Bioisis

"Modeling of SAM-1 Riboswitch (apo state)"

Experimental SAS Curve

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Low_res_thumbnail

Experimental Mass

30,100 Da

Experimental Details for BID:  1SAMRR
Experiment ID: 42    In addition, this experiment links to experiment(s):  43
Collected at: ALS BL 12.3.1
Contributors: Rambo, R.P. ,  Batey, R.T. ,  Stoddard, C.D. ,  Montange, R.K.
SAM-1 riboswitch binds a small metabolite (SAM). Crystal structures of bound and unbound states demonstrate nearly identical structures unlike the solution state where SAXS data showed two different states of the riboswitch. The riboswitch appears to occupy a single bound state in the presence of SAM identical to the crystal structure whereas in the absence of SAM, the riboswitch appears to occupy many conformational states. Based on the X-ray crystal structure, SAXS data in the absence of SAM-1 was used as a modeling constraint in torsion angle molecular dynamic simulations. Results demonstrate a scissoring of the riboswitch for opening the metabolite binding site.
RNA samples were prepared maximally at 2 to 3 mg/ml and serially diluted at four different concentrations.

Electron Pair Distribution

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       Dmax → 82 Å


Guinier Plot

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     Guinier Rg → 24.16 Å

Real Space Rg → 24.82 Å

The Guinier plot is used to estimate the radius of gyration, Rg, which is taken from the slope of a line observed at low scattering angles (typically in the range where q* Rg < 1.3). This should be in reasonable agreement with the real space Rg.


Kratky Plot

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The Kratky plot can be used to visually assess the degree of "unfoldedness" of a protein or RNA sample. The plot of a well-behaved folded protein approaches the baseline at high q values creating a parabolic shape.


Ensemble Model

A ENSEMBLE model was determined using the following:

Simulation MethodFIRST, FRODA and CNS 
Simulation Algorithmlimited torsion angle dynamics 
Ensemble Size5000 
Selection MethodGenetic Algorithm 
Member Size13 
Scoring Functionchi 
Score1.0 

Ensemble Fit

Med_res_ensemble_fit

The red line is the calculated SAXS profile from the ENSEMBLE model scaled to the experimental SAXS curve (cyan).

Supportive Diagnostic Figure for ENSEMBLE Selection

Diagnostic

Fig: Histogram analysis of EOM selection displaying four major distributions that represent four major structural classes of the SAM-1 riboswitch in solution (unbound). The varying conformational states suggest the riboswitch occupies many states in the unbound state.


Additional Experimental Details
Title

Modeling of SAM-1 Riboswitch (apo state)

Description

SAM-1 riboswitch binds a small metabolite (SAM). Crystal structures of bound and unbound states demonstrate nearly identical structures unlike the solution state where SAXS data showed two different states of the riboswitch. The riboswitch appears to occupy a single bound state in the presence of SAM identical to the crystal structure whereas in the absence of SAM, the riboswitch appears to occupy many conformational states. Based on the X-ray crystal structure, SAXS data in the absence of SAM-1 was used as a modeling constraint in torsion angle molecular dynamic simulations. Results demonstrate a scissoring of the riboswitch for opening the metabolite binding site.

Publication

Free state conformational sampling of the SAM-I riboswitch aptamer domain. Structure. 2010 Jul 14;18(7):787-97

Contributors

Rambo, R.P. ,  Batey, R.T. ,  Stoddard, C.D. ,  Montange, R.K.

Genomics and Proteomics

The experiment is composed of a single gene/ORF

Abbreviated name: SAM1RSWTCH

Annotation: SAM-1 aptamer domain from Thermoanearobacter tengocongensis metF-H2

GGCUUAUCAA GAGAGGUGGA GGGACUGGCC CGAGAAACCC GGCAACCAGA AAUGGUGCCG AAUUCCUGCA GCGGAAACGU UGAAAGAUGA GCCA
categoryResidue composition (%)
Nucleotides G (33.0) C (22.3) A (30.9) U (13.8) T (0.0)
Mass: 30,796 Da | Total residues: 94