Determination of macromolecular structures from anomalous diffraction of synchrotron radiation

Resonance between beams of x-ray waves and electronic transitions from bound atomic orbitals leads to a phenomenon known as anomalous scattering. This effect can be exploited in x-ray crystallographic studies on biological macromolecules by making diffraction measurements at selected wavelengths associated with a particular resonant transition. In this manner the problem of determining the three-dimensional structure of thousands of atoms is reduced to that of initially solving for a few anomalous scattering centers that can then be used as a reference for developing the entire structure. This method of multiwavelength anomalous diffraction has now been applied in a number of structure determinations. Optimal experiments require appropriate synchrotron instrumentation, careful experimental design, and sophisticated analytical procedures. There are rich opportunities for future applications.     

Structure of ribonuclease H phased at 2 A resolution by MAD analysis of the selenomethionyl protein

Ribonuclease H digests the RNA strand of duplex RNA.DNA hybrids into oligonucleotides. This activity is indispensable for retroviral infection and is involved in bacterial replication. The ribonuclease H from Escherichia coli is homologous with the retroviral proteins. The crystal structure of the E. coli enzyme reveals a distinctive alpha-beta tertiary fold. Analysis of the molecular model implicates a carboxyl triad in the catalytic mechanism and suggests a likely mode for the binding of RNA.DNA substrates. The structure was determined by the method of multiwavelength anomalous diffraction (MAD) with the use of synchrotron data from a crystal of the recombinant selenomethionyl protein.     

NMR structure of Mistic, a membrane-integrating protein for membrane protein expression

Although structure determination of soluble proteins has become routine, our understanding of membrane proteins has been limited by experimental bottlenecks in obtaining both sufficient yields of protein and ordered crystals. Mistic is an unusual Bacillus subtilis integral membrane protein that folds autonomously into the membrane, bypassing the cellular translocon machinery. Using paramagnetic probes, we determined by nuclear magnetic resonance (NMR) spectroscopy that the protein forms a helical bundle with a surprisingly polar lipid-facing surface. Additional experiments suggest that Mistic can be used for high-level production of other membrane proteins in their native conformations, including many eukaryotic proteins that have previously been intractable to bacterial expression.     

Protein crystal growth in microgravity

The crystals of most proteins or other biological macromolecules are poorly ordered and diffract to lower resolutions than those observed for most crystals of simple organic and inorganic compounds. Crystallization in the microgravity environment of space may improve crystal quality by eliminating convection effects near growing crystal surfaces. A series of 11 different protein crystal growth experiments was performed on U.S. space shuttle flight STS-26 in September 1988. The microgravity-grown crystals of gamma-interferon D1, porcine elastase, and isocitrate lyase are larger, display more uniform morphologies, and yield diffraction data to significantly higher resolutions than the best crystals of these proteins grown on Earth.     

Crystal structure of Cd,Zn metallothionein

The anomalous scattering data from five Cd in the native protein were used to determine the crystal structure of cadmium, zinc (Cd,Zn) metallothionein isoform II from rat liver. The structure of a 4-Cd cluster was solved by direct methods. A 2.3 A resolution electron density map was calculated by iterative single-wavelength anomalous scattering. The structure is folded into two domains. The amino terminal domain (beta) of residues 1 to 29 enfolds a three-metal cluster of one Cd and two Zn atoms coordinated by six terminal cysteine thiolate ligands and three bridging cysteine thiolates. The carboxyl terminal domain (alpha) of residues 30 to 61 enfolds a 4-Cd cluster coordinated by six terminal and five bridging cysteine thiolates. All seven metal sites have tetrahedral coordination geometry. The domains are roughly spherical, and the diameter is 15 to 20 A; there is limited contact between domains. The folding of alpha and beta is topologically similar but with opposite chirality. Redundant, short cysteine-containing sequences have similar roles in cluster formation in both alpha and beta.     

Discovery of a Frank-Kasper sigma phase in sphere-forming block copolymer melts

Sphere-forming block copolymers are known to self-assemble into body-centered cubic crystals near the order-disorder transition temperature. Small-angle x-ray scattering and transmission electron microscopy experiments on diblock and tetrablock copolymer melts have revealed an equilibrium phase characterized by a large tetragonal unit cell containing 30 microphase-separated spheres. This structure, referred to as the sigma (σ) phase by Frank and Kasper more than 50 years ago, nucleates and grows from the body-centered cubic phase similar to its occurrence in metal alloys and is a crystal approximant to dodecagonal quasicrystals. Formation of the σ phase in undiluted linear block copolymers (and certain branched dendrimers) appears to be mediated by macromolecular packing frustration, an entropic contribution to the interparticle interactions that control the sphere-packing geometry.     

High-resolution protein structure determination by serial femtosecond crystallography

Structure determination of proteins and other macromolecules has historically required the growth of high-quality crystals sufficiently large to diffract x-rays efficiently while withstanding radiation damage. We applied serial femtosecond crystallography (SFX) using an x-ray free-electron laser (XFEL) to obtain high-resolution structural information from microcrystals (less than 1 micrometer by 1 micrometer by 3 micrometers) of the well-characterized model protein lysozyme. The agreement with synchrotron data demonstrates the immediate relevance of SFX for analyzing the structure of the large group of difficult-to-crystallize molecules.     

X-ray Laue Diffraction from Protein Crystals

In conventional x-ray diffraction experiments on single crystals, essentially monochromatic x-rays are used. If polychromatic x-rays derived from a synchrotron radiation spectrum are used, they generate a Laue diffraction pattern. Laue patterns from single crystals of macromolecules can be obtained in less-than 1 second, and significant radiation damage does not occur over the course of an exposure. Integrated intensities are obtained without rotation of the crystal, and individual structure factors may be extracted for most reflections. The Laue technique thus offers advantages for the recording of diffraction patterns from short-lived structural intermediates; that is, for time-resolved crystallography.     

The impact of structural genomics: expectations and outcomes

Structural genomics (SG) projects aim to expand our structural knowledge of biological macromolecules while lowering the average costs of structure determination. We quantitatively analyzed the novelty, cost, and impact of structures solved by SG centers, and we contrast these results with traditional structural biology. The first structure identified in a protein family enables inference of the fold and of ancient relationships to other proteins; in the year ending 31 January 2005, about half of such structures were solved at a SG center rather than in a traditional laboratory. Furthermore, the cost of solving a structure at the most efficient SG center in the United States has dropped to one-quarter of the estimated cost of solving a structure by traditional methods. However, the efficiency of the top structural biology laboratories-even though they work on very challenging structures-is comparable to that of SG centers; moreover, traditional structural biology papers are cited significantly more often, suggesting greater current impact.     

Spin chirality, Berry phase, and anomalous Hall effect in a frustrated ferromagnet

An electron hopping on non-coplanar spin sites with spin chirality obtains a complex phase factor (Berry phase) in its quantum mechanical amplitude that acts as an internal magnetic field, and is predicted to manifest itself in the Hall effect when it is not cancelled. The present combined work of transport measurement, neutron scattering, and theoretical calculation provides evidence that the gigantic anomalous Hall effect observed in Nd2Mo2O7, a pyrochlore ferromagnet with geometrically frustrated lattice structure, is mostly due to the spin chirality and the associated Berry phase originating from the Mo spin tilting.     

Genetic and crystallographic studies of the 3',5'-exonucleolytic site of DNA polymerase I

Site-directed mutagenesis of the large fragment of DNA polymerase I (Klenow fragment) yielded two mutant proteins lacking 3',5'-exonuclease activity but having normal polymerase activity. Crystallographic analysis of the mutant proteins showed that neither had any alteration in protein structure other than the expected changes at the mutation sites. These results confirmed the presumed location of the exonuclease active site on the small domain of Klenow fragment and its physical separation from the polymerase active site. An anomalous scattering difference Fourier of a complex of the wild-type enzyme with divalent manganese ion and deoxythymidine monophosphate showed that the exonuclease active site has binding sites for two divalent metal ions. The properties of the mutant proteins suggest that one metal ion plays a role in substrate binding while the other is involved in catalysis of the exonuclease reaction.     

苏云金芽孢杆菌伴孢晶体的研究进展

伴胞晶体是苏云金芽孢杆菌(Bacillus thuringiensis,简称Bt)在芽孢形成过程中产生的一种蛋白晶体。作为生物杀虫农药之一,伴胞晶体/Bt由于具有高效、广谱、环保及生物安全等优势,在农林业中得到广泛应用。介绍了伴胞晶体的最新分类、命名情况,综述了其结构、杀虫机理及应用方面的研究进展,并对Bt生物农药的发展前景进行了展望。     

A structural probe of the doped holes in cuprate superconductors

An unresolved issue concerning cuprate superconductors is whether the distribution of carriers in the CuO2 plane is uniform or inhomogeneous. Because the carriers comprise a small fraction of the total charge density and may be rapidly fluctuating, modulations are difficult to detect directly. We demonstrate that in anomalous x-ray scattering at the oxygen K edge of the cuprates, the contribution of carriers to the scattering amplitude is selectively magnified 82 times. This enhances diffraction from the doped holes by more than 10(3), permitting direct structural analysis of the superconducting ground state. Scattering from thin films of La2CuO4+delta (superconducting transition temperature = 39 K) at temperature = 50 +/- 5 kelvin on the reciprocal space intervals (0,0,0.21) --> (0,0,1.21) and (0,0,0.6) --> (0.3,0,0.6) shows a rounding of the carrier density near the substrate suggestive of a depletion zone or similar effect. The structure factor for off-specular scattering was less than 3 x 10(-7) electrons, suggesting an absence of in-plane hole ordering in this material.     

LIII-Edge Anomalous X-ray Scattering by Cesium Measured with Synchrotron Radiation

Diffraction of monochromatized synchrotron radiation by crystals of cesium hydrogen tartrate has been used to measure the magnitude and phase of x-ray scattering for cesium near the LIII absorption edge. In this wavelength region the scattering amplitude of cesium is reduced by as much as 25 electrons per atom, compared to scattering of copper Kalpha x-rays. This change, which varies as a function of wavelength, affects the diffraction intensities in a manner similar to isomorphous substitution, and it is large enough to have promise for phase determination in the study of macromolecular structures. This experiment also demonstrates that accurate diffractometer measurements are possible with synchrotron radiation produced by an electron storage ring.     

Atomic resolution imaging of a carbon nanotube from diffraction intensities

Atomic imaging of three-dimensional structures has required a crystal in diffraction or a lens in electron imaging. Whereas diffraction achieves very high resolution by averaging over many cells, imaging gives localized structural information, such as the position of a single dopant atom. However, lens aberrations limit electron imaging resolution to about 1 angstrom. Resolution is reduced further by low contrast from weakscattering or from the limitations on electron dose for radiation-sensitive molecules. We show that both high resolution and high contrast can be achieved by imaging from diffraction with a nanometer-sized coherent electron beam. The phase problem is solved by oversampling and iterative phase retrieval. We apply this technique to image a double-wall carbon nanotube at 1-angstrom resolution, revealing the structure of two tubes of different helicities. Because the only requirement for imaging is a diffraction pattern sampled below the Nyquist frequency, our technique has the potential to image nonperiodic nanostructures, including biological macromolecules, at diffraction intensity-limited resolutions.     

Imaging doped holes in a cuprate superconductor with high-resolution Compton scattering

The high-temperature superconducting cuprate La(2-x)Sr(x)CuO(4) (LSCO) shows several phases ranging from antiferromagnetic insulator to metal with increasing hole doping. To understand how the nature of the hole state evolves with doping, we have carried out high-resolution Compton scattering measurements at room temperature together with first-principles electronic structure computations on a series of LSCO single crystals in which the hole doping level varies from the underdoped (UD) to the overdoped (OD) regime. Holes in the UD system are found to primarily populate the O 2p(x)/p(y) orbitals. In contrast, the character of holes in the OD system is very different in that these holes mostly enter Cu d orbitals. High-resolution Compton scattering provides a bulk-sensitive method for imaging the orbital character of dopants in complex materials.     

动态高压微射流在食品大分子改性方面的应用

动态高压微射流（DHPM）是一种新兴的物理改性技术,采用该技术处理食品大分子（蛋白质、酶、多糖、膳食纤维等）会改变这些大分子的结构和性质。DHPM 处理能有效地改善蛋白的溶解性、稳定性、起泡性、乳化性和致敏性等,提高酶的活性和稳定性,同时改变蛋白质和酶的结构和构象;DHPM处理能使非淀粉多 糖分子降解,改变其表观黏度、凝胶性能、吸水性、持水性、流动性、粒径等性质;而处理淀粉,则会改变淀粉结晶度,降低老化程度,改善糊化焓、膨胀性 能等;在膳食纤维方面,DHPM处理能改善膳食纤维水化性能,改变其微观形貌,提高表观黏度、屈服应力、可溶性膳食纤维含量等。因此,DHPM技术是一种改 性食品大分子的有效手段,本综述可为食品大分子资源的开发和改性提供理论参考。     

Pressure-induced amorphization and negative thermal expansion in ZrW2O8

It has recently been shown that zirconium tungstate (ZrW2O8) exhibits isotropic negative thermal expansion over its entire temperature range of stability. This rather unusual behavior makes this compound particularly suitable for testing model predictions of a connection between negative thermal expansion and pressure-induced amorphization. High-pressure x-ray diffraction and Raman scattering experiments showed that ZrW2O8 becomes progressively amorphous from 1.5 to 3.5 gigapascals. The amorphous phase was retained after pressure release, but the original crystalline phase returned after annealing at 923 kelvin. The results indicate a general trend between negative thermal expansion and pressure-induced amorphization in highly flexible framework structures.     

How fast-folding proteins fold

An outstanding challenge in the field of molecular biology has been to understand the process by which proteins fold into their characteristic three-dimensional structures. Here, we report the results of atomic-level molecular dynamics simulations, over periods ranging between 100 μs and 1 ms, that reveal a set of common principles underlying the folding of 12 structurally diverse proteins. In simulations conducted with a single physics-based energy function, the proteins, representing all three major structural classes, spontaneously and repeatedly fold to their experimentally determined native structures. Early in the folding process, the protein backbone adopts a nativelike topology while certain secondary structure elements and a small number of nonlocal contacts form. In most cases, folding follows a single dominant route in which elements of the native structure appear in an order highly correlated with their propensity to form in the unfolded state.     

New Opportunities in Synchrotron X-ray Crystallography

Several high-intensity synchrotron x-ray sources have been constructed over the past few years in the United States, West Germany, Great Britain, Japan, France, Italy, and the Soviet Union. Crystallographers have begun to use these facilities for experiments that take advantage of the characteristics of synchrotron radiation, namely, a broad distribution of wavelengths, high intensity, low divergence, strong polarization, and a pulsed time structure. In addition to more familiar diffraction experiments on single crystals and powdered samples, new types of crystallographic studies, for example, energy-dispersive and surface diffraction studies, have progressed rapidly with more general accessibility of synchrotron sources. These high-intensity sources allow diffraction experiments to be performed on very small crystals or on large biological molecules, and permit weak magnetic scattering to be detected Anomalous dispersion experiments can exploit he ability to vary the wavelength of the radiation, and the pulsed time structure of the beam makes possible fast time-resolved experiments. Because of the availability of synchrotron x-radiation, these and other kinds of experiments will be in the forefront of crystallographic research for the next several years.