核磁共振、X射线小角散射以及计算机模拟相结合构建生物大分子复合物的结构模型

近年来,结构生物学研究越来越注重生物大分子复合物的解析,因为许多重要生物学过程都离不开复合物的参与．溶液核磁共振是目前重要的结构解析方法之一．X射线小角散射(SAXS)作为一种新的结构生物学实验手段,近年来发展迅速．SAXS 能提供生物大分子复合物的较低分辨率结构信息,而核磁共振能解析复合物中各个亚基的原子分辨率结构．此外,通过核磁共振还能得到亚基之间的界面、取向以及距离信息．因此近年来通过计算机模拟,整合核磁共振和 SAXS 不同分辨率的结构信息,可以用来搭建生物大分子复合物的结构模型．该综述重点介绍这方面的研究进展．     

Transient protein softening during the working cycle of a molecular machine

Proper functioning of proteins usually requires a certain internal flexibility provided by stochastic structural fluctuations on the picosecond time scale. In contrast with conventional steady-state experiments, we report on a novel type of (laser-neutron) pump-probe experiment combining in situ activation of protein function with a time-dependent test of protein dynamics using quasielastic neutron scattering. A "transient protein softening" is shown to occur during the photocycle of bacteriorhodopsin as a direct proof for the functional significance of protein flexibility.     

Stiffness,resilience, compressibility

The flexibility of a protein is an important component of its functionality. We use nuclear resonance vibrational spectroscopy (NRVS) to quantify the flexibility of the heme iron environment in the electron-carrying protein cytochrome c by measuring the stiffness and the resilience. These quantities are sensitive to structural differences between the active sites of different proteins, as illustrated by a comparative analysis with myoglobin. The elasticity of the entire protein, on the other hand, can be probed quantitatively from NRVS and high energy-resolution inelastic X-ray scattering (IXS) measurements, an approach that we used to extract the bulk modulus of cytochrome c.     

Deriving the ultrastructure of α-crustacyanin using lower-resolution structural and biophysical methods

The low-resolution structure of α-crustacyanin has been determined to 30 ? resolution using negative-stain electron microscopy (EM) with single-particle averaging. The protein, which is an assembly of eight β-crustacyanin dimers, appears asymmetrical and rather open in layout. A model was built to the EM map using the X-ray crystallographic structure of β-crustacyanin guided by PISA interface analyses. The model has a theoretical sedimentation coefficient that matches well with the experimentally derived value from sedimentation velocity analytical ultracentrifugation. Additionally, the EM model has similarities to models calculated independently by rigid-body modelling to small-angle X-ray scattering (SAXS) data and extracted in silico from the β-crustacyanin crystal lattice. Theoretical X-ray scattering from each of these models is in reasonable agreement with the experimental SAXS data and together suggest an overall design for the α-crustacyanin assembly.     

Comparing proteins by their internal dynamics: Exploring structure–function relationships beyond static structural alignments

The growing interest for comparing protein internal dynamics owes much to the realisation that protein function can be accompanied or assisted by structural fluctuations and conformational changes. Analogously to the case of functional structural elements, those aspects of protein flexibility and dynamics that are functionally oriented should be subject to evolutionary conservation. Accordingly, dynamics-based protein comparisons or alignments could be used to detect protein relationships that are more elusive to sequence and structural alignments. Here we provide an account of the progress that has been made in recent years towards developing and applying general methods for comparing proteins in terms of their internal dynamics and advance the understanding of the structure–function relationship.     

Validation of protein structural models using THz spectroscopy: a promising approach to solve three-dimensional structures

Terahertz (THz) spectroscopy is a promising technique for the study of protein structure and internal flexibility. Here, we used THz spectroscopy and molecular modeling for bovine serum albumin (BSA) structure investigation. BSA molecule was built using homology modeling methods and 30 different more relaxed models were obtained by molecular dynamics simulations of the hydrated protein. As the experimental and simulated THz spectra are linear, we compared them by comparing the slopes of the lines that fit them. Six BSA structures had slope values in the range given by the slope of the experimental spectrum $\pm $  0.2 and a total of sixteen BSA structures had slope values in the 0.6 interval near the experimental slope value. BSA average structures were calculated over the six and the sixteen identified BSA molecules. Based on the similarity with the crystal structure of BSA, we validated the average structure calculated over the sixteen BSA conformations. The comparison with the crystal structure showed that the structure validated using THz spectroscopy is a coarse model of BSA, as its root-mean-square deviation relative to the crystal structure is 1.9 Å. The regions from our model that present the highest deviation from the crystal structure are exterior loops. The results presented here show that using THz spectroscopy and molecular modeling is a promising approach to determine the structure of proteins.     

利用小角X射线散射技术研究煤干馏过程中的孔隙结构变化

煤干馏是煤炭高效利用的重要途径之一。同步辐射小角X射线散射(Small Angle X-Ray Scattering,SAXS)技术是研究煤干馏过程中内部孔隙结构变化的常用手段。本文利用SAXS对山西余吾烟煤干馏过程(100~1200℃,温度梯度100℃)进行了表征,并对分形维数和粒径分布的变化进行了分析。结果发现煤干馏过程中,散射图像类似,具有各向同性特征,始终保持表面分形结构,且分形维数总体上呈现增大的趋势;随着煤干馏温度的升高,粒度分布发生了显著变化,充分反映了煤在不同干馏阶段的结构特征,对后续的煤炭研究具有一定的指导意义。     

Reply to a comment by S.N. Semenov and M.E. Schimpf on ‘Role of the velocity frame of reference in thermodiffusion in liquid mixtures’, Philosophical Magazine vol. 92, 705 (2012), by M. Eslamian,C.G. Jian and M.Z. Saghir.

A small angle X-ray scattering (SAXS) study of nanovoids in 99.988 and 99.995?at.% aluminium is presented. Absolute intensity calibration using a glassy carbon standard is used to extract the weak SAXS signature from nanovoids introduced by thermal quenching. SAXS analysis methods, including Guinier, Porod and Indirect Transform, are used to obtain values for the void–size, number distribution and volume fraction, as well as measures of the void-metal matrix interface structure in quenched aluminium samples. The SAXS analysis has identified a residual impurity effect on void formation and has been used to characterize trends in nanovoid size, number distribution and interface structure as a function of ageing time at elevated temperatures (artificial ageing). The work presented here, including identification of experimental tools that can be readily improved, demonstrates that SAXS studies are capable of providing precise characterization of nanovoid structure in aluminium. This level of information will be useful in developing phenomenological models of void nucleation and growth capable of linking atomic scale phenomena to macroscopic material properties.     

Interrogating the druggable genome with structural informatics

Summary Structural genomics projects are producing protein structure data at an unprecedented rate. In this paper, we present the Target Informatics Platform (TIP), a novel structural informatics approach for amplifying the rapidly expanding body of experimental protein structure information to enhance the discovery and optimization of small molecule protein modulators on a genomic scale. In TIP, existing experimental structure information is augmented using a homology modeling approach, and binding sites across multiple target families are compared using a clique detection algorithm. We report here a detailed analysis of the structural coverage for the set of druggable human targets, highlighting drug target families where the level of structural knowledge is currently quite high, as well as those areas where structural knowledge is sparse. Furthermore, we demonstrate the utility of TIP's intra- and inter-family binding site similarity analysis using a series of retrospective case studies. Our analysis underscores the utility of a structural informatics infrastructure for extracting drug discovery-relevant information from structural data, aiding researchers in the identification of lead discovery and optimization opportunities as well as potential “off-target” liabilities.     

The application of hybrid pixel detectors for in‐house SAXS instrumentation with a view to combined chromatographic operation

This study analyses the potential for laboratory‐based size‐exclusion chromatography (SEC) integrated small‐angle X‐ray scattering (SAXS) instrumentation to characterize protein complexes. Using a high‐brilliance home source in conjunction with a hybrid pixel X‐ray detector, the efficacy of SAXS data collection at pertinent protein concentrations and exposure times has been assessed. Scattering data from SOD1 and from the complex of SOD1 with its copper chaperone, using 10 min exposures, provided data quality in the range 0.03 < q < 0.25 Å^?1 that was sufficient to accurately assign radius of gyration, maximum dimension and molecular mass. These data demonstrate that a home source with integrated SEC–SAXS technology is feasible and would enable structural biologists studying systems containing transient protein complexes, or proteins prone to aggregation, to make advanced preparations in‐house for more effective use of limited synchrotron beam time.     

一款小角X射线散射原位测量专用简易样品加热装置

原位加热实验是同步辐射小角X射线散射领域的新热点。本文针对同步辐射小角X射线散射中需要加热并原位实时检测的实验而设计的一款样品简易原位加热专用装置,该装置主要由温控器、样品池、固定架组成,它具有结构简单、操作容易的特点。本文介绍该装置的基本结构、特点并采用该装置进行原位加热干燥褐煤实验,通过分析实验结果验证装置的可行性。     

Small Angle X-Ray Scattering of Hematite Aggregates

Small angle X-ray scattering has been utilised to study the structure of hematite aggregates. The small angle X-ray scattering (SAXS) spectra obtained provided insight into structure of the hematite aggregates and the size of the primary particles. The structural analysis results obtained by SAXS are consistent with previous results obtained from static light scattering studies. Both techniques indicate that the mass fractal dimensions of hematite aggregates are markedly higher than those obtained for other particle systems.     

Force-constant-decayed anisotropic network model: An improved method for predicting RNA flexibility

RNA is an important biological macromolecule, which plays an irreplaceable role in many life activities. RNA functions are largely determined by its tertiary structure and the intrinsic dynamics encoded in the structure. Thus, how to effective extract structure-encoded dynamics is of great significance for understanding RNA functions. Anisotropic network model (ANM) is an efficient method to investigate macromolecular dynamical properties, which has been widely used in protein studies. However, the performance of the conventional ANM in describing RNA flexibility is not as good as that on proteins. In this study, we proposed a new approach, named force-constant-decayed anisotropic network model (fcd-ANM), to improve the performance in investigating the dynamical properties encoded in RNA structures. In fcd-ANM, nucleotide pairs in RNA structure were connected by springs and the force constant of springs was decayed exponentially based on the separation distance to describe the differences in the inter-nucleotide interaction strength. The performance of fcd-ANM in predicting RNA flexibility was evaluated using a non-redundant structure database composed of 51 RNAs. The results indicate that fcd-ANM significantly outperforms the conventional ANM in reproducing the experimental B-factors of nucleotides in RNA structures, and the Pearson correlation coefficient between the predicted and experimental nucleotide B-factors was distinctly improved by 21.05% compared to the conventional ANM. Fcd-ANM can serve as a more effective method for analysis of RNA dynamical properties.     

Classification of ab initio models of proteins restored from small‐angle X‐ray scattering

In structure analyses of proteins in solution by using small‐angle X‐ray scattering (SAXS), the molecular models are restored by using ab initio molecular modeling algorithms. There can be variation among restored models owing to the loss of phase information in the scattering profiles, averaging with regard to the orientation of proteins against the direction of the incident X‐ray beam, and also conformational fluctuations. In many cases, a representative molecular model is obtained by averaging models restored in a number of ab initio calculations, which possibly provide nonrealistic models inconsistent with the biological and structural information about the target protein. Here, a protocol for classifying predicted models by multivariate analysis to select probable and realistic models is proposed. In the protocol, each structure model is represented as a point in a hyper‐dimensional space describing the shape of the model. Principal component analysis followed by the clustering method is applied to visualize the distribution of the points in the hyper‐dimensional space. Then, the classification provides an opportunity to exclude nonrealistic models. The feasibility of the protocol was examined through the application to the SAXS profiles of four proteins.     

Adsorption characterization of surfactant-templated ordered mesoporous silicas synthesized with and without hydrothermal treatment

This work reports a systematic study of ordered mesoporous silicas (OMSs) synthesized with and without hydrothermal treatment at 373 K for a series of surfactants of different alkyl chain length (from C10 to C18). For these samples nitrogen adsorption and small angle X-ray scattering (SAXS) data were measured to characterize their adsorption and surface properties. Namely, nitrogen adsorption isotherms were used to evaluate their specific surface area, pore volume and pore size distribution, whereas SAXS data provided information about their structural ordering. It is shown that while the room temperature synthesis afforded OMS samples with cubic MCM-48 structure, an additional 5-day hydrothermal treatment of these samples at 373 K caused their transformation to MCM-41 (two-dimensional hexagonal structure) and improved their pore uniformity, which was manifested by reducing the width of pore size distribution.     

Small angle X-ray scattering of the colloidal crystal

The monodisperse polystyrene spheres are assembled into the colloidal crystal on the glass substrate by vertical deposition method, which is aimed at the so-called photonic crystal applications. The structural information of the bulk colloidal crystal is crucial for understanding the crystal growth mechanism and developing the various applications of colloidal crystal. Small-angle X-ray scattering (SAXS) technique was used to obtain the bulk structure of the colloidal crystal at Beamline 1W2A of BSRF. It is found that the SAXS pattern is sensitive to the relative orientation between the colloidal sample and the incident X-ray direction. The crystal lattice was well distinguished and determined by the SAXS data.     

Nanoscale Structure of Urethane/Urea Elastomeric Films

The nanostructure of urethane/urea elastomeric membranes was investigated by small-angle X-ray scattering (SAXS) in order to establish relationships between their structure and mechanical properties. The networks were made up of polypropylene oxide (PPO) and polybutadiene (PB) segments. The structural differences were investigated in two types of membranes with the same composition but with different thermal treatment after casting. Type I was cured at 70–80 °C and type II at 20 °C. Both membranes showed similar phase separation by TEM, with nanodomains rich in PB or PPO and 25 nm dimensions. The main difference between type I and type II membranes was found by SAXS. The type I membrane spectra showed, besides a broad band at a 27-nm q value (modulus of the scattering vector), an extra band at 6 nm, which was not observed in the type II membrane. The SAXS spectra were interpreted in terms of PPO, PB soft segments, and urethane/urea links, as well as hard moiety segregation in the reaction medium. This additional segregation (q = 7 nm), although subtle, results in diverse mechanical behavior of in both membranes.     

小角X射线散射应用研究若干进展

文章从小角X射线散射（small-ande X-ray scattering，SAXS）的理论分析出发，结合国内外SAXS理论和应用研究的最新动态，总结了文章作者所在的研究小组近年来在SAXS技术用于材料微结构表征方面的研究成果。主要包括以下三个方面的内容：（1）有机／无机杂化材料中电子密度波动的研究；（2）弦长度分布函数材料的周期结构的研究；（3）纳米粉末晶化过程的研究。     

Flexibility of nucleic acids: From DNA to RNA

The structural flexibility of nucleic acids plays a key role in many fundamental life processes, such as gene replication and expression, DNA-protein recognition, and gene regulation. To obtain a thorough understanding of nucleic acid flexibility, extensive studies have been performed using various experimental methods and theoretical models. In this review, we will introduce the progress that has been made in understanding the flexibility of nucleic acids including DNAs and RNAs,and will emphasize the experimental findings and the effects of salt, temperature, and sequence. Finally, we will discuss the major unanswered questions in understanding the flexibility of nucleic acids.     

Software for the high-throughput collection of SAXS data using an enhanced Blu-Ice/DCS control system

Biological small-angle X-ray scattering (SAXS) provides powerful complementary data for macromolecular crystallography (MX) by defining shape, conformation and assembly in solution. Although SAXS is in principle the highest throughput technique for structural biology, data collection is limited in practice by current data collection software. Here the adaption of beamline control software, historically developed for MX beamlines, for the efficient operation and high-throughput data collection at synchrotron SAXS beamlines is reported. The Blu-Ice GUI and Distributed Control System (DCS) developed in the Macromolecular Crystallography Group at the Stanford Synchrotron Radiation Laboratory has been optimized, extended and enhanced to suit the specific needs of the biological SAXS endstation at the SIBYLS beamline at the Advanced Light Source. The customizations reported here provide a potential route for other SAXS beamlines in need of robust and efficient beamline control software. As a great deal of effort and optimization has gone into crystallographic software, the adaption and extension of crystallographic software may prove to be a general strategy to provide advanced SAXS software for the synchrotron community. In this way effort can be put into optimizing features for SAXS rather than reproducing those that have already been successfully implemented for the crystallographic community.