ACA 2002

Electron crystallography

Electron diffraction and imaging has a fascinating history in both biology and materials science. A special session on the topic was organized in conjunction with the presentation of the Patterson Award to Doug Dorset at the San Antonio, Texas ACA meeting. Doug provided an overview of the field, with emphasis on electron crystallography of organic crystals, as discussed in his comprehensive book. The symposia included talks by K. Downing on tubulin, B. Jap on two-dimensional crystallization techniques, C. Gilmore on the phase problem, J. Fryer on electron crystallography generally, and J. Spence and L. Marks on new methods. Application in the field of materials included mesoporous material (O. Terasaki), charge-density measurements by CBED imaging (Y. Zhu), electron nano-crystallography(J.M. Zuo), electron imaging from glasses (M. Treacy), and energy-filtered diffraction patterns from diatom frustules (Y. Kim).

Two things are driving this growth in electron diffraction and imaging methods - in biology, the urgent need to determine the shape of large macromolecules, at sub-nanometer resolution, which cannot be crystallised, and in materials science, the boom in nanoscience, for which the electron microscope provides the ideal probe.

John Spence

Electron microscopy of biological macromolecules

In addition to the day-long Symposium on Electron Crystallography that was organized in conjunction with this year’s Patterson Award to Doug Dorset, the Spring 2002 meeting featured three half-day sessions on Electron Microscopy of Biological Macromolecules. Electron microscopy of samples embedded in vitreous ice ('cryo-EM') has recently developed as a powerful way to visualize the three-dimensional assembly of macromolecules into various complexes and machines, and thus it is regarded as providing an important bridge between structural genomics and cell biology. A session focused on the quantitative interpretation of cryo-EM density maps included K. Namba’s presentation of a comprehensive and stunning survey of the bacterial flagellar apparatus and H. Li’s description of tubulin assembly. The second session moved on to report technology-developments that aim to automate many of the steps in cryo-EM that must currently be done 'by hand' and application to the pyruvate dehydrogenase complex from B. stearothermophilus, an 11 megadalton 'catalytic machine' by J. Milne and the changes in structure of GroEL chaperonin during its biochemical cycle by H. Saibil. The final session concerned work in which 2-D crystals are used in order to obtain 3-D density maps at high enough resolution to allow de novo modeling of the structure at atomic resolution. Other structures reported were: the membrane protein, bacteriorhodopsin (Y. Fujiyoshi), a bacterial oxylate (S. Subramaniam), and an aquaporin (A. Philippsen).

Bob Glaeser

Crystal structure determination

The Transactions Symposium dealt with Crystal Structure Determinations from Powder Diffraction. Powder patterns contain an enormous amount of information. However, a stumbling block to obtaining structural data was the lack of mathematical methods of peak shape duplication. It was not until the early 1980s that software began to appear that would allow for decomposition of the powder pattern to obtain accurate intensities. Since then, there has been great progress in both hardware and software for all aspects of structure determination and refinement. This symposium was convened to highlight these advances.

L. Cranswick and R. Shirley discussed methods of indexing the powder pattern. C. Giacovazzo described his EXPO, a much used program for ab initio structure solutions. Powder patterns with low angular resolution (H. Toraya), organic crystals affected by preferred orientation (M. Tremayne), metal terephthalates (J. Kaduk), and the use of high brightness synchrotron x-ray and high flux neutron sources to study powder samples under high pressure (J. Parise) were all addressed in the symposium. C. Hubbard discussed the use of the Powder Diffraction File (PDF) published by the Int’l Center for Diffraction Data (ICDD) to help establish phase identification, corrosion and decomposition products in the varied fields of mineralogy, materials science, manufacturing and pharmaceuticals. The solution of organic structures from powder data becomes very difficult when molecules are flexible or three or more are present in the asymmetric unit. Complex structures with poor resolution were addressed by C. Gilmore. P. Stephens discussed his use of the public domain Powder Structure Solution Program for the solution of structures of drugs. M. Deem described a Monte Carlo scheme to determine the crystal structure of all known zeolites. R. Grothe is developing a program for structure determination from anisotropic powder diffraction data collected on an area detector. By combining high-resolution synchrotron X-ray powder diffraction data and stereochemical restraints, it has been demonstrated that the Rietveld refinement of protein crystal structures is feasible (R. Von Dreel and P. Stephens).

Abraham Clearfield

New macromolecular crystals

In the microsymposium New Macromolecular Crystals, Techniques and Hardware, A. D’Arcy demonstrated that microbatch minimizes sample oxidation, makes temperature variation easier and allows increased reagent and sample concentrations by incorporating Si oil in the expermiment. He modifies the protein by complexation (adding an inhibitor), proteolysis, deglycosylation, and site directed mutagenesis. T. Tisone (Cartesian Technologies) described the use of non contact microfluidics and automation for protein crystallization. B. Haushalter (Parallel Synthesis Technologies) discussed nanoengineered surfaces for the epitaxial nucleation of protein crystals.

J. Jancarik discussed sparse matrix crystallization screens centrifugal filter devices for concentration and detection of monodispersity by dynamic light scattering (DLS). A. Hassell designed constructs using homologous models limited proteolysis, and the use of 'seeding' with good results, especially cross-seeding with apo forms to grow kinase-ligand complex crystals.

Bob Cudney

Protein folding and design

J. Richardson taught us a lesson in the importance of hydrogens in sidechain packing and introduced her new, interactive MAGE tools for evaluating and correcting mistakes in crystal structures (http://kinemage.biochem.d-uke.edu/molprobuty/).

Chris Bystrof

New methods in macromolecular crystallography

A number of groups are developing detectors that employ discrete semiconductor devices. Thin film transistor (TFT) technology produces a single, monolithic detective surface relatively inexpensively. E. Merritt described a procedure for comparing the validity of different techniques for modeling anisotropy in protein structure, which he applied to several refinements of anisotropic displacement parameters (ADP), translational, librational, and screw (TLS) motions at resolutions between 1 and 2Å. He concluded that the TLS method works well over this entire resolution range. The ADP method becomes preferable to TLS at resolutions higher than 1.6Å, but the precise resolution at which this happens is structure dependant. Both the ADP and TLS methods performed better than purely isotropic refinement. Several groups have tried to work at temperatures in the low teens (Kelvin) rather that at the ~100K accessible with liquid nitrogen. One successful method uses a cryostat that directs a fine, high-velocity stream of gaseous helium on the specimen.

Jim Fait

Scattering in nanoscience and nanotechnology

This symposium ranged from the fundamental to the applied and from purely inorganic to biological. Topics covered included research to understand the novel electronic properties of complex transition metal oxides (R. Osborn, Argonne), studies of nanoporous glassy materials in the encapsulation of radioactive waste (P. Thiyagarajan, Argonne), the characterization of the perfection of photolithographically created nanostructures (R. Jones, NIST), and videos of scattering from dancing nano-scale oil drops under shear (T. Mason, ExxonMobil). M. Firestone (Argonne) described how biomimetic lipid based complex fluids can be used as scaffolds for inorganic nanoparticles, and even functional proteins, and P. Sokol (Penn State) showed what inelastic neutron scattering can tell us about what happens in a buckytube full of hydrogen. The range of scattering techniques used was also varied, and the problem of scattering from nanostructured materials is leading to novel applications of scattering and ingenious extensions of scattering techniques. The varied techniques employed included wide angle scattering, small angle scattering, reflectography, x-ray circular dichroism, grazing incidence diffraction, and electron diffraction using nanometer sized beam-spots.

Simon J. L. Billinge and Paul Butler

Structural genomics: are the pieces ready yet?

The symposium discussed progress, challenges, and achievements in structural proteomics/genomics. C. Kissinger described a large scale high-throughput crystallography project with automated systems for protein production and structure determination. The system was applied to a set of bacterial targets yielding 53 new structures and 7 new folds. He said that the map-fitting step is the biggest hurdle to full automation.

A. Joachimiak emphasized the difficulty of optimizing crystallization conditions as a bottleneck in structure determination and described a robotic system for crystal mounting, including a prototype UV-light-based method for crystal centering. L. Tari presented a commercial view of how high-throughput crystallography can be used for structure-based drug design.

Case studies of structural proteomics described included a case in which sequence comparisons gave no information but the solved structure matched a methyl-transferase; in another, sequence and structure gave no obvious match but led to a good guess (NTP hydrolase); in others, neither sequence nor structure gave useful clues.

Tom Terwilliger

Reports and image taken from the ACA Newsletter, Fall 2002