Challenges in NMR-based structural genomics

Understanding the functions of the vast number of proteins encoded in many genomes that have been completely sequenced recently is the main challenge for biologists in the post-genomics era. Since the function of a protein is determined by its exact three-dimensional structure it is paramount to determine the 3D structures of all proteins. This need has driven structural biologists to undertake the structural genomics project aimed at determining the structures of all known proteins. Several centers for structural genomics studies have been established throughout the world. Nuclear magnetic resonance (NMR) spectroscopy has played a major role in determining protein structures in atomic details and in a physiologically relevant solution state. Since the number of new genes being discovered daily far exceeds the number of structures determined by both NMR and X-ray crystallography, a high-throughput method for speeding up the process of protein structure determination is essential for the success of the structural genomics effort. In this article we will describe NMR methods currently being employed for protein structure determination. We will also describe methods under development which may drastically increase the throughput, as well as point out areas where opportunities exist for biophysicists to make significant contribution in this important field.     

Towards dynamic structure of biological complexes at atomic resolution by cryo-EM

Cryo-electron microscopy makes use of transmission electron microscopy to image vitrified biological samples and reconstruct their three-dimensional structures from two-dimensional projections via computational approaches. After over40 years of development, this technique is now reaching its zenith and reforming the research paradigm of modern structural biology. It has been gradually taking over X-ray crystallography as the mainstream method. In this review, we briefly introduce the history of cryo-EM, recent technical development and its potential power to reveal dynamic structures. The technical barriers and possible approaches to tackle the upcoming challenges are discussed.     

Molecular modelling in structural biology

Molecular modelling is a powerful methodology for analysing the three dimensional structure of biological macromolecules. There are many ways in which molecular modelling methods have been used to address problems in structural biology. It is not widely appreciated that modelling methods are often an integral component of structure determination by NMR spectroscopy and X-ray crystallography. In this review we consider some of the numerous ways in which modelling can be used to interpret and rationalise experimental data and in constructing hypotheses that can be tested by experiment. Genome sequencing projects are producing a vast wealth of data describing the protein coding regions of the genome under study. However, only a minority of the protein sequences thus identified will have a clear sequence homology to a known protein. In such cases valuable three-dimensional models of the protein coding sequence can be constructed by homology modelling methods. Threading methods, which used specialised schemes to relate protein sequences to a library of known structures, have been shown to be able to identify the likely protein fold even in cases where there is no clear sequence homology. The number of protein sequences that cannot be assigned to a structural class by homology or threading methods, simply because they belong to a previously unidentified protein folding class, will decrease in the future as collaborative efforts in systematic structure determination begin to develop. For this reason, modelling methods are likely to become increasingly useful in the near future. The role of the blind prediction contests, such as the Critical Assessment of techniques for protein Structure Prediction (CASP), will be briefly discussed. Methods for modelling protein-ligand and protein-protein complexes are also described and examples of their applications given.     

Quantitative electron diffraction: From atoms to bonds

The development of new transmission electron microscopes with energy-filtering capability together with the increase in computer power over the past few years has enabled electron diffraction from inorganic crystals to become a quantitative and accurate science. With the introduction of new techniques and data analysis and with the advantage of a nanometre-sized probe, electron diffraction now rivals X-ray and neutron methods in many aspects of crystallography and solid-state physics. In this article, we have discussed two new developments which highlight the progress made in this area. First, a new method for ab-initio structure determination is explained and an example given to show its success. Second, energy-filtered diffraction patterns are used to refine the scattering potential of a crystal so that the bonding charge density can be reconstructed.     

NMR技术在蛋白质-蛋白质相互作用研究中的应用——泛素-蛋白水解酶体通路研究实例介绍

蛋白质-蛋白质相互作用在多种细胞内生理活动中发挥关键性作用,而蛋白质复合物结构信息的获得主要依赖于X-射线衍射技术和核磁共振技术2种主要技术手段的使用. 需要指出的是,虽然大部分蛋白质复合物的结构解析使用了X-射线衍射技术,然而在包括无法获得蛋白质复合物晶体、 蛋白质与蛋白质结合强度较弱以及蛋白质复合物系统具有复杂的动力学行为等几种情况下,核磁共振技术是可用于蛋白质复合物结构测定的唯一手段. 用于蛋白质-蛋白质相互作用研究的NMR技术主要有化学位移扰动分析、分子间NOE的检测、顺磁弛豫增强技术、残余偶极耦合检测技术等几种. 该文将结合这几种技术在泛素-蛋白水解酶体通路领域的应用实例对它们的工作原理以及可提供的信息做出总结介绍.     

Potential controlled surface aggregation of surfactants at electrode surfaces – A molecular view

By describing studies of three prototypical surfactants with similar hydrophobic tails but differently charged headgroups, this review provides a summary of the rich phase behavior of soluble surfactant molecules at electrified interfaces. With the use of electrochemical, scanning probe microscopy, and neutron scattering techniques we have been able to fully explore the adsorption and surface aggregation of these molecular systems. Furthermore, we have been able to provide compelling evidence of electric field-driven phase transitions in these surfactant films and their aggregated structures. Cumulatively, our results demonstrate that the electrical state of a surface (namely surface charge or applied potential) plays an integral role in determining the morphology of surfactants at solid interfaces. Unlike other aggregate shape determining factors such as the surfactant packing parameter, the electrical parameter can readily be adjusted in situ, providing a tunable means to control films of soft condensed matter.     

研究CTG和CCTG重复序列溶液结构的挑战

脱氧核糖核酸(DNA)重复序列的基因不稳定性和遗传神经退行性疾病有关. 通过在基因复制中形成异常结构,CTG和CCTG重复序列进行自我扩张,并会分别导致强直性肌营养不良类型1 (DM1) 和强直性肌营养不良类型2 (DM2). 尽管x-射线衍射晶体分析法在解决基因结构上是一个强大的技术,但是CTG和CCTG这些重复序列可能是因为其固有的灵活性,很难得到晶体,所以现在还没能成功鉴定其结构. 因此,核磁共振 (NMR) 光谱仍然是在单个脱氧核糖核酸层次上研究DNA结构的唯一技术. 最近,研究人员克服了包括由于重复系列导致的严重信号重叠、存在的多种构象及构象互换导致的光谱复杂性的挑战, 顺利运用核磁共振技术研究了CTG和CCTG重复序列的结构. 通过适当的样品设计、样品操作技术、次序修改、及/或单个脱氧核糖核酸替代实验,利用¹H 和³¹P核磁共振波谱法确定了CTG和 CCTG 重复序列的结构特点. 该综述回顾详细阐述上述研究中所采用的技术.     

Model building of a molluscan hemocyanin from X-ray solution scattering

Molluscan hemocyanins are proteins of a truly enormous size. Because of this, determination of their quaternary structure at high resolution cannot easily be obtained by standard methods such as X-ray crystallography and NMR. Therefore, different approaches, using several low-resolution techniques are currently necessary to understand hemocyanin structure. In this work a model of the Rapana venosa hemocyanin has been obtained from a template model and small angle X-ray scattering (SAXS) data. The template model was built from the electron density of the closely related Haliotis tuberculata hemocyanin and a computer program was written to fit this model to the SAXS data using the simulated annealing algorithm.     

Cohomology for Anyone

Crystallography has proven a rich source of ideas over several centuries. Among the many ways of looking at space groups, N. David Mermin has pioneered the Fourier-space approach. Recently, we have supplemented this approach with methods borrowed from algebraic topology. We now show what topology, which studies global properties of manifolds, has to do with crystallography. No mathematics is assumed beyond what the typical physics or crystallography student will have seen of group theory; in particular, the reader need not have any prior exposure to topology or to cohomology of groups.     

The application of neutron powder diffraction techniques for crystallographic studies at high pressures

Abstract

For phase transition studies, neutron powder diffraction offers a number of important advantages over x-ray based techniques, for example ab-initio structural determination. There are two distinct methods using either monochromatic angular dispersive geometry on a reactor cold source or time-of-flight energy-dispersive techniques requiring a pulsed neutron source. Both techniques offer comparable resolution but have differing advantages for high pressure studies. Recent studies illustrate the benefits of the two methods and the application of these to solve unknown crystal structures.     

Resonant elastic X-ray scattering in chemistry and materials science

The applications of anomalous scattering for locating metal atoms and discriminating between different elements has increased when optimised with synchrotron X-radiation.The on-resonance effect enhances the targeted elemental signal and allows small occupancies to be determined, including in situations of a mixed metal population at a single atomic site. Thus the applications of resonant elastic X-ray scattering in biological, inorganic and materials chemistry is being widely applied to single crystals, which is our emphasis, but also powders, fibres, solutions, amorphous and thin film states of matter. Recent developments have included the use of high photon energies (upto 100 keV) as well as softer X-rays (2 keV). The various instrument and technical capabilities have improved in the last 15 years. This ease of measurement of the resonant scattering signals along with absorption edge shifts indicates an expansion to the measurement of multiple data sets, to allow monitoring of redox changes. Whilst crystal structure determination in biological crystallography has been revolutionised by the MAD method, it is not a requirement for chemical or materials crystallography, as other phasing techniques are routine. Synchrotron source upgrades will allow nano-sized X-ray beams to be more widely available. The new X-ray lasers suggest new capabilities too.     

I19, the small‐molecule single‐crystal diffraction beamline at Diamond Light Source

The dedicated small‐molecule single‐crystal X‐ray diffraction beamline (I19) at Diamond Light Source has been operational and supporting users for over three years. I19 is a high‐flux tunable‐wavelength beamline and its key details are described in this article. Much of the work performed on the beamline involves structure determination from small and weakly diffracting crystals. Other experiments that have been supported to date include structural studies at high pressure, studies of metastable species, variable‐temperature crystallography, studies involving gas exchange in porous materials and structural characterizations that require analysis of the diffuse scattering between Bragg reflections. A range of sample environments to facilitate crystallographic studies under non‐ambient conditions are available as well as a number of options for automation. An indication of the scope of the science carried out on the beamline is provided by the range of highlights selected for this paper.     

在双螺旋结构的发现中物理学的作用与贡献

简述了薛定谔对基因物质的预言和德尔布吕克等人关于DNA是遗传物质的确定;介绍了DNA结构的X射线衍射分析,其中包括在X射线晶体学中的布拉格方程,螺旋结构的傅里叶变换,威尔金斯对双螺旋结构的实验验证;介绍了分子模型方法,其中包括键角与键长,杂化轨道理论,碳四面体型轨道的量子力学计算,在分子的空间构型中氢键的作用.     

微纳尺度俄歇电子能谱新技术开发及其应用进展

随着纳米结构材料的广泛应用,新型微纳尺度表征技术成为纳米科学技术发展的重要途径。本文基于局域电子信息全面性的思想,从俄歇电子能谱的原理出发,理论推导出俄歇价电子能谱的简明表述方式,确定俄歇价电子能谱与微观电子结构信息的内在联系和物理意义,建立了俄歇电子能谱探测微区一系列宏观参量的新技术。其中应力测量技术的空间分辨率可优于20nm,为微纳尺度力学测量的发展提供了重要的方法;非接触性的局域电学性质测量技术,超越了传统电学测量方法的思想框架,实现了无外场驱动的电荷密度分布、电场分布等本征电学性质表征、以及半导体异质结构整个空间区域的能带构造;结构相的测定技术,使纳米微区材料的晶体结构相识别成为可能;半导体微区导电类型测定技术,延续了非接触性电学测量的优点,并且能够灵活地探测分析复杂光电器件结构中不同区域的导电类型分布。通过实际应用于侧向外延GaN不同区域、AlxGa1-xN/GaN超晶格量子阱结构、ZnO纳米颗粒等纳米尺度复杂体系的微区宏观性质探测,获得应力/应变、电荷密度/电场、结构相以及导电类型及其分布等结果,验证了所建立的测量技术的有效性和可靠性。     

Electron microscopy study of incommensurate modulated structures in misfit ternary chalcogenides

The study of misfit structures by means of transmission electron microscopy and associated techniques is reviewed. It is complementary to X-ray crystallography and provides a local and direct view of the beautiful complexity of these systems. Three different types are presented in this work. Misfit columnar structures can be contemplated as a case of one-dimensional misfit structures while misfit layer structures are considered as two-dimensional misfit structures. Some extra dimensionality is added when these misfit layer structures wrap to give rise to tubular crystals. Electron microscopy/diffraction shows clearly in many examples the presence of structural modulations as weak satellite reflections that are very difficult to detect by X-ray diffraction methods. Besides, high-resolution images show the presence of stacking defects in some of these misfit layer structures.     

残留偶极耦合及其在蛋白质结构研究中的应用

近年来溶液中残留偶极耦合常数被用来获取生物大分子化学键之间相对取向等长程构象约束条件,用于计算或优化蛋白质及其复合物的三维空间结构. 介绍了用异核多维NMR技术测量残留偶极耦合常数的方法,及其在蛋白质结构计算中的一些应用：优化蛋白质溶液结构,评价蛋白质结构质量,确定蛋白质结构域取向,获取有关配体的构象和取向的信息,在缺乏NOE数据时构建蛋白质结构等.      

NMR Crystallography as a Novel Tool for the Understanding of the Mode of Action of Enzymes: SOD a Case Study

Nuclear magnetic resonance (NMR) crystallography is an approach for revealing molecular and supramolecular structures and molecular packing for systems where standard X-ray crystallography gives no results. It combines solid-state NMR techniques with chemical models and/or molecular dynamics and/or quantum chemical calculations. These techniques are often supported by other structure characterization methods. In the present review, recent results on the application of NMR crystallography for the investigation of the mode of action of superoxide dismutases are discussed. Studies of substrate–inhibitor complexes of human manganese and Streptomyces nickel superoxide dismutase are presented, which are chemical models of the transient enzyme–substrate complex. The review is completed by new, previously unpublished results, calculating an NMR structure of NiSOD model peptide-bound cyanide based on experimental restraints measured by us and derived from the literature and extended DFT calculations.     

Theory of14N nuclear quadrupole interaction in pyrrole,indole and carbazole

As a test of the accuracy of the Hartree-Fock Cluster procedure which is being increasingly used for the study of electronic structures and hyperfine properties of solid state systems, we have applied it to study the¹⁴N quadrupole coupling constants of the free molecules pyrrole, indole and carbazole, which have been measured by microwave spectroscopic techniques. Our results explain the experimental trends over the three molecules and provide very good quantitative agreement with experiment.     

核磁共振技术在生物研究中的应用

核磁共振(nuclear magnetic resonance,NMR)是以原子核自旋的共振跃迁为探测对象的谱学方法.当自旋量子数不为零的原子核处于外磁场中时,会引起能级的Zeeman分裂.若再施加能量等于Zeeman能级差的射频场,则会诱发原子核自旋的共振跃迁,这种现象即为核磁共振.核自旋的共振频率与原子的类型有关,且受原子所处化学和物理环境的影响.此外,NMR能量较低,不会影响探测对象(常为分子)的状态.因此,NMR能够在无损条件下提供多种具有原子和分子分辨的物质组成、结构、形态、动态变化等丰富信息.