scholarly journals Electrospray sample injection for single-particle imaging with X-ray lasers

2018 ◽  
Author(s):  
Johan Bielecki ◽  
Max F. Hantke ◽  
Benedikt J. Daurer ◽  
Hemanth K. N. Reddy ◽  
Dirk Hasse ◽  
...  
Keyword(s):  
Generation X ◽  
Room Temperature ◽  
Protein Structure Determination ◽  
High Temporal Resolution ◽  
X Ray ◽  
Free Sample ◽  
Large Particles ◽  
Particle Imaging ◽  
Single Particle Imaging ◽  
Initial Droplet Size

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 aerosolised molecules by non-volatile contaminants in the solution. The amount of contamination on the sample depends on the initial droplet-size during aerosolisation. 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 towards the ultimate goal of protein structure determination from imaging at room temperature and high temporal resolution.

scholarly journals Electrospray sample injection for single-particle imaging with x-ray lasers

2019 ◽  
Vol 5 (5) ◽  
pp. eaav8801 ◽  
Author(s):  
Johan Bielecki ◽  
Max F. Hantke ◽  
Benedikt J. Daurer ◽  
Hemanth K. N. Reddy ◽  
Dirk Hasse ◽  
...  
Keyword(s):  
Generation X ◽  
Room Temperature ◽  
Protein Structure Determination ◽  
High Temporal Resolution ◽  
X Ray ◽  
Free Sample ◽  
Large Particles ◽  
Particle Imaging ◽  
Single Particle Imaging ◽  
Initial Droplet Size

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.

scholarly journals Comparison of EMC and CM methods for orienting diffraction images in single-particle imaging experiments

IUCrJ ◽  
2021 ◽  
Vol 8 (6) ◽  
Author(s):  
Miklós Tegze ◽  
Gábor Bortel
Keyword(s):  
Intensity Distribution ◽  
Single Particle ◽  
Free Electron ◽  
Free Electron Laser ◽  
Biological Molecules ◽  
Crucial Step ◽  
X Ray ◽  
Diffraction Patterns ◽  
Particle Imaging ◽  
Single Particle Imaging

In single-particle imaging (SPI) experiments, diffraction patterns of identical particles are recorded. The particles are injected into the X-ray free-electron laser (XFEL) beam in random orientations. The crucial step of the data processing of SPI is finding the orientations of the recorded diffraction patterns in reciprocal space and reconstructing the 3D intensity distribution. Here, two orientation methods are compared: the expansion maximization compression (EMC) algorithm and the correlation maximization (CM) algorithm. To investigate the efficiency, reliability and accuracy of the methods at various XFEL pulse fluences, simulated diffraction patterns of biological molecules are used.

scholarly journals Current Status of Single Particle Imaging with X-ray Lasers

2018 ◽  
Vol 8 (1) ◽  
pp. 132 ◽  
Author(s):  
Zhibin Sun ◽  
Jiadong Fan ◽  
Haoyuan Li ◽  
Huaidong Jiang
Keyword(s):  
Single Particle ◽  
Current Status ◽  
X Ray ◽  
Particle Imaging ◽  
Single Particle Imaging

Incorporating particle symmetry into orientation determination in single-particle imaging

2018 ◽  
Vol 74 (5) ◽  
pp. 512-517
Author(s):  
Miklós Tegze ◽  
Gábor Bortel
Keyword(s):  
Three Dimensional ◽  
Symmetric Case ◽  
X Ray Diffraction ◽  
X Ray ◽  
Coherent Diffraction Imaging ◽  
Diffraction Imaging ◽  
Diffraction Patterns ◽  
Particle Imaging ◽  
Single Particle Imaging ◽  
Orientation Determination

In coherent-diffraction-imaging experiments X-ray diffraction patterns of identical particles are recorded. The particles are injected into the X-ray free-electron laser (XFEL) beam in random orientations. If the particle has symmetry, finding the orientation of a pattern can be ambiguous. With some modifications, the correlation-maximization method can find the relative orientations of the diffraction patterns for the case of symmetric particles as well. After convergence, the correlation maps show the symmetry of the particle and can be used to determine the symmetry elements and their orientations. The C factor, slightly modified for the symmetric case, can indicate the consistency of the assembled three-dimensional intensity distribution.

scholarly journals Start-to-end simulation of single-particle imaging using ultra-short pulses at the European X-ray Free-Electron Laser

IUCrJ ◽  
2017 ◽  
Vol 4 (5) ◽  
pp. 560-568 ◽  
Author(s):  
Carsten Fortmann-Grote ◽  
Alexey Buzmakov ◽  
Zoltan Jurek ◽  
Ne-Te Duane Loh ◽  
Liubov Samoylova ◽  
...  
Keyword(s):  
Pulse Duration ◽  
Diffraction Data ◽  
Single Particle ◽  
Free Electron ◽  
Free Electron Laser ◽  
Structural Information ◽  
X Ray ◽  
Particle Imaging ◽  
Single Particle Imaging ◽  
The Impact

Single-particle imaging with X-ray free-electron lasers (XFELs) has the potential to provide structural information at atomic resolution for non-crystalline biomolecules. This potential exists because ultra-short intense pulses can produce interpretable diffraction data notwithstanding radiation damage. This paper explores the impact of pulse duration on the interpretability of diffraction data using comprehensive and realistic simulations of an imaging experiment at the European X-ray Free-Electron Laser. It is found that the optimal pulse duration for molecules with a few thousand atoms at 5 keV lies between 3 and 9 fs.

scholarly journals Abstract P-1: Analysis of Tick-borne Encephalitis Virus Single-particle Imaging on X-ray Free-electron Laser

2021 ◽  
Vol 11 (Suppl_1) ◽  
pp. S11-S11
Author(s):  
Grigoriy Armeev ◽  
Alexey Shaytan ◽  
Mikhail Vorovich ◽  
Alexey Egorov ◽  
Aydar Ishmukhametov ◽  
...  
Keyword(s):  
Electron Density ◽  
Single Particle ◽  
Free Electron ◽  
Free Electron Laser ◽  
Encephalitis Virus ◽  
X Ray ◽  
Tick Borne Encephalitis ◽  
Tick Borne Encephalitis Virus ◽  
Particle Imaging ◽  
Single Particle Imaging

Background: Tick-borne encephalitis virus (TBEV) is a dangerous human pathogen which envelope structure is already known from cryoEM study. TBEV mature viral particle size (~50 nm in diameter) makes it suitable for single-particle imaging (SPI) on X-ray free-electron laser (XFEL). XFEL SPI studies are at the early stages of development; thus, a well-described and conformationally homogeneous sample is required to develop approaches for experimental setup and data analysis. Here we present the image analysis results of data collected in October 2019 during the European XFEL experiment #2316. Methods: The detector was placed at 1.62 m from the injector; photon energy was around 6 keV, pulse energy 4 mJ, beam diameter ~ 500 nm. All runs were processed to detect hits with threshold filter (5th percentile of lit pixels) and further filtered to omit low-intensity images and images that lack detector modules. Filtered hits were background and geometry corrected with SPImage library and custom python scripts. Then hits were azimuthally integrated using PyFAI library. Scattering profiles were further clustered using the affinity propagation algorithm with cosine similarity metric in log space. Extracted classes were used to build averaged images. All hit profiles were fitted with model scattering to estimate the diameter of the particle. Simulated diffraction patterns were prepared using Condor from the cryoEM electron density map (EMDB ID 3752). Results: During the analysis after the filtering, only 276 clean and bright hits were collected per 135 min of injection (from 27287 hits detected via lit pixels threshold). Thus the hit rate was around ~ 2 hits/min, which is expected to rise in the future. The majority of hits correspond to the 40-50 nm particles (Fig. 1a), which is expected for TBEV. However, the exact size may vary due to solvent evaporation, ion condensation, and possible variability in the sample. Conclusion: The averaged images and their scattering profiles correlate with the simulated scattering patterns, though not ideally (Fig. 1 bc). Such discrepancy is expected due to the absence of electron density in the center of modeled viral structures.

scholarly journals The SwissFEL soft X-ray free-electron laser beamline: Athos

2019 ◽  
Vol 26 (4) ◽  
pp. 1073-1084 ◽  
Author(s):  
Rafael Abela ◽  
Arturo Alarcon ◽  
Jürgen Alex ◽  
Christopher Arrell ◽  
Vladimir Arsov ◽  
...  
Keyword(s):  
Free Electron ◽  
Free Electron Laser ◽  
Pulse Length ◽  
X Ray ◽  
Materials Research ◽  
Transport Line ◽  
Beam Parameters ◽  
X Architecture ◽  
Particle Imaging ◽  
Single Particle Imaging

The SwissFEL soft X-ray free-electron laser (FEL) beamline Athos will be ready for user operation in 2021. Its design includes a novel layout of alternating magnetic chicanes and short undulator segments. Together with the APPLE X architecture of undulators, the Athos branch can be operated in different modes producing FEL beams with unique characteristics ranging from attosecond pulse length to high-power modes. Further space has been reserved for upgrades including modulators and an external seeding laser for better timing control. All of these schemes rely on state-of-the-art technologies described in this overview. The optical transport line distributing the FEL beam to the experimental stations was designed with the whole range of beam parameters in mind. Currently two experimental stations, one for condensed matter and quantum materials research and a second one for atomic, molecular and optical physics, chemical sciences and ultrafast single-particle imaging, are being laid out such that they can profit from the unique soft X-ray pulses produced in the Athos branch in an optimal way.

Nanocrystalline protein domains via salting-out

2021 ◽  
Vol 77 (11) ◽  
Author(s):  
Daniel G. Greene ◽  
Shannon Modla ◽  
Stanley I. Sandler ◽  
Norman J. Wagner ◽  
Abraham M. Lenhoff
Keyword(s):  
Generation X ◽  
Protein Structure Determination ◽  
Biological Macromolecules ◽  
Bulk Crystals ◽  
Salting Out ◽  
X Ray ◽  
Mesoscale Structure ◽  
Protein Gels ◽  
Amorphous Structures ◽  
The Rich

Protein salting-out is a well established phenomenon that in many cases leads to amorphous structures and protein gels, which are usually not considered to be useful for protein structure determination. Here, microstructural measurements of several different salted-out protein dense phases are reported, including of lysozyme, ribonuclease A and an IgG1, showing that salted-out protein gels unexpectedly contain highly ordered protein nanostructures that assemble hierarchically to create the gel. The nanocrystalline domains are approximately 10–100 nm in size, are shown to have structures commensurate with those of bulk crystals and grow on time scales in the order of an hour to a day. Beyond revealing the rich, hierarchical nanoscale to mesoscale structure of protein gels, the nanocrystals that these phases contain are candidates for structural biology on next-generation X-ray free-electron lasers, which may enable the study of biological macromolecules that are difficult or impossible to crystallize in bulk.

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