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(Re)discovering missing physics in Rosetta, through RNA
Rhiju Das
Baker lab
Understanding structure
Watson & Crick (1953)
Beyond a single structure?
Work with Pehr Harbury and
Rebecca Fenn (Stanford)
“Remeasuring the Double
Helix”
The basic idea
Thomas Young (1773-1829)
= /d
d
The basic idea
Thomas Young (1773-1829)
A new x-ray rulerSugar coated gold clusters
86 Å = 68 Å + 2 x 9 Å
The double helix as a length standard
15bp
10bp
25bp
20bp
35bp
30bp
15bp
10bp
25bp20bp
35bp
30bp
The double helix as a length standard
Rise per base pair:3.29±0.07 Å
Crystal avg:3.32±0.19 Å
Wait a minute…
15bp
10bp
25bp20bp
35bp
30bp
Variance in length of 10 bp duplex:
1.4 Å2 (predicted)
8.5 Å2 (measured)
Variance in length of 35 bp duplex:
4.8 Å2 (predicted)
51.0 Å2 (measured)
Wait a minute…
15bp
10bp
25bp20bp
35bp
30bp
Conventional model[elastic rod, S=1000 pN]
Best fit elastic rod model Quadratic fit
What does it mean?Uncorrelated
(drunken walk)
variance length
Correlated (marching in
lockstep)
variance length2
Are you sure? (How a rotation project got stretched 7 years)
1. Statistical errors2. Sample impurities3. Multiple DNA’s per gold particle4. Truncation products from oligo
synthesis5. Perturbations of DNA by gold
probes6. Poorer signal-to-noise in longer
constructs7. End-effects8. Long-range electrostatics
between gold probes
Are you sure? (How a rotation project got stretched 7 years)
1. Statistical errors2. Sample impurities3. Multiple DNA’s per gold particle4. Truncation products from oligo
synthesis5. Perturbations of DNA by gold
probes6. Poorer signal-to-noise in longer
constructs7. End-effects8. Long-range electrostatics
between gold probes
Are you sure? (How a rotation project got stretched 7 years)
1. Statistical errors2. Sample impurities3. Multiple DNA’s per gold particle4. Truncation products from oligo
synthesis5. Perturbations of DNA by gold
probes6. Poorer signal-to-noise in longer
constructs7. End-effects8. Long-range electrostatics
between gold probes
Are you sure? (How a rotation project got stretched 7 years)
1. Statistical errors2. Sample impurities3. Multiple DNA’s per gold particle4. Truncation products from oligo
synthesis5. Perturbations of DNA by gold
probes6. Poorer signal-to-noise in longer
constructs7. End-effects8. Long-range electrostatics
between gold probes
Are you sure? (How a rotation project got stretched 7 years)
1. Statistical errors2. Sample impurities3. Multiple DNA’s per gold particle4. Truncation products from oligo
synthesis5. Perturbations of DNA by gold
probes6. Poorer signal-to-noise in longer
constructs7. End-effects8. Long-range electrostatics
between gold probes
Are you sure? (How a rotation project got stretched 7 years)
1. Statistical errors2. Sample impurities3. Multiple DNA’s per gold particle4. Truncation products from oligo
synthesis5. Perturbations of DNA by gold
probes6. Poorer signal-to-noise in longer
constructs7. End-effects8. Long-range electrostatics
between gold probes
Are you sure? (How a rotation project got stretched 7 years)
1. Statistical errors2. Sample impurities3. Multiple DNA’s per gold particle4. Truncation products from oligo
synthesis5. Perturbations of DNA by gold
probes6. Poorer signal-to-noise in longer
constructs7. End-effects8. Long-range electrostatics
between gold probes
10 mM NaCl100 mM NaCl1 M NaCl -- indistinguishable variances
Why hasn’t this been detected before?
• As for crystallography, single molecule FRET, electron paramagnetic resonance:
• The “stiffness” of DNA was previously measured by single molecule measurements under high tension.
Wanted: a mechanism for correlations
Remeasuring the double helix
A new x-ray ruler for measuring distance distributions
[see also work of Alivisatos+colleagues]
Unexpected and unexplained
Soft, correlated stretching fluctuations in DNA.
[see also: unusually high “kink” rate (Widom), twist/stretch coupling (Bustamante), more]
Acknowledgments
Pehr Harbury
Becky Fenn
Josh Silverman
Peter Walker
Tim Fenn
Gold adviceChris Ackerson
DNA adviceBuzz BaldwinEd BoasZev BryantStirling ChurchmanAlex DunnDave HalpinJim HavranekDan HerschlagLance MartinBruce RobinsonMickey SchurrAlyssa SmithRebecca WeisingerJarret Wrenn
SAXS adviceYu BaiSeb DoniachKazuki ItoJan LipfertIan MillettSoenke SeifertP. ThiyagarajanHiro Tsuruta
Rosetta advicePhil BradleyDavid Baker
Data collected at:SSRLAPS
Where do the X-rays come from?
s =
SAXS data looks like this.
-0.1 -0.05 0 0.05 0.1s (Å-1)
0 50 100 150R (Å)
Fouriertransform
SAXS data looks like this.
-0.1 -0.05 0 0.05 0.1s (Å-1)
Fouriertransform
0 50 100 150R (Å)
Since the biomolecule scatters, how to extract probe-probe interference?
– – +
Need to subtract out:• Biomolecule scattering• Biomolecule-probe interference• (Probe scattering)