自由电子激光在生物学中的应用

同步辐射的发展和应用已经极大的推动了自然科学包括生物学的巨大发展,其中结构生物学更是离不开X射线衍射分析,小角散射等。X射线自由电子激光(XFEL)相比同步辐射具有更高强度,完全相干等特点,被称为第四代光源。科学家已经利用XFEL实现了尺度约为1微米的蛋白质晶体的高分辨率结构解析,并且也实现了单颗粒的病毒的低分辨重构。未来,XFEL将会为生物学的发展打开一扇新的大门。     

Serial femtosecond crystallography at the SACLA: breakthrough to dynamic structural biology

X-ray crystallography visualizes the world at the atomic level. It has been used as the most powerful technique for observing the three-dimensional structures of biological macromolecules and has pioneered structural biology. To determine a crystal structure with high resolution, it was traditionally required to prepare large crystals (> 200 μm). Later, synchrotron radiation facilities, such as SPring-8, that produce powerful X-rays were built. They enabled users to obtain good quality X-ray diffraction images even with smaller crystals (ca. 200–50 μm). In recent years, one of the most important technological innovations in structural biology has been the development of X-ray free electron lasers (XFELs). The SPring-8 Angstrom Compact free electron LAser (SACLA) in Japan generates the XFEL beam by accelerating electrons to relativistic speeds and directing them through in-vacuum, short-period undulators. Since user operation started in 2012, we have been involved in the development of serial femtosecond crystallography (SFX) measurement systems using XFEL at the SACLA. The SACLA generates X-rays a billion times brighter than SPring-8. The extremely bright XFEL pulses enable data collection with microcrystals (ca. 50–1 μm). Although many molecular analysis techniques exist, SFX is the only technique that can visualize radiation-damage-free structures of biological macromolecules at room temperature in atomic resolution and fast time resolution. Here, we review the achievements of the SACLA-SFX Project in the past 5 years. In particular, we focus on: (1) the measurement system for SFX; (2) experimental phasing by SFX; (3) enzyme chemistry based on damage-free room-temperature structures; and (4) molecular movie taken by time-resolved SFX.     

IPET and FETR: experimental approach for studying molecular structure dynamics by cryo-electron tomography of a single-molecule structure

The dynamic personalities and structural heterogeneity of proteins are essential for proper functioning. Structural determination of dynamic/heterogeneous proteins is limited by conventional approaches of X-ray and electron microscopy (EM) of single-particle reconstruction that require an average from thousands to millions different molecules. Cryo-electron tomography (cryoET) is an approach to determine three-dimensional (3D) reconstruction of a single and unique biological object such as bacteria and cells, by imaging the object from a series of tilting angles. However, cconventional reconstruction methods use large-size whole-micrographs that are limited by reconstruction resolution (lower than 20 Å), especially for small and low-symmetric molecule (<400 kDa). In this study, we demonstrated the adverse effects from image distortion and the measuring tilt-errors (including tilt-axis and tilt-angle errors) both play a major role in limiting the reconstruction resolution. Therefore, we developed a “focused electron tomography reconstruction” (FETR) algorithm to improve the resolution by decreasing the reconstructing image size so that it contains only a single-instance protein. FETR can tolerate certain levels of image-distortion and measuring tilt-errors, and can also precisely determine the translational parameters via an iterative refinement process that contains a series of automatically generated dynamic filters and masks. To describe this method, a set of simulated cryoET images was employed; to validate this approach, the real experimental images from negative-staining and cryoET were used. Since this approach can obtain the structure of a single-instance molecule/particle, we named it individual-particle electron tomography (IPET) as a new robust strategy/approach that does not require a pre-given initial model, class averaging of multiple molecules or an extended ordered lattice, but can tolerate small tilt-errors for high-resolution single “snapshot” molecule structure determination. Thus, FETR/IPET provides a completely new opportunity for a single-molecule structure determination, and could be used to study the dynamic character and equilibrium fluctuation of macromolecules.     

A 11.5 A single particle reconstruction of GroEL using EMAN.

Single-particle analysis has become an increasingly important method for structural determination of large macromolecular assemblies. GroEL is an 800 kDa molecular chaperone, which, along with its co-chaperonin GroES, promotes protein folding both in vitro and in the bacterial cell. EMAN is a single-particle analysis software package, which was first publicly distributed in 2000. We present a three-dimensional reconstruction of native naked GroEL to approximately 11.5 A performed entirely with EMAN. We demonstrate that the single-particle reconstruction, X-ray scattering data and X-ray crystal structure all agree well at this resolution. These results validate the specific methods of image restoration, reconstruction and evaluation techniques implemented in EMAN. It also demonstrates that the single-particle reconstruction technique and X-ray crystallography will yield consistent structure factors, even at low resolution, when image restoration is performed correctly. A detailed comparison of the single-particle and X-ray structures exhibits some small variations in the equatorial domain of the molecule, likely due to the absence of crystal packing forces in the single-particle reconstruction.     

Microfocal X-ray CT imaging and pulmonary arterial distensibility in excised rat lungs

The objective of this study was to develop an X-ray computed tomographic method for measuring pulmonary arterial dimensions and locations within the intact rat lung. Lungs were removed from rats and their pulmonary arterial trees were filled with perfluorooctyl bromide to enhance X-ray absorbance. The lungs were rotated within the cone of the X-ray beam projected from a microfocal X-ray source onto an image intensifier, and 360 images were obtained at 1 degrees increments. The three-dimensional image volumes were reconstructed with isotropic resolution using a cone beam reconstruction algorithm. The vessel diameters were obtained by fitting a functional form to the image of the vessel circular cross section. The functional form was chosen to take into account the point spread function of the image acquisition and reconstruction system. The diameter measurements obtained over a range of vascular pressures were used to characterize the distensibility of the rat pulmonary arteries. The distensibility coefficient alpha [defined by D(P) = D(0)(1 + alphaP), where D(P) is the diameter at intravascular pressure (P)] was approximately 2.8% mmHg and independent of vessel diameter in the diameter range (about 100 to 2,000 mm) studied.     

Time-resolved studies of metalloproteins using X-ray free electron laser radiation at SACLA

BackgroundThe invention of the X-ray free-electron laser (XFEL) has provided unprecedented new opportunities for structural biology. The advantage of XFEL is an intense pulse of X-rays and a very short pulse duration (<10 fs) promising a damage-free and time-resolved crystallography approach.Scope of reviewRecent time-resolved crystallographic analyses in XFEL facility SACLA are reviewed. Specifically, metalloproteins involved in the essential reactions of bioenergy conversion including photosystem II, cytochrome c oxidase and nitric oxide reductase are described.Major conclusionsXFEL with pump-probe techniques successfully visualized the process of the reaction and the dynamics of a protein. Since the active center of metalloproteins is very sensitive to the X-ray radiation, damage-free structures obtained by XFEL are essential to draw mechanistic conclusions. Methods and tools for sample delivery and reaction initiation are key for successful measurement of the time-resolved data.General significanceXFEL is at the center of approaches to gain insight into complex mechanism of structural dynamics and the reactions catalyzed by biological macromolecules. Further development has been carried out to expand the application of time-resolved X-ray crystallography. This article is part of a Special Issue entitled Novel measurement techniques for visualizing ‘live’ protein molecules.     

7 ? resolution in protein two-dimensional-crystal X-ray diffraction at Linac Coherent Light Source

Membrane proteins arranged as two-dimensional crystals in the lipid environment provide close-to-physiological structural information, which is essential for understanding the molecular mechanisms of protein function. Previously, X-ray diffraction from individual two-dimensional crystals did not represent a suitable investigational tool because of radiation damage. The recent availability of ultrashort pulses from X-ray free-electron lasers (XFELs) has now provided a means to outrun the damage. Here, we report on measurements performed at the Linac Coherent Light Source XFEL on bacteriorhodopsin two-dimensional crystals mounted on a solid support and kept at room temperature. By merging data from about a dozen single crystal diffraction images, we unambiguously identified the diffraction peaks to a resolution of 7 Å, thus improving the observable resolution with respect to that achievable from a single pattern alone. This indicates that a larger dataset will allow for reliable quantification of peak intensities, and in turn a corresponding increase in the resolution. The presented results pave the way for further XFEL studies on two-dimensional crystals, which may include pump–probe experiments at subpicosecond time resolution.     

Structural Comparison of Cephalopod Hemocyanins: Phylogenetic Significance

Hemocyanins, the respiratory molecules of cephalopod mollusks, are hollow cylinders with five internal arches. Three hemocyanins representative of three orders of cephalopods (Benthoctopus species, Octopoda; Vampyroteuthis infernalis, Vampyromorpha; Sepia officinalis, Sepioidea) were subjected to cryoelectron microscopy and three-dimensional (3D) reconstruction. The structure of Benthoctopus hemocyanin, solved at 26.4-A resolution, possesses arches comprising two identical functional units. The similarity between these functional units and the structure recently observed in X-ray crystallography for Octopus by Cuff et al. (J. Mol. Biol., 1998, 232, 522-529) allows the identification of their N- and C-terminal domains in the 3D reconstruction volume. Conversely, arches present in the 3D reconstruction volume of Sepia hemocyanin (21.8 A resolution) contain four functional units that are disposed differently. The strong resemblance between the reconstruction volumes of Vampyroteuthis (21.4-A resolution) and Benthoctopus hemocyanins suggests that Sepioidea diverged from a group containing Octopoda and Vampyromorpha.     

Opportunities and challenges for time-resolved studies of protein structural dynamics at X-ray free-electron lasers

X-ray free-electron lasers (XFELs) are revolutionary X-ray sources. Their time structure, providing X-ray pulses of a few tens of femtoseconds in duration; and their extreme peak brilliance, delivering approximately 10¹² X-ray photons per pulse and facilitating sub-micrometre focusing, distinguish XFEL sources from synchrotron radiation. In this opinion piece, I argue that these properties of XFEL radiation will facilitate new discoveries in life science. I reason that time-resolved serial femtosecond crystallography and time-resolved wide angle X-ray scattering are promising areas of scientific investigation that will be advanced by XFEL capabilities, allowing new scientific questions to be addressed that are not accessible using established methods at storage ring facilities. These questions include visualizing ultrafast protein structural dynamics on the femtosecond to picosecond time-scale, as well as time-resolved diffraction studies of non-cyclic reactions. I argue that these emerging opportunities will stimulate a renaissance of interest in time-resolved structural biochemistry.     

Image reconstruction reveals the complex molecular organization of adenovirus

The three-dimensional structure of adenovirus has been determined by image reconstruction from cryo-electron micrographs. Comparison with the high resolution X-ray crystal structure of hexon, the major capsid protein, enabled an unusually detailed interpretation of the density map and confirmed the validity of the reconstruction. The hexon packing in the capsid shows more extensive intermolecular interfaces between facets than previously proposed. The reconstruction provides the first three-dimensional visualization of the vertex proteins, including the penton base and its associated protruding fiber. Three minor capsid proteins that stabilize and modulate capsomer interactions are revealed. One of these components stabilizes the group-of-nine hexons in the center of each facet and the other two bridge hexons in adjacent facets. The strategic positions of these proteins highlight the importance of cementing proteins in stabilizing a complex assembly.     

染色体三维结构重建方法研究

染色体三维结构重构问题是近年生物领域中基因组学的热点研究问题,是以二维交互频率数据为基础来预测其三维空间结构。最新相关实验表明染色质的三维空间结构对于基因表达、调控等方面都具有重要意义。而Hi-c数据能利用染色质交互信息形成二维接触矩阵重构出染色体三维结构。本综述以染色体三维结构重建方法为研究对象,通过对染色体三维结构重建方法进行比较分析,综述了目前基于Hi-c数据在染色体三维结构重建中的经典方法,系统介绍了染色体三维结构重建技术的发展脉络,以促进染色体三维结构重建的进一步研究。     

Single-particle image reconstruction of a tetramer of HIV integrase bound to DNA

The HIV integrase enzyme (IN) catalyzes the initial DNA breaking and joining reactions that integrate viral DNA in the host chromosome. Structures for individual IN domains have been determined by X-ray crystallography and NMR spectroscopy, but the structure of the complete IN-DNA complex has remained elusive. Homogeneous complexes of IN tetramers were assembled on DNA three-way junction substrates designed to resemble integration intermediates. Electron microscopy and single-particle image analysis of these complexes yielded a three-dimensional reconstruction at approximately 27 A resolution. The map of the IN-DNA complex displays four lobes of density approximately 50 A in diameter. Three of the lobes form a roughly triangular base with a central channel approximately 20 A in diameter. The fourth lobe is centered between two lobes and extends approximately 40 A above the base. We propose that the central channel tethers the target DNA, and two of the lobes may bind the ends of the viral DNA. The asymmetry of the complex is a feature not incorporated in previous structural models and potentially provides the first view of an asymmetric reaction intermediate.     

Automated three-dimensional reconstruction of keyhole limpet hemocyanin type 1

We have reconstructed a three-dimensional map of keyhole limpet hemocyanin isoform 1 (KLH1), using our automated data collection software, Leginon, integrated with particle selection algorithms, and the SPIDER reconstruction package. KLH1, a 7.9 MDa macromolecule, is an extracellular respiratory pigment composed of two asymmetric decamers, and presents an overall D(5) point-group symmetry. The reconstruction is in agreement with previous data published on molluscan hemocyanins. The reconstructed map (11.3A resolution, 3sigma criterion) was used to fit an available X-ray crystallography structure of Octopus dofleini Odg, solved at 2.3A [J. Mol. Biol. 278 (4) (1998) 855], with satisfactory results. The results validate the approach of automating the cryoEM process and demonstrate that the quality of the images acquired and the particles selected is comparable to those obtained using manual methods. Several problems remain to be solved however before these results can be generalized.     

Scaling structure factor amplitudes in electron cryomicroscopy using X-Ray solution scattering

The structure factors derived from electron cryomicroscopic images are modified by the contrast transfer function of the microscope's objective lens and other influences. The phases of the structure factors can be corrected in a straightforward way when the positions of the contrast transfer function rings are determined. However, corrected amplitudes are also essential to yield an accurate distribution of mass in the reconstruction. The correct scale factors for the amplitudes are difficult to evaluate for data that are merged from many different micrographs. We opt to use X-ray solution scattering intensity from a concentrated suspension of the specimen to correct the amplitudes of the spherically averaged structure factors. When this approach is applied to the three-dimensional image data of ice-embedded acrosomal bundles, the core of a filament in a three-dimensional reconstruction of the acrosomal bundle becomes denser and matches more closely the outer density ascribed to scruin.     

Partitivirus structure reveals a 120-subunit, helix-rich capsid with distinctive surface arches formed by quasisymmetric coat-protein dimers

Two distinct partitiviruses, Penicillium stoloniferum viruses S and F, can be isolated from the fungus Penicillium stoloniferum. The bisegmented dsRNA genomes of these viruses are separately packaged in icosahedral capsids containing 120 coat-protein subunits. We used transmission electron cryomicroscopy and three-dimensional image reconstruction to determine the structure of Penicillium stoloniferum virus S at 7.3 A resolution. The capsid, approximately 350 A in outer diameter, contains 12 pentons, each of which is topped by five arched protrusions. Each of these protrusions is, in turn, formed by a quasisymmetric dimer of coat protein, for a total of 60 such dimers per particle. The density map shows numerous tubular features, characteristic of alpha helices and consistent with secondary structure predictions for the coat protein. This three-dimensional structure of a virus from the family Partitiviridae exhibits both similarities to and differences from the so-called "T = 2" capsids of other dsRNA viruses.     

Features of reovirus outer capsid protein mu1 revealed by electron cryomicroscopy and image reconstruction of the virion at 7.0 Angstrom resolution

Reovirus is a useful model for addressing the molecular basis of membrane penetration by one of the larger nonenveloped animal viruses. We now report the structure of the reovirus virion at approximately 7.0 A resolution as obtained by electron cryomicroscopy and three-dimensional image reconstruction. Several features of the myristoylated outer capsid protein mu1, not seen in a previous X-ray crystal structure of the mu1-sigma3 heterohexamer, are evident in the virion. These features appear to be important for stabilizing the outer capsid, regulating the conformational changes in mu1 that accompany perforation of target membranes, and contributing directly to membrane penetration during cell entry.     

Three-dimensional reconstruction of the connector of bacteriophage phi 29 at 1.8 nm resolution

The three-dimensional reconstruction of the connector of bacteriophage phi 29 has been obtained from tilt series of negatively stained tetragonal ordered aggregates under low-dose conditions and up to a resolution of (1/1.8) nm-1. These connectors are built up as dodecamers of only one structural polypeptide (p10). Two connectors form the crystal unit cell, each one facing in the opposite direction with respect to the plane of the crystal and partially overlapping. The main features of the two connectors that build the unit cell were essentially the same, although they were negatively stained in slightly different ways, probably due to their situations with respect to the carbon-coated support grid. The main features of the phi 29 connector structure revealed by this three-dimensional reconstruction are: the existence of two clearly defined domains, one with a diameter of around 14 nm and the other narrower (diameter approximately equal to 7.5 nm); an inner hole running all along the structure (around 7 to 8 nm in height) with a cylindrical profile and an average diameter of 4 nm; a general 6-fold symmetry along the whole structure and a 12-fold one in the wider domain; a clockwise twist of the more contrasted regions of both domains from the narrower towards the wider domain (the direction of DNA encapsidation). These features are compatible with an active role for the connector in the process of DNA packaging.     

Three-dimensional structure by cryo-electron microscopy of YvcC, an homodimeric ATP-binding cassette transporter from Bacillus subtilis

YvcC, a multidrug transporter from Bacillus subtilis, is a member of the ATP-binding cassette superfamily, highly homologous to each half of human multidrug-resistance P-glycoprotein and to several other bacterial half-ABC transporters. Here, the purified recombinant histidine-tagged YvcC has been reconstituted into a lipid bilayer. Controlled and partial detergent removal from YvcC-lipid micelles allowed the production of particularly interesting lipid-detergent-YvcC ring-shaped particles, about 40 nm in diameter, well suited for single particle analysis by cryo-electron microscopy. Furthermore, binding of these histidine-tagged ring-shaped particles to lipid layers functionalized with a Ni(2+)-chelating head group generated a preferential perpendicular orientation, eliminating the missing cone in the final three-dimensional reconstruction. From such analysis, a computed volume has been determined to 2.5 nm resolution giving a detailed insight into the structural organization of this half-ABC transporter within a membrane. The repetitive unit in the ring-shaped particles is consistent with a homodimeric organization of YvcC. Each subunit was composed of three domains: a 5 nm height transmembrane region, a stalk of about 4 nm in height and 2 nm in diameter, and a cytoplasmic lobe of about 5-6 nm in diameter. The latest domain, which fitted with the reported X-ray structure of HisP, was identified as the nucleotide-binding domain (NBD). The 3D reconstruction of the YvcC homodimer well compared with the very recent X-ray crystallographic data on the MsbA homodimer from Escherichia coli, supporting the existence of a central open chamber between the two subunits constituting the homodimer. In addition, the 3D reconstruction of YvcC embedded in a membrane revealed an asymmetric organization of the two NBDs sites within the homodimer, as well as a dimeric interaction between two homodimers.     

The three-dimensional structure of Infectious flacherie virus capsid determined by cryo-electron microscopy

Cryo-electron microscopy and image reconstruction were used to determine the three-dimensional structure of Infectious flacherie virus (IFV). 5047 particles were selected for the final reconstruction. The FSC curve showed that the resolution of this capsid structure was 18 Å. The structure is a psuedo T=3 (P=3) icosahedral capsid with a diameter of 302.4 Å and a single shell thickness of 15 Å. The density map showed that IFV has a smooth surface without any prominent protrude or depression. Comparison of the IFV structure with those of the insect picorna-like virus-Cricket paralysis virus (CrPV)and human picornavirus-Human rhinovirus 14 (HRV 14) revealed that the IFV structure resembles the CrPV structure. The “Rossmann canyon” is absent in both IFV and CrPV particles. The polypeptide topology of IFV VP2, IFV VP3 was predicted and the subunit location at the capsid surface was further analyzed.