Neutron Laue macromolecular crystallography

Recent progress in neutron protein crystallography such as the use of the Laue technique and improved neutron optics and detector technologies have dramatically improved the speed and precision with which neutron protein structures can now be determined. These studies are providing unique and complementary insights on hydrogen and hydration in protein crystal structures that are not available from X-ray structures alone. Parallel improvements in modern molecular biology now allow fully (per)deuterated protein samples to be produced for neutron scattering that essentially eradicate the large-and ultimately limiting-hydrogen incoherent scattering background that has hampered such studies in the past. High quality neutron data can now be collected to near atomic resolution (approximately 2.0 A) for proteins of up to approximately 50 kDa molecular weight using crystals of volume approximately 0.1 mm3 on the Laue diffractometer at ILL. The ability to flash-cool and collect high resolution neutron data from protein crystals at cryogenic temperature (15 K) has opened the way for kinetic crystallography on freeze trapped systems. Current instrument developments now promise to reduce crystal volume requirements by a further order of magnitude, making neutron protein crystallography a more accessible and routine technique.     

Rational protein crystallization by mutational surface engineering

Protein crystallization constitutes a limiting step in structure determination by X-ray diffraction. Even if single crystals are available, inadequate physical quality may seriously limit the resolution of the available data and consequently the accuracy of the atomic model. Recent studies show that targeted mutagenesis of surface patches containing residues with large flexible side chains and their replacement with smaller amino acids lead to effective preparation of X-ray quality crystals of proteins otherwise recalcitrant to crystallization. Furthermore, this technique can also be used to obtain crystals of superior quality as compared to those grown for the wild-type protein, sometimes increasing the effective resolution by as much as 1 A or more. Several recent examples of this new methodology suggest that the method has the potential to become a routine tool in protein crystallography.     

Resolution-by-proxy: a simple measure for assessing and comparing the overall quality of NMR protein structures

In protein X-ray crystallography, resolution is often used as a good indicator of structural quality. Diffraction resolution of protein crystals correlates well with the number of X-ray observables that are used in structure generation and, therefore, with protein coordinate errors. In protein NMR, there is no parameter identical to X-ray resolution. Instead, resolution is often used as a synonym of NMR model quality. Resolution of NMR structures is often deduced from ensemble precision, torsion angle normality and number of distance restraints per residue. The lack of common techniques to assess the resolution of X-ray and NMR structures complicates the comparison of structures solved by these two methods. This problem is sometimes approached by calculating "equivalent resolution" from structure quality metrics. However, existing protocols do not offer a comprehensive assessment of protein structure as they calculate equivalent resolution from a relatively small number (<5) of protein parameters. Here, we report a development of a protocol that calculates equivalent resolution from 25 measurable protein features. This new method offers better performance (correlation coefficient of 0.92, mean absolute error of 0.28 ?) than existing predictors of equivalent resolution. Because the method uses coordinate data as a proxy for X-ray diffraction data, we call this measure "Resolution-by-Proxy" or ResProx. We demonstrate that ResProx can be used to identify under-restrained, poorly refined or inaccurate NMR structures, and can discover structural defects that the other equivalent resolution methods cannot detect. The ResProx web server is available at http://www.resprox.ca.     

Difficult macromolecular structures determined using X-ray diffraction techniques

Macromolecular crystallography has been, for the last few decades, the main source of structural information of biological macromolecular systems and it is one of the most powerful techniques for the analysis of enzyme mechanisms and macromolecular interactions at the atomic level. In addition, it is also an extremely powerful tool for drug design. Recent technological and methodological developments in macromolecular X-ray crystallography have allowed solving structures that until recently were considered difficult or even impossible, such as structures at atomic or subatomic resolution or large macromolecular complexes and assemblies at low resolution. These developments have also helped to solve the 3D-structure of macromolecules from twin crystals. Recently, this technique complemented with cryo-electron microscopy and neutron crystallography has provided the structure of large macromolecular machines with great precision allowing understanding of the mechanisms of their function.     

生物高分辨电子显微学进展

生物高分辨电子显微学是近年来发展起来的一种可与X射线晶体学相媲美的测定生物大分子高分辨结构的方法．它克服了一些限制X射线晶体学应用的困难,可以直接对非晶体状态的生物大分子或仅能形成二维晶体的蛋白进行结构测定．这一技术主要包括高分辨电子显微象的获得与电子显微象解析．文章就这一技术应用中的一些问题：自然结构的保持、辐射损伤、低衬度、低信噪比等进行了讨论．     

Characterization of perdeuterated high-potential iron-sulfur protein with high-resolution X-ray crystallography

Perdeuteration in neutron crystallography is an effective method for determining the positions of hydrogen atoms in proteins. However, there is shortage of evidence that the high-resolution details of perdeuterated proteins are consistent with those of the nondeuterated proteins. In this study, we determined the X-ray structure of perdeuterated high-potential iron-sulfur protein (HiPIP) at a high resolution of 0.85 å resolution. The comparison of the nondeuterated and perdeuterated structures of HiPIP revealed slight differences between the two structures. The spectroscopic and spectroelectrochemical studies also showed that perdeuterated HiPIP has approximately the same characteristics as nondeuterated HiPIP. These results further emphasize the suitability of using perdeuterated proteins in the high-resolution neutron crystallography.     

Monitoring and validating active site redox states in protein crystals

High resolution protein crystallography using synchrotron radiation is one of the most powerful tools in modern biology. Improvements in resolution have arisen from the use of X-ray beamlines with higher brightness and flux and the development of advanced detectors. However, it is increasingly recognised that the benefits brought by these advances have an associated cost, namely deleterious effects of X-ray radiation on the sample (radiation damage). In particular, X-ray induced reduction and damage to redox centres has been shown to occur much more rapidly than other radiation damage effects, such as loss of resolution or damage to disulphide bridges. Selection of an appropriate combination of in-situ single crystal spectroscopies during crystallographic experiments, such as UV-visible absorption and X-ray absorption spectroscopy (XAFS), allows for effective monitoring of redox states in protein crystals in parallel with structure determination. Such approaches are also essential in cases where catalytic intermediate species are generated by exposure to the X-ray beam. In this article, we provide a number of examples in which multiple single crystal spectroscopies have been key to understanding the redox status of Fe and Cu centres in crystal structures. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State.     

Crystal structure of S. aureus YlaN, an essential leucine rich protein involved in the control of cell shape

The crystal structure of a conserved leucine rich protein, YlaN, from Staphylococcus aureus has been determined by X-ray crystallography to 2.3 A resolution. Whilst the precise function of S. aureus YlaN is unknown its homologue in B. subtilis has been shown to be essential for cell survival and is thought to be involved in controlling cell shape. The structure of S. aureus YlaN provides the first view of its protein family, which reveals that it is a novel homodimer whose subunit architecture is comprised of an antiparallel 3 helix bundle reminiscent of the helical arrangements seen in leucine zipper proteins. Analysis of the pattern of sequence conservation on the structure has led to the identification of two connected solvent exposed patches of conserved residues in each subunit located at one end of but on opposite faces of the molecule. We suggest that YlaN has a binding role in the cell rather than a catalytic function and a search for its ligand is underway to accelerate its exploitation as a target for antibiotic discovery.     

High-resolution membrane protein structure by joint calculations with solid-state NMR and X-ray experimental data

X-ray diffraction and nuclear magnetic resonance spectroscopy (NMR) are the staple methods for revealing atomic structures of proteins. Since crystals of biomolecular assemblies and membrane proteins often diffract weakly and such large systems encroach upon the molecular tumbling limit of solution NMR, new methods are essential to extend structures of such systems to high resolution. Here we present a method that incorporates solid-state NMR restraints alongside of X-ray reflections to the conventional model building and refinement steps of structure calculations. Using the 3.7 Å crystal structure of the integral membrane protein complex DsbB-DsbA as a test case yielded a significantly improved backbone precision of 0.92 Å in the transmembrane region, a 58% enhancement from using X-ray reflections alone. Furthermore, addition of solid-state NMR restraints greatly improved the overall quality of the structure by promoting 22% of DsbB transmembrane residues into the most favored regions of Ramachandran space in comparison to the crystal structure. This method is widely applicable to any protein system where X-ray data are available, and is particularly useful for the study of weakly diffracting crystals.     

Macromolecular electron microscopy in the era of structural genomics

Macromolecular machines carry out many cellular functions. Cryo-electron microscopy (cryo-EM) is emerging as a powerful method for studying the structure, assembly and dynamics of such macromolecules, and their interactions with substrates. With resolutions still improving, ‘single-particle’ analyses are already depicting secondary structure. Moreover, cryo-EM can be combined in several ways with X-ray diffraction to enhance the resolution of cryo-EM and the applicability of crystallography. Electron tomography holds promise for visualizing machines at work inside cells.     

Rapid increase of near atomic resolution virus capsid structures determined by cryo-electron microscopy

The recent technological advances in electron microscopes, detectors, as well as image processing and reconstruction software have brought single particle cryo-electron microscopy (cryo-EM) into prominence for determining structures of bio-molecules at near atomic resolution. This has been particularly true for virus capsids, ribosomes, and other large assemblies, which have been the ideal specimens for structural studies by cryo-EM approaches. An analysis of time series metadata of virus structures on the methods of structure determination, resolution of the structures, and size of the virus particles revealed a rapid increase in the virus structures determined by cryo-EM at near atomic resolution since 2010. In addition, the data highlight the median resolution (～3.0?Å) and size (～310.0?Å in diameter) of the virus particles determined by X-ray crystallography while no such limits exist for cryo-EM structures, which have a median diameter of 508?Å. Notably, cryo-EM virus structures in the last four years have a median resolution of 3.9?Å. Taken together with minimal sample requirements, not needing diffraction quality crystals, and being able to achieve similar resolutions of the crystal structures makes cryo-EM the method of choice for current and future virus capsid structure determinations.     

First-principles calculations of protein circular dichroism in the far-ultraviolet and beyond

Understanding the relationship between the amino acid sequence of a protein and its unique, compact three-dimensional structure is one of the grand challenges in molecular biophysics. One exciting approach to the protein-folding problem is fast time-resolved spectroscopy in the ultra-violet (UV). Time-resolved electronic circular dichroism (CD) spectroscopy offers resolution on a nanosecond (or faster) timescale, but does not provide the spatial resolution of techniques like X-ray crystallography or NMR. There is a need to underpin fast timescale spectroscopic studies of protein folding with a stronger theoretical foundation. We review some recent studies in this regard and briefly highlight how modern quantum chemical models of aromatic groups have improved the accuracy of calculations of protein CD spectra near-UV. On the other side of the far-UV, we describe calculations indicating that charge-transfer transitions are likely to be responsible for bands observed in the vacuum UV in protein CD.     

Fragment-based phase extension for three-dimensional structure determination of membrane proteins by electron crystallography

In electron crystallography, membrane protein structure is determined from two-dimensional crystals where the protein is embedded in a membrane. Once large and well-ordered 2D crystals are grown, one of the bottlenecks in electron crystallography is the collection of image data to directly provide experimental phases to high resolution. Here, we describe an approach to bypass this bottleneck, eliminating the need for high-resolution imaging. We use the strengths of electron crystallography in rapidly obtaining accurate experimental phase information from low-resolution images and accurate high-resolution amplitude information from electron diffraction. The low-resolution experimental phases were used for the placement of α helix fragments and extended to high resolution using phases from the fragments. Phases were further improved by density modifications followed by fragment expansion and structure refinement against the high-resolution diffraction data. Using this approach, structures of three membrane proteins were determined rapidly and accurately to atomic resolution without high-resolution image data.     

Analytical ultracentrifugation in structural biology

Researchers in the field of structural biology, especially X-ray crystallography and protein nuclear magnetic resonance, are interested in knowing as much as possible about the state of their target protein in solution. Not only is this knowledge relevant to studies of biological function, it also facilitates determination of a protein structure using homogeneous monodisperse protein samples. A researcher faced with a new protein to study will have many questions even after that protein has been purified. Analytical ultracentrifugation (AUC) can provide all of this information readily from a small sample in a non-destructive way, without the need for labeling, enabling structure determination experiments without any wasting time and material on uncharacterized samples. In this article, I use examples to illustrate how AUC can contribute to protein structural analysis. Integrating information from a variety of biophysical experimental methods, such as X-ray crystallography, small angle X-ray scattering, electrospray ionization-mass spectrometry, AUC allows a more complete understanding of the structure and function of biomacromolecules.     

蛋白质结构与相互作用研究新方法——交联质谱技术

蛋白质的空间结构信息以及蛋白质间的相互作用信息对于研究蛋白质的功能有重要意义.研究蛋白质结构与相互作用的传统技术,如核磁共振技术、X射线晶体衍射技术等,对于蛋白质的纯度、结晶性和绝对量均有比较高的要求,限制了其广泛应用.交联质谱技术是近十多年来发展起来的新技术,它将质谱技术与交联技术相结合,在研究蛋白质结构与相互作用方面具有速度快、成本小、蛋白质各方面性状要求低等优势.本文就交联质谱技术各个环节的技术方法加以综述,包括交联质谱实验分离富集技术、常见交联剂特性、交联质谱数据库搜索算法、结果验证研究和交联质谱技术的应用等方面,并展望了该研究方向未来的发展.     

Crystallographic phases through genetic engineering: experiences with colicin A

Colicins are antibiotic proteins that kill sensitive Escherichia coli cells. The structure of the pore-forming fragment of colicin A has been solved to 2.5 A resolution using the techniques of X-ray crystallography and genetic engineering. Site-directed mutagenesis was used to construct a number of cysteine-containing mutant proteins, one of which yielded an excellent mercurial derivative. Our experiences suggest strategies for obtaining useful heavy-atom derivatives for protein crystallography using genetic engineering techniques.