Future directions in research onbiomolecular structure

NSLS-II Workshop

July 17,2007

Molecular structure and dynamics in biology

1. Where are we?2. Where are we going?3. How will NSLS-II (and similar installations) help us get there?

The molecular biological sciences:

1. Structure

2. Information transfer: “Molecular Biology” and “Systems’ Biology”

The molecular biological sciences:

1. Structure

2. Information transfer: “Molecular Biology” and “Systems’ Biology”

Structural biology in the twentieth century

1953 1961 1977 1998 2000

DNA Protein Virus Ion channel Ribosome

Molecules:

Cells:

1950’s:

NMRX-ray

EMOpt.

10 Å

100 Å

10,000 Å

Chemistry,genetic

“engineering”

c-Src

kinase

c-Src tyrosine kinase

QuickTime™ and aYUV420 codec decompressor

are needed to see this picture.

HIV-1 envelope glycoprotein

Nitrogenase

Howard & Rees, 200650 Å

F1 ATPaseSource of intracellular energy

Reinisch et al, 2000

Reovirus core

100 Å

R. Kornberg & coworkers, 2001

Yeast RNA polymerase II

25 A

Cate & co-workers, 2005

Limitations of crystallographyfor structure determination:

Inhomogeneity, even modest,is generally incompatible with

crystallization

Viral entry via the endosome

Fotin et al, 2004a100 Å

Anatomy of a clathrin coat

Triskelion = 3 x (Heavy Chain + Light Chain)

N

CC

N

proximal

knee

distal

linker

terminal domain

Clathrin lattice

ankle

NMRX-ray

EMOpt.

10 Å

100 Å

10,000 Å

Chemistry,genetic

“engineering”

QuickTime™ and aCinepak decompressor

are needed to see this picture.

~1 m

clathrin

reovirus

“Molecular movies”: to link live-cell dynamics and molecular structure.

The goal is a data-based dynamic picturerather than simply an imaginative animation

How will we get the requisite atomic-resolutionsnapshots of various substructures?

X-ray crystallography will continue to bethe principal method, and adequate progresswill depend on being able to get good datafrom very small and weakly diffractingcrystals

What are the critical technical problems?

Signal-to-noise: Signal is restricted by damage Noise is determined by characteristics of the sample (and by the extent to which the measurements can minimize it)

Sources of noise1. Scatter from interstitial solvent in crystal2. Scatter from surrounding solvent and mount3. Beam-path scatter4. Detector a. Pixels too large b. Detector noise

What is needed to optimize datacollection from such crystals?

1. Very small beam2. Positionally very stable beam3. Very low divergence4. Suitably precise sample handling instruments5. Large detectors with very small pixel sizes to match

3.5 ÅDengue sE trimerP3221 a=b=159Å c=145Å1° rotation D=450 mm

Small and weakly diffracting crystals

For a protein crystal, damage from inelastic scatter ~ Bragg photon/unit cell (Sliz et al, 2003. Structure 11:13-19)

Example: 20x20x20 3 crystal with 100x100x100 Å3 cellAbout 500 photons/reflection if you “burn up” crystal (inpractice, long-range order disappears much sooner).

Data from multiple crystals can be scaled and merged

Summary

1. “Molecular movies” are a goal of structural cell biology2. The fundamental elements of cellular molecular movies will continue to be provided by x-ray crystallography3. Critical barriers: the x-ray optical precision needed to make many accurate measurements from small crystals and new kinds of beamline instrumentation4. NSLS-II appears to have many of the characteristics suitable for surmounting these barriers

Sources of noise

1. Scatter from interstitial solvent in crystal2. Scatter from surrounding solvent and mount3. Beam-path scatter4. Detector a. Pixels too large b. Detector noise