Phasing coherently illuminated nanocrystals bounded by partial unit cells

With the use of highly coherent femtosecond X-ray pulses from a free-electron laser, it is possible to record protein nanocrystal diffraction patterns with far more information than is present in conventional crystallographic diffraction data. It has been suggested that diffraction phases may be retrieved from such data via iterative algorithms, without the use of a priori information and without restrictions on resolution. Here, we investigate the extension of this approach to nanocrystals with edge terminations that produce partial unit cells, and hence cannot be described by a common repeating unit cell. In this situation, the phase problem described in previous work must be reformulated. We demonstrate an approximate solution to this phase problem for crystals with random edge terminations.     

Femtosecond nanocrystallography using X-ray lasers for membrane protein structure determination

The invention of free electron X-ray lasers has opened a new era for membrane protein structure determination with the recent first proof-of-principle of the new concept of femtosecond nanocrystallography. Structure determination is based on thousands of diffraction snapshots that are collected on a fully hydrated stream of nanocrystals. This review provides a summary of the method and describes how femtosecond X-ray crystallography overcomes the radiation-damage problem in X-ray crystallography, avoids the need for growth and freezing of large single crystals while offering a new method for direct digital phase determination by making use of the fully coherent nature of the X-ray beam. We briefly review the possibilities for time-resolved crystallography, and the potential for making 'molecular movies' of membrane proteins at work.     

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。     

Diffraction before destruction

X-ray free-electron lasers have opened up the possibility of structure determination of protein crystals at room temperature, free of radiation damage. The femtosecond-duration pulses of these sources enable diffraction signals to be collected from samples at doses of 1000 MGy or higher. The sample is vaporized by the intense pulse, but not before the scattering that gives rise to the diffraction pattern takes place. Consequently, only a single flash diffraction pattern can be recorded from a crystal, giving rise to the method of serial crystallography where tens of thousands of patterns are collected from individual crystals that flow across the beam and the patterns are indexed and aggregated into a set of structure factors. The high-dose tolerance and the many-crystal averaging approach allow data to be collected from much smaller crystals than have been examined at synchrotron radiation facilities, even from radiation-sensitive samples. Here, we review the interaction of intense femtosecond X-ray pulses with materials and discuss the implications for structure determination. We identify various dose regimes and conclude that the strongest achievable signals for a given sample are attained at the highest possible dose rates, from highest possible pulse intensities.     

Fine structure in near-field and far-field laser diffraction patterns from skeletal muscle fibers.

Regions of muscle fibers that are many sarcomeres in length and uniform with regard to striation spacing, curvature, and tilt have been observed by light microscopy. We have investigated the possibility that these sarcomere domains can explain the fine structure in optical diffraction patterns of skeletal muscle fibers. We studied near-field and far-field diffraction patterns with respect to fiber translation and to masking of the laser beam. The position of diffracted light in the near-field pattern depends on sarcomere length and position of the diffracting regions within the laser beam. When a muscle fiber was translated longitudinally through a fixed laser beam, the fine structural lines in the near-field diffraction pattern moved in the same direction and by the same amount as the fiber movement. Translation of the muscle fiber did not result in fine structure movement in the far-field pattern. As the laser beam was incrementally masked from one side, some fine structural lines in both the near-field and far-field diffraction patterns changed in intensity while others remained the same. Eventually, all the fine structural lines broadened and decreased in intensity. Often a fine structural line increased in intensity or a dark area in the diffraction pattern became brighter as the laser beam was restricted. From these results we conclude that the fine structure in the laser diffraction pattern is due to localized and relatively uniform regions of sarcomeres (domains) and to cross interference among light rays scattered by different domains.     

High-resolution structure of viruses from random diffraction snapshots

The advent of the X-ray free-electron laser (XFEL) has made it possible to record diffraction snapshots of biological entities injected into the X-ray beam before the onset of radiation damage. Algorithmic means must then be used to determine the snapshot orientations and thence the three-dimensional structure of the object. Existing Bayesian approaches are limited in reconstruction resolution typically to 1/10 of the object diameter, with the computational expense increasing as the eighth power of the ratio of diameter to resolution. We present an approach capable of exploiting object symmetries to recover three-dimensional structure to high resolution, and thus reconstruct the structure of the satellite tobacco necrosis virus to atomic level. Our approach offers the highest reconstruction resolution for XFEL snapshots to date and provides a potentially powerful alternative route for analysis of data from crystalline and nano-crystalline objects.     

Crystalline fibril structure of type II collagen in lamprey notochord sheath

We report here the existence of a crystalline molecular packing of type II collagen in the fibrils of the lamprey notochord sheath. This is the first finding of a crystalline structure in any collagen other than type I.The lamprey notochord sheath has a composition similar to that of cartilage, with type II collagen, a minor collagen component with 1α, 2α and 3α chains, and cartilage-like proteoglycan. The high degree of orientation of fibrils in the notochord makes it possible to use X-ray diffraction to determine collagen fibril organization in this type II-containing tissue. The low angle equatorial scattering shows the fibrils are all about 17 nm in diameter and have an average center-to-center separation of 31 nm. These results are supported by electron microscope observations. A set of broad equatorial diffraction maxima at higher angles represents the sampling of the collagen molecular transform by a limited crystalline lattice, extending over a lateral dimension close to the diameter of one fibril. This indicates that each 17 nm fibril contains a crystalline array of molecules and, although a unit cell is difficult to determine because of the broad overlapping reflections, it is clear that the quasi-hexagonal triclinic unit cell of type I collagen in rat tail tendon is not consistent with the data. The meridional diffraction pattern showed 26 orders with the characteristic 67 nm periodicity found for tendon. However, the intensities of these reflections differ markedly from those found for tendon and cannot be explained by an unmodified gap/ overlap model within each 67 nm period. Both X-ray diffraction and electron microscope data indicate a low degree of contrast along the fibril axis and are consistent with a periodic binding of a non-collagenous component in such a way as to obscure the gap region.     

Changes in the molecular packing of fibrillin microfibrils during extension indicate intrafibrillar and interfibrillar reorganization in elastic response

Fibrillin-rich microfibrils are the major structural components of the extracellular matrix that provide elasticity in a majority of connective tissues. The basis of elastic properties lies in the organization of fibrillin molecules, which, unfortunately, is still poorly understood. An X-ray diffraction study of hydrated fibrillin-rich microfibrils from zonular filaments has been conducted to give an insight into the molecular structure of microfibrils in intact tissue. A series of measurements was taken during controlled tissue extension to observe alterations in the lateral packing of microfibrils. Computer-generated simulated patterns were used to fit the experimental X-ray scattering data and to obtain the fibril diameter and lateral distance between the fibrils. The results suggest a nonlinear correlation between external strain and decrease in fibril diameter and lateral spacing. This was accompanied by a nonlinear increase in axial periodicity and a structure with a 160-nm periodicity, which is reported here for the first time using X-ray diffraction. These changes may reflect the unraveling of fibrillin from the complex folded arrangement into a linear structure. This finding supports a pleating model where fibrillin molecules are highly folded within the microfibrils; more importantly, the connection is made between the interaction of individual microfibrils and the change in their suprafibrillar coherent organization during extension. We suggest that the intermediate states observed in our study reflect sequential unfolding of fibrillin and can explain the process of its reversible unraveling.     

The structure of ferritin cores determined by electron nanodiffraction

Electron nanodiffraction, with a 100-keV electron beam less than 1 nm in diameter, has been used to obtain single-crystal diffraction patterns from individual iron-containing cores of ferritin molecules. We show that, while a majority of the cores have a hexagonal structure somewhat similar to the major phase in the mineral ferrihydrite, as previously assumed, several minor phases are present including some that are similar in structure to the iron oxides magnetite and hematite and also some composed of highly disordered material. In general, each core consists of one single crystal of one phase.     

X-ray structure determination of the glycine cleavage system protein H of Mycobacterium tuberculosis using an inverse Compton synchrotron X-ray source

Structural genomics discovery projects require ready access to both X-ray diffraction and NMR spectroscopy which support the collection of experimental data needed to solve large numbers of novel protein structures. The most productive X-ray crystal structure determination laboratories make extensive use of tunable synchrotron X-ray light to solve novel structures by anomalous diffraction methods. This requires that frozen cryo-protected crystals be shipped to large multi acre synchrotron facilities for data collection. In this paper we report on the development and use of the first laboratory-scale synchrotron light source capable of performing many of the state-of-the-art synchrotron applications in X-ray science. This Compact Light Source is a first-in-class device that uses inverse Compton scattering to generate X-rays of sufficient flux, tunable wavelength and beam size to allow high-resolution X-ray diffraction data collection from protein crystals. We report on benchmarking tests of X-ray diffraction data collection with hen egg white lysozyme, and the successful high-resolution X-ray structure determination of the Glycine cleavage system protein H from Mycobacterium tuberculosis using diffraction data collected with the Compact Light Source X-ray beam.     

Coherent scattering in multi-harmonic light microscopy

By focusing a pulsed laser beam into a sample, harmonic up-conversion can be generated as well as multi-photon excited fluorescence. Whereas multi-photon excited fluorescence microscopy is well established, the use of multi-harmonic generation for three-dimensional image contrast is very recent. Both techniques can provide similar resolution and, for adequate radiating source density, comparable signal levels, allowing them to be combined in a single versatile instrument. However, harmonic generation differs fundamentally from fluorescence generation in that it is coherent and produces radiation patterns that are highly sensitive to phase. As such, multi-harmonic generation microscopy provides a unique window into molecular spatial organization that is inaccessible to fluorescence.     

X-ray diffraction and electron microscope studies on the structure of bacterial F pili.

F pili are hollow cylinders with 80 Å outer diameter and 20 Å inner diameter. Both X-ray fibre diffraction and optical diffraction of electron micrographs show a strong layer-line corresponding to a spacing of 32 Å, to which a J₄ Bessel function is assigned on the basis of the optical diffraction. X-ray diffraction patterns show near-meridional intensity on a layer-line corresponding to a spacing of 12.8 Å, to which a J₁ Bessel function is assigned. Mass per length measurements on unstained specimens in the scanning transmission electron microscope give 3000 daltons/Å, indicating that the 11,200 dalton pilin subunits are 3.7 Å apart along the axial direction of the pili. These observations show that the pilus structure can be represented as four coaxial helices of pitch 128 Å with the pilin subunits elongated and overlapping along the line of these helices. Each of these helices of subunits is translated axially with respect to its neighbour, to give a basic helix of 3.6 units per turn of 12.8 Å pitch. Radial electron density calculations indicate a 50 Å diameter girdle of hydrophobic amino acids between the inner and outer diameters of the protein shell. A molecular model of the structure at low resolution is presented.     

Lipidic phase membrane protein serial femtosecond crystallography

X-ray free electron laser (X-FEL)-based serial femtosecond crystallography is an emerging method with potential to rapidly advance the challenging field of membrane protein structural biology. Here we recorded interpretable diffraction data from micrometer-sized lipidic sponge phase crystals of the Blastochloris viridis photosynthetic reaction center delivered into an X-FEL beam using a sponge phase micro-jet.     

Ferrochelatase activity in wild-type and mutant strains of Spirillum itersonii. Solubilization with chaotropic reagents

New low-angle X-ray diffraction data have been obtained from nerve myelin after rehydration. The X-ray patterns show the first six orders of diffraction of a lamellar repeat unit of about 100 Å. Direct methods of structure analysis have been used to determine uniquely the phases of the first three orders of diffraction. The electron density profile of rehydrated nerve myelin has been obtained on an absolute electron density scale and is compared with the electron density profile of normal nerve myelin at the same resolution of 16–17 Å. Possible electron-density profiles of rehydrated nerve myelin at a resolution of 8 Å are shown.     

Physical properties of glycosyl diacylglycerols. 2. X-ray diffraction studies of a homologous series of 1,2-Di-O-acyl-3-O-(alpha-D-glucopyranosyl)-sn-glycerols

X-ray diffraction methods were used to characterize the thermotropic polymorphism exhibited by aqueous dispersions of a homologous series of 1,2-O-acyl-3-O-(alpha-D-glucopyranosyl)-sn-glycerols. Upon cooling from temperatures at which the acyl chains of these lipids are melted, all of these compounds form structures that exhibit both low-angle and wide-angle diffraction patterns consistent with the formation of lamellar L beta gel phases. After a suitable protocol of low-temperature annealing, complex diffraction patterns consistent with the formation of highly ordered, lamellar, crystal-like phases are obtained. These patterns are similar for all of the compounds studied, suggesting that the unit cell structure is invariant. The assumption that the unit cell structure is invariant permits the assignment of phases to the diffraction orders, thereby making possible the construction of electron density profiles. These electron density profiles indicate that the crystal-like phases of these lipids are poorly hydrated structures with the hydrocarbon chains inclined at 35 degrees to the bilayer normal. The diffraction patterns of the crystal-like phases of these lipids changed abruptly at the calorimetrically determined phase transition temperatures to those characteristic of either lamellar liquid crystalline phases (N less than or equal to 17) or inverted nonbilayer phases. With these X-ray diffraction data we demonstrate that, at elevated temperatures, the shorter chain homologues (N less than or equal to 16) form cubic phases of the Pn3m space group, whereas the longer chain compounds form inverted hexagonal phases.     

Microscopic phase reconstruction of cervical exfoliated cell under partially coherent illumination

Basic coherent diffraction imaging methods strongly rely on having a highly coherent illumination in order to reconstruct the phase accurately. However, regardless of considering the turbulent transport medium, the instability of the system or the generation mechanism of the light source, partially coherent illumination is more common in real case. In this paper, we proposed an efficient microscopic phase imaging method to study normal and abnormal cervical exfoliated cells. By applying three phase modulations in a single point of the sample's transmitted field, the phase can be retrieved with correspoding three intensities under partially coherent illumination. Compared with intensity map, we can efficiently and clearly judge the proportion of high density shrinking abnormal cells from the phase distributions, which provides a confident analysis and evaluation basis for early medical diagnosis of cervical cancer. This study also has potential applications in noninvasive optical imaging of dynamic biological tissues.     

Temporal coding: the relevance of classical activity, its relation to pattern frequency bands, and a remark on recoding of excitatory drive into phase shifts

We consider an oscillatory network model that is obtained as complex-valued generalization of the classical Cohen-Grossberg-Hopfield (CGH) model. Apart from a synchronizing mechanism, a stronger and/or more coherent input to a unit in the network implies a higher phase velocity of this unit. This constitutes the desynchronizing mechanism, referred to as acceleration. The units' activity of the classical model translates into the amplitudes of the phase model oscillators. This allows to associate classical and temporal coding with amplitude and phase dynamics, respectively. We discuss how the two dynamics act together to achieve the unambiguous pattern recognition that avoids the superposition problem. With respect to coherence, dominating patterns may take coherent states also if only a subset of its units is on-state. The competition for coherence, introduced by acceleration, realizes a kind of feature counting that identifies the dominating pattern as the pattern with the most on-state units. This dominating but possibly only partially active pattern may take a coherent state with a frequency level that is related to the number of on-state units. We also speculate on neurophysiological findings, related to observed phase differences between optimally and suboptimally activated neurons, that may indicate the presence of acceleration.     

Signatures of DNA flexibility, interactions and sequence-related structural variations in classical X-ray diffraction patterns

The theory of X-ray diffraction from ideal, rigid helices allowed Watson and Crick to unravel the DNA structure, thereby elucidating functions encoded in it. Yet, as we know now, the DNA double helix is neither ideal nor rigid. Its structure varies with the base pair sequence. Its flexibility leads to thermal fluctuations and allows molecules to adapt their structure to optimize their intermolecular interactions. In addition to the double helix symmetry revealed by Watson and Crick, classical X-ray diffraction patterns of DNA contain information about the flexibility, interactions and sequence-related variations encoded within the helical structure. To extract this information, we have developed a new diffraction theory that accounts for these effects. We show how double helix non-ideality and fluctuations broaden the diffraction peaks. Meridional intensity profiles of the peaks at the first three helical layer lines reveal information about structural adaptation and intermolecular interactions. The meridional width of the fifth layer line peaks is inversely proportional to the helical coherence length that characterizes sequence-related and thermal variations in the double helix structure. Analysis of measured fiber diffraction patterns based on this theory yields important parameters that control DNA structure, packing and function.     

Direct Determination of the Lamellar Structure of Peripheral Nerve Myelin at Moderate Resolution (7 Å)

Low-angle X-ray diffraction patterns have been recorded from normal nerve and nerve swollen in glycerol solutions. The new X-ray data have a resolution of 7 Å. Direct methods of structure analysis which include deconvolution of the auto-correlation function and sampling theorem reconstructions have been used in the interpretation of the X-ray data. Phases have been assigned to the first 12 orders of diffraction from normal nerve. Fourier syntheses at a resolution of 7 Å are described and an absolute electron density scale is derived. A possible molecular interpretation of the electron density profile is given.     

Atomic structure of a single large biomolecule from diffraction patterns of random orientations

The short and intense pulses of the new X-ray free electron lasers, now operational or under construction, may make possible diffraction experiments on single molecule-sized objects with high resolution, before radiation damage destroys the sample. In a single molecule imaging (SMI) experiment thousands of diffraction patterns of single molecules with random orientations are recorded. One of the most challenging problems of SMI is how to assemble these noisy patterns of unknown orientations into a consistent single set of diffraction data. Here we present a new method which can solve the orientation problem of SMI efficiently even for large biological molecules and in the presence of noise. We show on simulated diffraction patterns of a large protein molecule, how the orientations of the patterns can be found and the structure to atomic resolution can be solved. The concept of our algorithm could be also applied to experiments where images of an object are recorded in unknown orientations and/or positions like in cryoEM or tomography.