A versatile liquid-jet setup for the European XFEL.

The SPB/SFX instrument of the European XFEL provides unique possibilities for high-throughput serial femtosecond crystallography. This publication presents the liquid-jet sample delivery setup of this instrument. The setup is compatible with state-of-the-art gas dynamic virtual nozzle systems as well as high-viscosity extruders and provides space and flexibility for other liquid injection devices and future upgrades. The liquid jets are confined in a differentially pumped catcher assembly and can be replaced within a couple of minutes through a load-lock. A two-microscope imaging system allows visual control of the jets from two perspectives.

Fractal morphology, imaging and mass spectrometry of single aerosol particles in flight.

The morphology of micrometre-size particulate matter is of critical importance in fields ranging from toxicology to climate science, yet these properties are surprisingly difficult to measure in the particles' native environment. Electron microscopy requires collection of particles on a substrate; visible light scattering provides insufficient resolution; and X-ray synchrotron studies have been limited to ensembles of particles. Here we demonstrate an in situ method for imaging individual sub-micrometre particles to nanometre resolution in their native environment, using intense, coherent X-ray pulses from the Linac Coherent Light Source free-electron laser. We introduced individual aerosol particles into the pulsed X-ray beam, which is sufficiently intense that diffraction from individual particles can be measured for morphological analysis. At the same time, ion fragments ejected from the beam were analysed using mass spectrometry, to determine the composition of single aerosol particles. Our results show the extent of internal dilation symmetry of individual soot particles subject to non-equilibrium aggregation, and the surprisingly large variability in their fractal dimensions. More broadly, our methods can be extended to resolve both static and dynamic morphology of general ensembles of disordered particles. Such general morphology has implications in topics such as solvent accessibilities in proteins, vibrational energy transfer by the hydrodynamic interaction of amino acids, and large-scale production of nanoscale structures by flame synthesis.

Demonstration of simultaneous experiments using thin crystal multiplexing at the Linac Coherent Light Source.

Multiplexing of the Linac Coherent Light Source beam was demonstrated for hard X-rays by spectral division using a near-perfect diamond thin-crystal monochromator operating in the Bragg geometry. The wavefront and coherence properties of both the reflected and transmitted beams were well preserved, thus allowing simultaneous measurements at two separate instruments. In this report, the structure determination of a prototypical protein was performed using serial femtosecond crystallography simultaneously with a femtosecond time-resolved XANES studies of photoexcited spin transition dynamics in an iron spin-crossover system. The results of both experiments using the multiplexed beams are similar to those obtained separately, using a dedicated beam, with no significant differences in quality.

Femtosecond dark-field imaging with an X-ray free electron laser.

The emergence of femtosecond diffractive imaging with X-ray lasers has enabled pioneering structural studies of isolated particles, such as viruses, at nanometer length scales. However, the issue of missing low frequency data significantly limits the potential of X-ray lasers to reveal sub-nanometer details of micrometer-sized samples. We have developed a new technique of dark-field coherent diffractive imaging to simultaneously overcome the missing data issue and enable us to harness the unique contrast mechanisms available in dark-field microscopy. Images of airborne particulate matter (soot) up to two microns in length were obtained using single-shot diffraction patterns obtained at the Linac Coherent Light Source, four times the size of objects previously imaged in similar experiments. This technique opens the door to femtosecond diffractive imaging of a wide range of micrometer-sized materials that exhibit irreproducible complexity down to the nanoscale, including airborne particulate matter, small cells, bacteria and gold-labeled biological samples.

Mechanism and dynamics of fatty acid photodecarboxylase.

Fatty acid photodecarboxylase (FAP) is a photoenzyme with potential green chemistry applications. By combining static, time-resolved, and cryotrapping spectroscopy and crystallography as well as computation, we characterized Chlorella variabilis FAP reaction intermediates on time scales from subpicoseconds to milliseconds. High-resolution crystal structures from synchrotron and free electron laser x-ray sources highlighted an unusual bent shape of the oxidized flavin chromophore. We demonstrate that decarboxylation occurs directly upon reduction of the excited flavin by the fatty acid substrate. Along with flavin reoxidation by the alkyl radical intermediate, a major fraction of the cleaved carbon dioxide unexpectedly transformed in 100 nanoseconds, most likely into bicarbonate. This reaction is orders of magnitude faster than in solution. Two strictly conserved residues, R451 and C432, are essential for substrate stabilization and functional charge transfer.

Cryptotomography: reconstructing 3D Fourier intensities from randomly oriented single-shot diffraction patterns.

We reconstructed the 3D Fourier intensity distribution of monodisperse prolate nanoparticles using single-shot 2D coherent diffraction patterns collected at DESY's FLASH facility when a bright, coherent, ultrafast x-ray pulse intercepted individual particles of random, unmeasured orientations. This first experimental demonstration of cryptotomography extended the expansion-maximization-compression framework to accommodate unmeasured fluctuations in photon fluence and loss of data due to saturation or background scatter. This work is an important step towards realizing single-shot diffraction imaging of single biomolecules.

In cellulo crystallization of Trypanosoma brucei IMP dehydrogenase enables the identification of genuine co-factors.

Sleeping sickness is a fatal disease caused by the protozoan parasite Trypanosoma brucei (Tb). Inosine-5'-monophosphate dehydrogenase (IMPDH) has been proposed as a potential drug target, since it maintains the balance between guanylate deoxynucleotide and ribonucleotide levels that is pivotal for the parasite. Here we report the structure of TbIMPDH at room temperature utilizing free-electron laser radiation on crystals grown in living insect cells. The 2.80 Å resolution structure reveals the presence of ATP and GMP at the canonical sites of the Bateman domains, the latter in a so far unknown coordination mode. Consistent with previously reported IMPDH complexes harboring guanosine nucleotides at the second canonical site, TbIMPDH forms a compact oligomer structure, supporting a nucleotide-controlled conformational switch that allosterically modulates the catalytic activity. The oligomeric TbIMPDH structure we present here reveals the potential of in cellulo crystallization to identify genuine allosteric co-factors from a natural reservoir of specific compounds.

Amino-terminal polypeptides of vimentin are responsible for the changes in nuclear architecture associated with human immunodeficiency virus type 1 protease activity in tissue culture cells.

Electron microscopy of human skin fibroblasts syringe-loaded with human immunodeficiency virus type 1 protease (HIV-1 PR) revealed several effects on nuclear architecture. The most dramatic is a change from a spherical nuclear morphology to one with multiple lobes or deep invaginations. The nuclear matrix collapses or remains only as a peripheral rudiment, with individual elements thicker than in control cells. Chromatin organization and distribution is also perturbed. Attempts to identify a major nuclear protein whose cleavage by the protease might be responsible for these alterations were unsuccessful. Similar changes were observed in SW 13 T3 M [vimentin(+)] cells, whereas no changes were observed in SW 13 [vimentin(-)] cells after microinjection of protease. Treatment of SW 13 [vimentin(-)] cells, preinjected with vimentin to establish an intermediate filament network, with HIV-1 PR resulted in alterations in chromatin staining and distribution, but not in nuclear shape. These same changes were produced in SW 13 [vimentin(-)] cells after the injection of a mixture of vimentin peptides, produced by the cleavage of vimentin to completion by HIV-1 PR in vitro. Similar experiments with 16 purified peptides derived from wild-type or mutant vimentin proteins and five synthetic peptides demonstrated that exclusively N-terminal peptides were capable of altering chromatin distribution. Furthermore, two separate regions of the N-terminal head domain are primarily responsible for perturbing nuclear architecture. The ability of HIV-1 to affect nuclear organization via the liberation of vimentin peptides may play an important role in HIV-1-associated cytopathogenesis and carcinogenesis.

Ultrastructural analysis of cytoplasmic intermediate filaments and the nuclear lamina in the mouse plasmacytoma cell line MPC-11 after the induction of vimentin synthesis.

We examined cytoplasmic intermediate filaments (IFs) and the nuclear lamina in cells of the mouse plasmacytoma cell line MPC-11 (lacking both IF proteins and lamins A and C) after induction of vimentin synthesis with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) by means of whole-mount immunogold electron microscopy (IEM). The technique of IEM was modified to allow analysis of the cytoskeleton and nuclear lamina of cells grown in suspension culture employing antibodies against vimentin and lamin B. IEM showed that newly synthesized vimentin assembled into IFs which formed anastomosing networks throughout the cytoplasm, radiating primarily from the nucleus. The filaments decorated by gold-conjugated antibodies appeared to make contact with the lipid-depleted nuclear envelope residue either by directly terminating on it or through an indirect link via short fibers of varying diameter. Some filaments terminated on the subunits of the nuclear pore complexes but they did not pass through the pores. In the absence of lamins A and C, lamin B formed a nuclear lamina consisting of a globular-filamentous network anchoring the nuclear pore complexes.

Non-viral cellular substrates for human immunodeficiency virus type 1 protease.

A computer search revealed 10 proteins with homology to the sequence we originally identified in vimentin as the site of cleavage by human immunodeficiency virus type 1 (HIV-1) protease. Of these 10 proteins (actin, alpha-actinin, spectrin, tropomyosins, vinculin, dystrophin, MAP-2, villin, TRK-1 and Ig mu-chain), we show that 4 of the first 5 were cleaved in vitro by this protease, as are MAP-1 and -2 [(1990) J. Gen. Virol. 71, 1985-1991]. In these proteins, cleavage is not restricted to a single motif, but occurs at many sites. However, cleavage is not random, since 9 other proteins including the cytoskeletal proteins filamin and band 4.1 are not cleaved in the in vitro assay. Thus, the ability of HIV-1 protease to cleave specific components of the cytoskeleton may be an important, although as yet unevaluated aspect of the life cycle of this retrovirus and/or may directly contribute to the pathogenesis observed during infection.

Selective binding of specific mouse genomic DNA fragments by mouse vimentin filaments in vitro.

Mouse vimentin intermediate filaments (IFs) reconstituted in vitro were analyzed for their capacity to select certain DNA sequences from a mixture of about 500-bp-long fragments of total mouse genomic DNA. The fragments preferentially bound by the IFs and enriched by several cycles of affinity binding and polymerase chain reaction (PCR) amplification were cloned and sequenced. In general, they were G-rich and highly repetitive in that they often contained Gn, (GT)n, and (GA)n repeat elements. Other, more complex repeat sequences were identified as well. Apart from the capacity to adopt a Z-DNA and triple helix configuration under superhelical tension, many fragments were potentially able to form cruciform structures and contained consensus binding sites for various transcription factors. All of these sequence elements are known to occur in introns and 5'/3'-flanking regions of genes and to play roles in DNA transcription, recombination and replication. A FASTA search of the EMBL data bank indeed revealed that sequences homologous to the mouse repetitive DNA fragments are commonly associated with gene-regulatory elements. Unexpectedly, vimentin IFs also bound a large number of apparently overlapping, AT-rich DNA fragments that could be aligned into a composite sequence highly homologous to the 234-bp consensus centromere repeat sequence of gamma-satellite DNA. Previous experiments have shown a high affinity of vimentin for G-rich, repetitive telomere DNA sequences, superhelical DNA, and core histones. Taken together, these data support the hypothesis that, after penetration of the double nuclear membrane via an as yet unidentified mechanism, vimentin IFs cooperatively fix repetitive DNA sequence elements in a differentiation-specific manner in the nuclear periphery subjacent to the nuclear lamina and thus participate in the organization of chromatin and in the control of transcription, replication, and recombination processes. This includes aspects of global regulation of gene expression such as the position effects associated with translocation of genes to heterochromatic centromere and telomere regions of the chromosomes.

Role of the intermediate filament protein vimentin in delaying senescence and in the spontaneous immortalization of mouse embryo fibroblasts.

Because knockout of the vimentin gene in mice did not produce an immediately obvious, overt, or lethal specific phenotype, the conjecture was made that the mutation affects some subtle cellular functions whose loss manifests itself only when the mutant animals are exposed to stress. In order to substantiate this idea in a tractable in vitro system, primary embryo fibroblasts from wildtype (V(+/+)) and vimentin-knockout (V(-/-)) mice were compared with regard to their growth behavior under the pseudophysiologic conditions of conventional cell culture. Whereas in the course of serial transfer, the V(+/+) fibroblasts progressively reduced their growth potential, passed through a growth minimum around passage 12 (crisis), and, as immortalized cells, resumed faster growth, the V(-/-) fibroblasts also cut down their growth rate but much earlier, and they either did not immortalize or did so at an almost undetectable rate. Cells withdrawing from the cell cycle showed increased concentrations of reactive oxygen species and signs of oxidative damage: enlarged and flattened morphology, large nuclear volume, reinforced stress fiber system as a result of increased contents of actin and associated proteins, prominent extracellular matrix, and perinuclear masses of pathological forms of mitochondria with low membrane potential. The differences in the cell cycle behavior of the V(+/+) and V(-/-) cells in conjunction with the morphologic changes observed in mitotically arrested cells suggests a protective function of vimentin against oxidative cell damage. Because vimentin exhibits affinity for and forms crosslinkage products with recombinogenic nuclear as well as mitochondrial DNA in intact cells, it is credible to postulate that vimentin plays a role in the recombinogenic repair of oxidative damage inflicted on the nuclear and mitochondrial genome throughout the cells' replicative lifespan. Recombinational events mediated by vimentin also appear to take place when the cells pass through the genetically unstable state of crisis to attain immortality. The residual immortalization potential of V(-/-) fibroblasts might be attributable to their capacity to synthesize, in place of vimentin, the tetrameric form of a lacZ fusion protein carrying, in addition to a nuclear localization signal, the N-terminal 59 amino acids of vimentin and thus its DNA-binding site. On the basis of these results and considerations, a major biologic role of vimentin may be to protect animals during development and postnatal life against genetic damage and, because of its contribution to the plasticity of the genome, to allow them to respond to environmental challenges.

Isolation of SDS-stable complexes of the intermediate filament protein vimentin with repetitive, mobile, nuclear matrix attachment region, and mitochondrial DNA sequence elements from cultured mouse and human fibroblasts.

Crosslinkage of vimentin to DNA in mouse L929 cells by formaldehyde and isolation of SDS-stable DNA-vimentin complexes from normal L929 cells and mouse and human embryo fibroblasts indicated close spatial relations between these components in the intact cell. The adducts, obtained by immunoprecipitation with anti-vimentin antibody, contained substantial quantities, not only of repetitive and mobile sequence elements such as centromeric satellite DNA, telomere DNA, microsatellites and minisatellites, long and short interspersed nucleotide elements, and retroposons, but also of mitochondrial (mt) DNA. Because the SDS-stable complexes could be isolated with distinctly higher yields from oxidatively stressed, senescent fibroblasts and were dissociated by boiling, they possibly arose from accidental condensation reactions mediated by unsaturated and dialdehydes, products of free radical-induced lipid peroxidation. They can therefore be considered vestiges of a general interaction of vimentin with cellular DNA. The sequence patterns of their DNA fragments were similar to those of extrachromosomal circular and linear DNA, including retroviral elements, markers and enhancers of genomic instability that also occur in the cytoplasm and are able to transport vimentin into the nucleus. Many of the fragments were also remarkably similar to AT-rich nuclear matrix attachment regions (MARs) in that they contained, in addition to various mobile elements, a palette of typical MAR motifs. With its tendency to multimerize and to interact with single-stranded and supercoiled DNA, vimentin thus behaves like a nuclear matrix protein and may as such participate in a variety of nuclear matrix-associated processes such as replication, recombination, repair, and transcription of DNA. These activities seem to be extendible to the mitochondrial compartment, as vimentin was also crosslinked to mtDNA, preferentially to its D-loop and hypervariable main control region. These sites are prone to point and deletion mutations and, like nuclear MARs, are associated with the cyto-karyomatrix. Moreover, as a developmentally regulated and tissue-specific cyto-karyomatrix protein, vimentin may contribute to the organization of chromatin, including centromeric and telomeric heterochromatin at the nuclear periphery, with all its consequences for genomic activities during embryogenesis and in adulthood of vertebrates. However, because of its high affinity for hypervariable, recombinogenic DNA sequences, vimentin is proposed to play a major role in both the preservation and the evolution of the nuclear and mitochondrial genome.

Binding of nucleic acids to intermediate filaments of the vimentin type and their effects on filament formation and stability.

Guanine-rich polynucleotides such as poly(dG), oligo(dG)12-18 or poly(rG) were shown to exert a strong inhibitory effect on vimentin filament assembly and also to cause disintegration of preformed filaments in vitro. Gold-labeled oligo(dG)25 was preferentially localized at the physical ends of the aggregation and disaggregation products and at sites along filaments with a basic periodicity of 22.7 nm. Similar effects were observed with heat-denatured eukaryotic nuclear DNA or total rRNA, although these nucleic acids could affect filament formation and structure only at ionic strengths lower than physiological. However, whenever filaments were formed or stayed intact, they appeared associated with the nucleic acids. These electron microscopic observations were corroborated by sucrose gradient analysis of complexes obtained from preformed vimentin filaments and radioactively labeled heteroduplexes. Among the duplexes of the DNA type, particularly poly(dG).poly(dC), and, of those of the RNA type, preferentially poly(rA).poly(rU), were carried by the filaments with high efficiency into the pellet fraction. Single-stranded 18S and 28S rRNA interacted only weakly with vimentin filaments. Nevertheless, in a mechanically undisturbed environment, vimentin filaments could be densely decorated with intact 40S and 60S ribosomal subunits as revealed by electron microscopy. These results indicate that, in contrast to single-stranded nucleic acids with their compact random coil configuration, double-stranded nucleic acids with their elongated and flexible shape have the capability to stably interact with the helically arranged, surface-exposed amino-terminal polypeptide chains of vimentin filaments. Such interactions might be of physiological relevance in regard to the transport and positioning of nucleic acids and nucleoprotein particles in the various compartments of eukaryotic cells. Conversely, nucleic acids might be capable of affecting the cytoplasmic organization of vimentin filament networks through their filament-destabilizing potentials.

Pepstatin A: polymerization of an oligopeptide.

Pepstatin A, a pentapeptide with the molecular weight of 686, is a naturally occurring inhibitor of aspartyl proteases secreted by Streptomyces species. Above a critical concentration of 0.1 mM at low ionic strength and neutral pH, it can polymerize into filaments which may extend over several micrometers. After negative staining, these filaments show a helical substructure with characteristic diameters ranging from 6 to 12 nm. Selected images at higher magnification suggest the filaments are composed of two intertwined 6 nm strands. This is in agreement with the optical diffraction analysis which additionally established a periodic pitch of 25 nm for the helical intertwining. Rotary shadowing of the pepstatin A filaments clearly demonstrated the right-handedness of the helical twist. In physiological salt solution or at higher concentrations of pepstatin A, a variety of higher order structures were observed, including ribbons, sheets and cylinders with both regular and twisted or irregular geometries. Pepstatin A can interact with intermediate filament subunit proteins. These proteins possess a long, alpha-helical rod domain that forms coiled-coil dimers, which through both hydrophobic and ionic interactions form tetramers which, in turn, in the presence of physiological salt concentrations, polymerize into the 10 nm intermediate filaments. In the absence of salt, pepstatin A and intermediate filament proteins polymerize into long filaments with a rough surface and a diameter of 15-17 nm. This polymerization appears to be primarily driven by nonionic interactions between pepstatin A and polymerization-competent forms of intermediate filament proteins, resulting in a composite filament. Polymerization-incompetent proteolytic fragments of vimentin, lacking portions of the head and/or tail domain, failed to copolymerize with pepstatin A into long filaments under these conditions. These peptides, as well as bovine serum albumin, were found to stick to the surface of pepstatin A filaments, ribbons and sheets. Independent evidence for direct association of pepstatin A with intermediate filament subunit proteins was provided not only by electron microscopy but also by UV difference spectra. Pepstatin A loses its ability to inhibit the aspartyl protease of the human immunodeficiency virus type 1 following polymerization into the higher order structures described here. The amazing fact that pepstatin A can spontaneously self-associate to form very large polymers seems to be a more rare event for such small peptides. The other examples of synthetic or naturally occurring oligopeptides discussed in this review which are able to polymerize into higher order structures possess a common property, their hydrophobicity, often manifested by clusters of valine or isoleucine residues.(ABSTRACT TRUNCATED AT 400 WORDS)

Potential role of the viral protease in human immunodeficiency virus type 1 associated pathogenesis.

Infection with the human immunodeficiency virus type 1 (HIV-1) results in a variety of pathological changes culminating in the acquired immune deficiency syndrome (AIDS). While most of these changes can readily be accounted for either by direct effects of HIV-1 on the immune system or by indirect effects of secondary infectious agents as a result of faulty immune surveillance, the direct cause for a number of disease states, including some neuropathies, myopathies, nephropathy, thrombocytopenia, wasting syndromes and increased incidence of cancers (primarily lymphoma) has remained an enigma. We have recently shown that the HIV-1 protease, a viral encoded enzyme necessary for virus maturation and infectivity, can cleave a variety of host cell cytoskeletal proteins in vitro. Potential substrates for the HIV-1 protease are found in all of the cell types affected in these unexplained diseases. Recent proposals suggest that elements of the cytoskeleton may play an important role in the regulation of large scale genetic regulation. We propose that some of the degenerative changes associated with infection by HIV-1 are a direct consequence of cleavage of host cell cytoskeletal proteins, which in turn may be responsible for the increased incidence of cancer in HIV-1 infected individuals as a result of the perturbation of the regulation of gene expression by cytoskeletal components.

Single-particle structure determination by correlations of snapshot X-ray diffraction patterns.

Diffractive imaging with free-electron lasers allows structure determination from ensembles of weakly scattering identical nanoparticles. The ultra-short, ultra-bright X-ray pulses provide snapshots of the randomly oriented particles frozen in time, and terminate before the onset of structural damage. As signal strength diminishes for small particles, the synthesis of a three-dimensional diffraction volume requires simultaneous involvement of all data. Here we report the first application of a three-dimensional spatial frequency correlation analysis to carry out this synthesis from noisy single-particle femtosecond X-ray diffraction patterns of nearly identical samples in random and unknown orientations, collected at the Linac Coherent Light Source. Our demonstration uses unsupported test particles created via aerosol self-assembly, and composed of two polystyrene spheres of equal diameter. The correlation analysis avoids the need for orientation determination entirely. This method may be applied to the structural determination of biological macromolecules in solution.

Intermediate filament and related proteins: potential activators of nucleosomes during transcription initiation and elongation?

Intermediate filament (IF) protein tetramers contain two DNA- and core-histone-binding motifs in rotational symmetry in one and the same structural entity. We propose that IF protein oligomers might displace histone octamers from nucleosomes in the process of transcription initiation and elongation, to deposit them transiently on their alpha-helical coiled-coil domains. We further propose that structurally related proteins of the karyoskeleton, constructed from an alpha-helical domain capable of coiled-coil formation and a basic DNA-binding region adjacent to it, may be similarly involved in nucleosome activation. These proteins would function as auxiliary factors that disrupt nucleosomal structure to permit transcription and other DNA-dependent processes to proceed expiditiously.