Matching Items (18)

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Experimental strategies for imaging bioparticles with femtosecond hard X-ray pulses

Description

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

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 ∼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 ∼35 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[superscript 12] photons per µm[superscript 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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Created

Date Created
  • 2017-04-07

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Open data set of live cyanobacterial cells imaged using an X-ray laser

Description

Structural studies on living cells by conventional methods are limited to low resolution because radiation damage kills cells long before the necessary dose for high resolution can be delivered. X-ray

Structural studies on living cells by conventional methods are limited to low resolution because radiation damage kills cells long before the necessary dose for high resolution can be delivered. X-ray free-electron lasers circumvent this problem by outrunning key damage processes with an ultra-short and extremely bright coherent X-ray pulse. Diffraction-before-destruction experiments provide high-resolution data from cells that are alive when the femtosecond X-ray pulse traverses the sample. This paper presents two data sets from micron-sized cyanobacteria obtained at the Linac Coherent Light Source, containing a total of 199,000 diffraction patterns. Utilizing this type of diffraction data will require the development of new analysis methods and algorithms for studying structure and structural variability in large populations of cells and to create abstract models. Such studies will allow us to understand living cells and populations of cells in new ways. New X-ray lasers, like the European XFEL, will produce billions of pulses per day, and could open new areas in structural sciences.

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Created

Date Created
  • 2016-08-01

Ion Traps for Single Particle Imaging

Description

X-ray Free Electron Lasers (XFELs) are used for diffractive x-ray imaging of the structure of many biological particles, such as viruses and proteins. The ultimate goal for XFEL-based microscopy is

X-ray Free Electron Lasers (XFELs) are used for diffractive x-ray imaging of the structure of many biological particles, such as viruses and proteins. The ultimate goal for XFEL-based microscopy is atomic resolution images of non-crystalline particles. However, data collection efficiency as well as the limited amount of measurement time given annually to researchers, such high-resolution images are presently impossible to attain. Here, we consider two potential solutions to the single-particle hit rate problem; the first looks at applying static electric fields to existing aerodynamic particle injectors, and the second looks at the viability of using time-varying electric fields associated with ion traps to create high-density regions of particles. For the static solution, we looked at applying a constant electric potential to the nozzle, as well as applying a high voltage to a ring electrode in close proximity to a grounded nozzle. We considered the breakdown field strength of the helium gas used to determine how closely the ring electrode could be placed without creating an arc that could potentially destroy expensive equipment. Then, we considered the possibility of using electrodynamic ion traps to increase particle densities. We first characterized how charged particles behave in oscillating electric fields using a simple electrode geometry. Using the general results from this, we then constructed a rudimentary ion trap to test if our experiment agreed with the theory. Finally, we conducted a literature review to determine what particle densities other scientists have been able to measure using ion traps. We then compared existing ion traps to what we expect from the nozzle injectors to determine which method may be the better solution.

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Created

Date Created
  • 2017-05

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Coherent soft X-ray diffraction imaging of coliphage PR772 at the Linac coherent light source

Description

Single-particle diffraction from X-ray Free Electron Lasers offers the potential for molecular structure determination without the need for crystallization. In an effort to further develop the technique, we present a

Single-particle diffraction from X-ray Free Electron Lasers offers the potential for molecular structure determination without the need for crystallization. In an effort to further develop the technique, we present a dataset of coherent soft X-ray diffraction images of Coliphage PR772 virus, collected at the Atomic Molecular Optics (AMO) beamline with pnCCD detectors in the LAMP instrument at the Linac Coherent Light Source. The diameter of PR772 ranges from 65–70 nm, which is considerably smaller than the previously reported ~600 nm diameter Mimivirus. This reflects continued progress in XFEL-based single-particle imaging towards the single molecular imaging regime. The data set contains significantly more single particle hits than collected in previous experiments, enabling the development of improved statistical analysis, reconstruction algorithms, and quantitative metrics to determine resolution and self-consistency.

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Created

Date Created
  • 2017-06-27

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Expression, purification and crystallization of CTB-MPR, a candidate mucosal vaccine component against HIV-1

Description

CTB-MPR is a fusion protein between the B subunit of cholera toxin (CTB) and the membrane-proximal region of gp41 (MPR), the transmembrane envelope protein of Human immunodeficiency virus 1 (HIV-1),

CTB-MPR is a fusion protein between the B subunit of cholera toxin (CTB) and the membrane-proximal region of gp41 (MPR), the transmembrane envelope protein of Human immunodeficiency virus 1 (HIV-1), and has previously been shown to induce the production of anti-HIV-1 antibodies with antiviral functions. To further improve the design of this candidate vaccine, X-ray crystallography experiments were performed to obtain structural information about this fusion protein. Several variants of CTB-MPR were designed, constructed and recombinantly expressed in Escherichia coli. The first variant contained a flexible GPGP linker between CTB and MPR, and yielded crystals that diffracted to a resolution of 2.3 Å, but only the CTB region was detected in the electron-density map. A second variant, in which the CTB was directly attached to MPR, was shown to destabilize pentamer formation. A third construct containing a polyalanine linker between CTB and MPR proved to stabilize the pentameric form of the protein during purification. The purification procedure was shown to produce a homogeneously pure and monodisperse sample for crystallization. Initial crystallization experiments led to pseudo-crystals which were ordered in only two dimensions and were disordered in the third dimension. Nanocrystals obtained using the same precipitant showed promising X-ray diffraction to 5 Å resolution in femtosecond nanocrystallography experiments at the Linac Coherent Light Source at the SLAC National Accelerator Laboratory. The results demonstrate the utility of femtosecond X-ray crystallography to enable structural analysis based on nano/microcrystals of a protein for which no macroscopic crystals ordered in three dimensions have been observed before.

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Created

Date Created
  • 2014-08-20

Serial time-resolved crystallography of photosystem II using a femtosecond X-ray laser

Description

Photosynthesis, a process catalysed by plants, algae and cyanobacteria converts sunlight to energy thus sustaining all higher life on Earth. Two large membrane protein complexes, photosystem I and II (PSI

Photosynthesis, a process catalysed by plants, algae and cyanobacteria converts sunlight to energy thus sustaining all higher life on Earth. Two large membrane protein complexes, photosystem I and II (PSI and PSII), act in series to catalyse the light-driven reactions in photosynthesis. PSII catalyses the light-driven water splitting process, which maintains the Earth’s oxygenic atmosphere. In this process, the oxygen-evolving complex (OEC) of PSII cycles through five states, S[subscript 0] to S[subscript 4], in which four electrons are sequentially extracted from the OEC in four light-driven charge-separation events. Here we describe time resolved experiments on PSII nano/microcrystals from Thermosynechococcus elongatus performed with the recently developed technique of serial femtosecond crystallography. Structures have been determined from PSII in the dark S[subscript 1] state and after double laser excitation (putative S[subscript 3] state) at 5 and 5.5 Å resolution, respectively. The results provide evidence that PSII undergoes significant conformational changes at the electron acceptor side and at the Mn[subscript 4]CaO[subscript 5] core of the OEC. These include an elongation of the metal cluster, accompanied by changes in the protein environment, which could allow for binding of the second substrate water molecule between the more distant protruding Mn (referred to as the ‘dangler’ Mn) and the Mn[subscript 3]CaO[subscript x] cubane in the S[subscript 2] to S[subscript 3] transition, as predicted by spectroscopic and computational studies. This work shows the great potential for time-resolved serial femtosecond crystallography for investigation of catalytic processes in biomolecules.

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Created

Date Created
  • 2014-09-11

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The Electrodynamic Ion Trap

Description

In this experiment an Electrodynamic Ion Ring Trap was constructed and tested. Due to the nature of Electrostatic fields, the setup required an oscillating voltage source to stably trap the

In this experiment an Electrodynamic Ion Ring Trap was constructed and tested. Due to the nature of Electrostatic fields, the setup required an oscillating voltage source to stably trap the particles. It was built in a safe manner, The power supply was kept in a project box to avoid incidental contact, and was connected to a small copper wire in the shape of a ring. The maximum voltage that could be experienced via incidental contact was well within safe ranges a 0.3mA. Within minutes of its completion the trap was able to trap small Lycopodium powder spores mass of approximately 1.7*10^{-11}kg in clusters of 15-30 for long timescales. The oscillations of these spores were observed to be roughly 1.01mm at their maximum, and in an attempt to understand the dynamics of the Ion Trap, a concept called the pseudo-potential of the trap was used. This method proved fairly inaccurate, involving much estimation and using a static field estimation of 9.39*10^8 N\C and a charge estimate on the particles of ~1e, a maximum oscillation distance of 1.37m was calculated. Though the derived static field strength was not far off from the field strength required to achieve the correct oscillation distance (Percent error of 9.92%, the small discrepancy caused major calculation errors. The trap's intended purpose however was to eventually trap protein molecules for mapping via XFEL laser, and after its successful construction that goal is fairly achievable. The trap was also housed in a vacuum chamber so that it could be more effectively implemented with the XFEL.

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Created

Date Created
  • 2019-05

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Double-flow Focused Liquid Injector for Efficient Serial Femtosecond Crystallography

Description

Serial femtosecond crystallography requires reliable and efficient delivery of fresh crystals across the beam of an X-ray free-electron laser over the course of an experiment. We introduce a double-flow focusing

Serial femtosecond crystallography requires reliable and efficient delivery of fresh crystals across the beam of an X-ray free-electron laser over the course of an experiment. We introduce a double-flow focusing nozzle to meet this challenge, with significantly reduced sample consumption, while improving jet stability over previous generations of nozzles. We demonstrate its use to determine the first room-temperature structure of RNA polymerase II at high resolution, revealing new structural details. Moreover, the double flow-focusing nozzles were successfully tested with three other protein samples and the first room temperature structure of an extradiol ring-cleaving dioxygenase was solved by utilizing the improved operation and characteristics of these devices.

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Created

Date Created
  • 2017-03-16

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Coherent Diffraction of Single Rice Dwarf Virus Particles Using Hard X-rays at the Linac Coherent Light Source

Description

Single particle diffractive imaging data from Rice Dwarf Virus (RDV) were recorded using the Coherent X-ray Imaging (CXI) instrument at the Linac Coherent Light Source (LCLS). RDV was chosen as

Single particle diffractive imaging data from Rice Dwarf Virus (RDV) were recorded using the Coherent X-ray Imaging (CXI) instrument at the Linac Coherent Light Source (LCLS). RDV was chosen as it is a well-characterized model system, useful for proof-of-principle experiments, system optimization and algorithm development. RDV, an icosahedral virus of about 70 nm in diameter, was aerosolized and injected into the approximately 0.1 μm diameter focused hard X-ray beam at the CXI instrument of LCLS. Diffraction patterns from RDV with signal to 5.9 Ångström were recorded. The diffraction data are available through the Coherent X-ray Imaging Data Bank (CXIDB) as a resource for algorithm development, the contents of which are described here.

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Created

Date Created
  • 2016-08-01

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Diffraction Data of Core-shell Nanoparticles from an X-ray Free Electron Laser

Description

X-ray free-electron lasers provide novel opportunities to conduct single particle analysis on nanoscale particles. Coherent diffractive imaging experiments were performed at the Linac Coherent Light Source (LCLS), SLAC National Laboratory,

X-ray free-electron lasers provide novel opportunities to conduct single particle analysis on nanoscale particles. Coherent diffractive imaging experiments were performed at the Linac Coherent Light Source (LCLS), SLAC National Laboratory, exposing single inorganic core-shell nanoparticles to femtosecond hard-X-ray pulses. Each facetted nanoparticle consisted of a crystalline gold core and a differently shaped palladium shell. Scattered intensities were observed up to about 7 nm resolution. Analysis of the scattering patterns revealed the size distribution of the samples, which is consistent with that obtained from direct real-space imaging by electron microscopy. Scattering patterns resulting from single particles were selected and compiled into a dataset which can be valuable for algorithm developments in single particle scattering research.

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Created

Date Created
  • 2017-04-11