Future directions in research on biomolecular 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 DNA Protein Virus Ion channel Ribosome Molecules: Cells: 1950’s:

NMR X- ray EM Opt. 10 Å 100 Å 10,000 Å Chemistry, genetic “engineering”

c-Src kinase

c-Src tyrosine kinase

HIV-1 envelope glycoprotein

Nitrogenase Howard & Rees, Å

F1 ATPase Source 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 crystallography for structure determination: Inhomogeneity, even modest, is generally incompatible with crystallization

Viral entry via the endosome

Fotin et al, 2004a 100 Å

Anatomy of a clathrin coat Triskelion = 3 x (Heavy Chain + Light Chain) N C C N proximal knee distal linker terminal domain Clathrin lattice ankle

NMR X- ray EM Opt. 10 Å 100 Å 10,000 Å Chemistry, genetic “engineering”

~1  m clathrin reovirus

“Molecular movies”: to link live-cell dynamics and molecular structure. The goal is a data-based dynamic picture rather than simply an imaginative animation

How will we get the requisite atomic-resolution snapshots of various substructures? X-ray crystallography will continue to be the principal method, and adequate progress will depend on being able to get good data from very small and weakly diffracting crystals

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 noise 1. Scatter from interstitial solvent in crystal 2. Scatter from surrounding solvent and mount 3. Beam-path scatter 4. Detector a. Pixels too large b. Detector noise

What is needed to optimize data collection from such crystals? 1.Very small beam 2.Positionally very stable beam 3.Very low divergence 4.Suitably precise sample handling instruments 5.Large detectors with very small pixel sizes to match

3.5 Å Dengue sE trimer P a=b=159Å c=145Å 1° rotation D=450 mm

Small and weakly diffracting crystals For a protein crystal, damage from inelastic scatter  ~ Bragg photon/unit cell (Sliz et al, Structure 11:13-19) Example: 20x20x20  3 crystal with 100x100x100 Å 3 cell About 500 photons/reflection if you “burn up” crystal (in practice, 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 biology 2.The fundamental elements of cellular molecular movies will continue to be provided by x-ray crystallography 3.Critical barriers: the x-ray optical precision needed to make many accurate measurements from small crystals and new kinds of beamline instrumentation 4.NSLS-II appears to have many of the characteristics suitable for surmounting these barriers

Sources of noise 1. Scatter from interstitial solvent in crystal 2. Scatter from surrounding solvent and mount 3. Beam-path scatter 4. Detector a. Pixels too large b. Detector noise