Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 Apr 30;2(4):041601.
doi: 10.1063/1.4919740. eCollection 2015 Jul.

Single-particle structure determination by X-ray free-electron lasers: Possibilities and challenges

A Hosseinizadeh  1 , A Dashti  1 , P Schwander  1 , R Fung  1 , A Ourmazd  1
Affiliations

Single-particle structure determination by X-ray free-electron lasers: Possibilities and challenges

A Hosseinizadeh et al. Struct Dyn. .

Abstract

Single-particle structure recovery without crystals or radiation damage is a revolutionary possibility offered by X-ray free-electron lasers, but it involves formidable experimental and data-analytical challenges. Many of these difficulties were encountered during the development of cryogenic electron microscopy of biological systems. Electron microscopy of biological entities has now reached a spatial resolution of about 0.3 nm, with a rapidly emerging capability to map discrete and continuous conformational changes and the energy landscapes of biomolecular machines. Nonetheless, single-particle imaging by X-ray free-electron lasers remains important for a range of applications, including the study of large "electron-opaque" objects and time-resolved examination of key biological processes at physiological temperatures. After summarizing the state of the art in the study of structure and conformations by cryogenic electron microscopy, we identify the primary opportunities and challenges facing X-ray-based single-particle approaches, and possible means for circumventing them.

PubMed Disclaimer

Figures

FIG. 1.
FIG. 1.
(a) 3D structure of ribosome shown in three standard views. (b) Energy landscape of ribosome. (Low-energy regions appear blue.) Conformational changes occurring between each of the seven numbered points along the trajectory are shown in letters. Movies showing the continuous conformational changes along the low-energy trajectory are available in Dashti et al. (2014). Reprinted with permission from Dashti et al., Proc. Natl. Acad. Sci. U. S. A. 111(49), 17492–17497 (2014). Copyright 2014 by PNAS.
FIG. 2.
FIG. 2.
Some artifacts in a typical XFEL diffraction pattern from a large virus, obtained with a liquid-jet injector. Features marked (a) and (b) are due to the scattering from injector nozzle. Features (c) and (d) stem from movements in the position of the liquid jet containing the sample. Dark lines marked (e) represent the effect of electronic noise and dead pixels.
FIG. 3.
FIG. 3.
Representative selection of snapshots showing shot-to-shot variations in effects extraneous to the particle under observation.
FIG. 4.
FIG. 4.
(a) Manifold of simulated diffraction snapshots from an icosahedral virus. Each point in this plot represents a diffraction pattern. (b) 2D representation of the manifold of the experimental XFEL snapshots from a large icosahedral virus.
FIG. 5.
FIG. 5.
The total intensity of the snapshots (vertical axis) is correlated with the position along the parabolic manifold [arc length of parabola in Fig. 4(b)].
FIG. 6.
FIG. 6.
(a) A typical raw XFEL diffraction pattern obtained from a biological object injected by a continuous liquid jet. (b) The flat-field correction for this snapshot deduced from the collection of diffraction patterns themselves. (c) Corrected snapshot after dynamic flat-fielding.
See this image and copyright information in PMC

References

    1. Amunts, A. , Brown, A. , Bai, X.-C. , Llácer, J. L. , Hussain, T. , Emsley, P. , Long, F. , Murshudov, G. , Scheres, S. H. W. , and Ramakrishnan, V. , “ Structure of the yeast mitochondrial large ribosomal subunit,” Science 343(6178), 1485–1489 (2014).10.1126/science.1249410 - DOI - PMC - PubMed
    1. Aquila, A. , Barty, A. , Bostedt, C. , Boutet, S. , Carini, G. , dePonte, D. , Drell, P. , Doniach, S. , Downing, K. H. , Earnest, T. , Elmlund, H. , Elser, V. , Gühr, M. , Hajdu, J. , Hastings, J. , Hau-Riege, S. P. , Huang, Z. , Lattman, E. E. , Maia, F. R. N. C. , Marchesini, S. , Ourmazd, A. , Pellegrini, C. , Santra, R. , Schlichting, I. , Schroer, C. , Spence, J. C. H. , Vartanyants, I. A. , Wakatsuki, S. , Weis, W. I. , and Williams, G. J. , “ The linac coherent light source single particle imaging road map,” Struct. Dyn. 2, 041701 (2015).10.1063/1.4918726 - DOI - PMC - PubMed
    1. Dashti, A. , Schwander, P. , Langlois, R. , Fung, R. , Li, W. , Hosseinizadeh, A. , Liao, H. Y. , Pallesen, J. , Sharma, G. , Stupina, V. A. , Simon, A. E. , Dinman, J. D. , Frank, J. , and Ourmazd, A. , “ Trajectories of the ribosome as a brownian nanomachine,” Proc. Natl. Acad. Sci. U. S. A. 111(49), 17492–17497 (2014).10.1073/pnas.1419276111 - DOI - PMC - PubMed
    1. Ekeberg, T. et al., “ Three-dimensional reconstruction of the giant mimivirus particle with an X-ray free electron laser,” Phys. Rev. Lett. 114, 098102 (2015).10.1103/PhysRevLett.114.098102 - DOI - PubMed
    1. Fischer, N. , Konevega, A. L. , Wintermeyer, W. , Rodnina, M. V. , and Stark, H. , “ Ribosome dynamics and tRNA movement by time-resolved electron cryomicroscopy,” Nature 466(7304), 329–333 (2010).10.1038/nature09206 - DOI - PubMed