| 1. |
- Bielecki, Johan, 1982, et al.
(författare)
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Electrospray sample injection for single-particle imaging with x-ray lasers
- 2019
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Ingår i: Science advances. - : American Association for the Advancement of Science (AAAS). - 2375-2548. ; 5:5
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Tidskriftsartikel (refereegranskat)abstract
- The possibility of imaging single proteins constitutes an exciting challenge for x-ray lasers. Despite encouraging results on large particles, imaging small particles has proven to be difficult for two reasons: not quite high enough pulse intensity from currently available x-ray lasers and, as we demonstrate here, contamination of the aerosolized molecules by nonvolatile contaminants in the solution. The amount of contamination on the sample depends on the initial droplet size during aerosolization. Here, we show that, with our electrospray injector, we can decrease the size of aerosol droplets and demonstrate virtually contaminant-free sample delivery of organelles, small virions, and proteins. The results presented here, together with the increased performance of next-generation x-ray lasers, constitute an important stepping stone toward the ultimate goal of protein structure determination from imaging at room temperature and high temporal resolution.
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| 2. |
- Chihara, Akane, et al.
(författare)
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A novel capsid protein network allows the characteristic internal membrane structure of Marseilleviridae giant viruses
- 2022
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Ingår i: Scientific Reports. - : Springer Nature. - 2045-2322. ; 12:1
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Tidskriftsartikel (refereegranskat)abstract
- Marseilleviridae is a family of giant viruses, showing a characteristic internal membrane with extrusions underneath the icosahedral vertices. However, such large objects, with a maximum diameter of 250 nm are technically difficult to examine at sub-nanometre resolution by cryo-electron microscopy. Here, we tested the utility of 1 MV high-voltage cryo-EM (cryo-HVEM) for single particle structural analysis (SPA) of giant viruses using tokyovirus, a species of Marseilleviridae, and revealed the capsid structure at 7.7 & Aring; resolution. The capsid enclosing the viral DNA consisted primarily of four layers: (1) major capsid proteins (MCPs) and penton proteins, (2) minor capsid proteins (mCPs), (3) scaffold protein components (ScPCs), and (4) internal membrane. The mCPs showed a novel capsid lattice consisting of eight protein components. ScPCs connecting the icosahedral vertices supported the formation of the membrane extrusions, and possibly act like tape measure proteins reported in other giant viruses. The density on top of the MCP trimer was suggested to include glycoproteins. This is the first attempt at cryo-HVEM SPA. We found the primary limitations to be the lack of automated data acquisition and software support for collection and processing and thus achievable resolution. However, the results pave the way for using cryo-HVEM for structural analysis of larger biological specimens.
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| 3. |
- Daurer, Benedikt J., et al.
(författare)
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Experimental strategies for imaging bioparticles with femtosecond hard X-ray pulses
- 2017
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Ingår i: IUCrJ. - : INT UNION CRYSTALLOGRAPHY. - 2052-2525. ; 4, s. 251-262
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Tidskriftsartikel (refereegranskat)abstract
- This study explores the capabilities of the Coherent X-ray Imaging Instrument at the Linac Coherent Light Source to image small biological samples. The weak signal from small samples puts a significant demand on the experiment. Aerosolized Omono River virus particles of similar to 40 nm in diameter were injected into the submicrometre X-ray focus at a reduced pressure. Diffraction patterns were recorded on two area detectors. The statistical nature of the measurements from many individual particles provided information about the intensity profile of the X-ray beam, phase variations in the wavefront and the size distribution of the injected particles. The results point to a wider than expected size distribution (from similar to 35 to similar to 300 nm in diameter). This is likely to be owing to nonvolatile contaminants from larger droplets during aerosolization and droplet evaporation. The results suggest that the concentration of nonvolatile contaminants and the ratio between the volumes of the initial droplet and the sample particles is critical in such studies. The maximum beam intensity in the focus was found to be 1.9 * 10(12) photons per mu m(2) per pulse. The full-width of the focus at half-maximum was estimated to be 500 nm (assuming 20% beamline transmission), and this width is larger than expected. Under these conditions, the diffraction signal from a sample-sized particle remained above the average background to a resolution of 4.25 nm. The results suggest that reducing the size of the initial droplets during aerosolization is necessary to bring small particles into the scope of detailed structural studies with X-ray lasers.
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| 4. |
- Gorkhover, Tais, et al.
(författare)
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Femtosecond X-ray Fourier holography imaging of free-flying nanoparticles
- 2018
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Ingår i: Nature Photonics. - : Springer Science and Business Media LLC. - 1749-4885 .- 1749-4893. ; 12:3, s. 150-153
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Tidskriftsartikel (refereegranskat)abstract
- Ultrafast X-ray imaging on individual fragile specimens such as aerosols 1 , metastable particles 2 , superfluid quantum systems 3 and live biospecimens 4 provides high-resolution information that is inaccessible with conventional imaging techniques. Coherent X-ray diffractive imaging, however, suffers from intrinsic loss of phase, and therefore structure recovery is often complicated and not always uniquely defined 4,5 . Here, we introduce the method of in-flight holography, where we use nanoclusters as reference X-ray scatterers to encode relative phase information into diffraction patterns of a virus. The resulting hologram contains an unambiguous three-dimensional map of a virus and two nanoclusters with the highest lateral resolution so far achieved via single shot X-ray holography. Our approach unlocks the benefits of holography for ultrafast X-ray imaging of nanoscale, non-periodic systems and paves the way to direct observation of complex electron dynamics down to the attosecond timescale.
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| 5. |
- Haldar, Sourav, et al.
(författare)
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Precise Triggering and Chemical Control of Single-Virus Fusion within Endosomes
- 2021
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Ingår i: Journal of Virology. - : American Society for Microbiology. - 0022-538X .- 1098-5514. ; 95:1
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Tidskriftsartikel (refereegranskat)abstract
- Many enveloped viruses infect cells within endocytic compartments. The pH drop that accompanies endosomal maturation, often in conjunction with proteolysis, triggers viral proteins to insert into the endosomal membrane and drive fusion. Fusion dynamics have been studied by tracking viruses within living cells, which limits the precision with which fusion can be synchronized and controlled, and reconstituting viral fusion to synthetic membranes, which introduces nonphysiological membrane curvature and composition. To overcome these limitations, we report chemically controllable triggering of single-virus fusion within endosomes. We isolated influenza (A/Aichi/68; H3N2) virus:endosome conjugates from cells, immobilized them in a microfluidic flow cell, and rapidly and controllably triggered fusion. Observed lipid-mixing kinetics were surprisingly similar to those of influenza virus fusion with model membranes of opposite curvature: 80% of single-virus events had indistinguishable kinetics. This result suggests that endosomal membrane curvature is not a key permissive feature for viral entry, at least lipid mixing. The assay preserved endosomal membrane asymmetry and protein composition, providing a platform to test how cellular restriction factors and altered endosomal trafficking affect viral membrane fusion.IMPORTANCE Many enveloped viruses infect cells via fusion to endosomes, but controlling this process within living cells has been challenging. We studied the fusion of influenza virus virions to endosomes in a chemically controllable manner. Extracting virus: endosome conjugates from cells and exogenously triggering fusion permits precise study of virus:endosome fusion kinetics. Surprisingly, endosomal curvature does not grossly alter fusion kinetics, although membrane deformability does. This supports a model for influenza virus entry where cells restrict or permit membrane fusion by changing deformability, for instance, using interferon-induced proteins.
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| 6. |
- Konold, Patrick E., et al.
(författare)
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Microsecond time-resolved X-ray scattering by utilizing MHz repetition rate at second-generation XFELs
- 2024
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Ingår i: NATURE METHODS. - : NATURE PORTFOLIO. - 1548-7091 .- 1548-7105.
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Tidskriftsartikel (refereegranskat)abstract
- Detecting microsecond structural perturbations in biomolecules has wide relevance in biology, chemistry and medicine. Here we show how MHz repetition rates at X-ray free-electron lasers can be used to produce microsecond time-series of protein scattering with exceptionally low noise levels of 0.001%. We demonstrate the approach by examining J alpha helix unfolding of a light-oxygen-voltage photosensory domain. This time-resolved acquisition strategy is easy to implement and widely applicable for direct observation of structural dynamics of many biochemical processes. The MHz repetition rates available at second-generation X-ray free-electron lasers enable the collection of microsecond time-resolved X-ray scattering data with exceptionally low noise, providing insights into protein structural dynamics.
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| 7. |
- Konold, Patrick, et al.
(författare)
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Microsecond time-resolved X-ray scattering by utilizing MHz repetition rate at second-generation XFELs
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Detecting microsecond structural perturbations in biomolecules has wide relevance inbiology, chemistry, and medicine. Here, we show how MHz repetition rates at X-ray freeelectron lasers (XFELs) can be used to produce microsecond time-series of proteinscattering with exceptionally low noise levels of 0.001%. We demonstrate the approach byderiving new mechanistic insight into Jɑ helix unfolding of a Light-Oxygen-Voltage (LOV)photosensory domain. This time-resolved acquisition strategy is easy to implement andwidely applicable for direct observation of structural dynamics of many biochemicalprocesses.
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| 8. |
- Kurimoto, Eiji, et al.
(författare)
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Crystal structure of human proteasome assembly chaperone PAC4 involved in proteasome formation
- 2017
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Ingår i: Protein Science. - : WILEY. - 0961-8368 .- 1469-896X. ; 26:5, s. 1080-1085
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Tidskriftsartikel (refereegranskat)abstract
- The 26S proteasome is a large protein complex, responsible for degradation of ubiquinated proteins in eukaryotic cells. Eukaryotic proteasome formation is a highly ordered process that is assisted by several assembly chaperones. The assembly of its catalytic 20S core particle depends on at least five proteasome-specific chaperones, i.e., proteasome-assembling chaperons 1-4 (PAC1-4) and proteasome maturation protein (POMP). The orthologues of yeast assembly chaperones have been structurally characterized, whereas most mammalian assembly chaperones are not. In the present study, we determined a crystal structure of human PAC4 at 1.90-angstrom resolution. Our crystallographic data identify a hydrophobic surface that is surrounded by charged residues. The hydrophobic surface is complementary to that of its binding partner, PAC3. The surface also exhibits charge complementarity with the proteasomal 4-5 subunits. This will provide insights into human proteasome-assembling chaperones as potential anticancer drug targets.
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| 9. |
- Kördel, Mikael, et al.
(författare)
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Biological Laboratory X-ray Microscopy
- 2018
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Ingår i: Microscopy and Microanalysis. - 1431-9276 .- 1435-8115. ; 24:S2, s. 346-347
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Tidskriftsartikel (refereegranskat)
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| 10. |
- Lundholm, Ida V., et al.
(författare)
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Considerations for three-dimensional image reconstruction from experimental data in coherent diffractive imaging
- 2018
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Ingår i: IUCrJ. - : International Union of Crystallography. - 2052-2525. ; 5, s. 531-541
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Tidskriftsartikel (refereegranskat)abstract
- Diffraction before destruction using X-ray free-electron lasers (XFELs) has the potential to determine radiation-damage-free structures without the need for crystallization. This article presents the three-dimensional reconstruction of the Melbournevirus from single-particle X-ray diffraction patterns collected at the LINAC Coherent Light Source (LCLS) as well as reconstructions from simulated data exploring the consequences of different kinds of experimental sources of noise. The reconstruction from experimental data suffers from a strong artifact in the center of the particle. This could be reproduced with simulated data by adding experimental background to the diffraction patterns. In those simulations, the relative density of the artifact increases linearly with background strength. This suggests that the artifact originates from the Fourier transform of the relatively flat background, concentrating all power in a central feature of limited extent. We support these findings by significantly reducing the artifact through background removal before the phase-retrieval step. Large amounts of blurring in the diffraction patterns were also found to introduce diffuse artifacts, which could easily be mistaken as biologically relevant features. Other sources of noise such as sample heterogeneity and variation of pulse energy did not significantly degrade the quality of the reconstructions. Larger data volumes, made possible by the recent inauguration of high repetition-rate XFELs, allow for increased signal-to-background ratio and provide a way to minimize these artifacts. The anticipated development of three-dimensional Fourier-volume-assembly algorithms which are background aware is an alternative and complementary solution, which maximizes the use of data.
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