAXS measurements were conducted with a fixed protein concentration of 4 mg/ml. When appropriate, DNA was added to the sample at an equimolar concentration as the MSH2/MSH3 complex. DNA binding was assayed through non-denaturing gel electrophoresis. Each component (protein or DNA) of the final sample (at least 16 μL) was added at a volume of 4 μL or greater. Samples were collected by the automated SAXS data collection pipeline at the SIBYLS beamline in Berkeley CA. Sample volumes of 16 μL were used. Buffer blanks were collected prior to each sample. Four exposures were collected for each sample (0.5, 1, 2 and 5 seconds). Comparison between exposures were made to ensure radiation damaged results were not analyzed. Merging the four exposures and excluding radiation damage optimized signal to noise. The X-ray pathlength was 1.5 mm. The experimental X-ray energy was 12 keV. Data were recorded on a MAR165 detector placed concentric with the beam. The full q range captured was 0.01 < q < 0.3 Å-1. The DNA used had 18 base pairs with one strand having an additional central sequence of (CA)x4 that formed the loop.

File description:

Each SAXS profile has MutSß. In addition there are samples with either ADP, ATP, AMPpMP or ATPgammaS. This set is also collected with DNA. The file names are self explanatory with components listed.

Data collection technique:

HT-SAXS

Wavelength

Sample to Detector Distance


Created: 2022-07-08

Updated: 2022-07-08

Submitting Author:

Greg Hura

Lawrence Berkeley National Laboratory, The SIBYLS Beamline

United States of America

[email protected]

Collaborators:

Project Leader:

Greg Hura

[email protected]


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Complete Set of SAXS Data Files:

The complete SAXS dataset is downloadable as a zip file.


Sample SAXS Data Files (total 0):

These are individual SAXS data files that are included in the complete dataset, but are available separately for quick download as examples of what is contained in the zip file.


Supplemental Data and Supporting Materials (total 0):

These data and materials may include X-ray crystal structure coordinates, multi-angle light scattering data, .etc. It may also include additional details or methods pertaining to the SAXS experiments, or the researcher's interpretations of the results.


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SAXS Similarity SAXS FrameSlice


Samples:

  • Macromolecule 1: MutSß
    • Sample Full Name: His-tagged human MutSß (MSH2/MSH3)
    • Sample Type: Protein
    • Source Organism:
    • Source Organism NCBI Taxonomy ID:
    • Expression System:
    • Expression NCBI Taxonomy ID:
    • Sequence or Chemical Formula:
  • Macromolecule 2: (CA)4 loop
    • Sample Full Name: DNA with a central unpaired loop
    • Sample Type: DNA
    • Source Organism:
    • Source Organism NCBI Taxonomy ID:
    • Expression System:
    • Expression NCBI Taxonomy ID:
    • Sequence or Chemical Formula: