(Re)discovering missing physics in Rosetta, through RNA Rhiju Das Baker lab

(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)