Solving protein nanocrystals by cryo-EM diffraction: Multiple scattering artifacts

Ganesh Subramanian, Shibom Basu, Haiguang Liu, Jian Min Zuo, John Spence

    Research output: Contribution to journalArticle

    16 Citations (Scopus)

    Abstract

    The maximum thickness permissible within the single-scattering approximation for the determination of the structure of perfectly ordered protein microcrystals by transmission electron diffraction is estimated for tetragonal hen-egg lysozyme protein crystals using several approaches. Multislice simulations are performed for many diffraction conditions and beam energies to determine the validity domain of the required single-scattering approximation and hence the limit on crystal thickness. The effects of erroneous experimental structure factor amplitudes on the charge density map for lysozyme are noted and their threshold limits calculated. The maximum thickness of lysozyme permissible under the single-scattering approximation is also estimated using R-factor analysis. Successful reconstruction of density maps is found to result mainly from the use of the phase information provided by modeling based on the protein data base through molecular replacement (MR), which dominates the effect of poor quality electron diffraction data at thicknesses larger than about 200 Å. For perfectly ordered protein nanocrystals, a maximum thickness of about 1000 Å is predicted at 200 keV if MR can be used, using R-factor analysis performed over a subset of the simulated diffracted beams. The effects of crystal bending, mosaicity (which has recently been directly imaged by cryo-EM) and secondary scattering are discussed. Structure-independent tests for single-scattering and new microfluidic methods for growing and sorting nanocrystals by size are reviewed.

    Original languageEnglish (US)
    Pages (from-to)87-93
    Number of pages7
    JournalUltramicroscopy
    Volume148
    DOIs
    StatePublished - Jan 1 2015

    Fingerprint

    Multiple scattering
    Nanocrystals
    artifacts
    nanocrystals
    Diffraction
    Scattering
    R388
    proteins
    Proteins
    lysozyme
    scattering
    diffraction
    factor analysis
    Enzymes
    Factor analysis
    Muramidase
    Electron diffraction
    Crystals
    electron diffraction
    approximation

    Keywords

    • Charge density map
    • Electron diffraction
    • Lysozyme
    • Multiple scattering
    • Protein crystallography
    • R-factor

    ASJC Scopus subject areas

    • Atomic and Molecular Physics, and Optics
    • Instrumentation
    • Electronic, Optical and Magnetic Materials

    Cite this

    Solving protein nanocrystals by cryo-EM diffraction : Multiple scattering artifacts. / Subramanian, Ganesh; Basu, Shibom; Liu, Haiguang; Zuo, Jian Min; Spence, John.

    In: Ultramicroscopy, Vol. 148, 01.01.2015, p. 87-93.

    Research output: Contribution to journalArticle

    Subramanian, Ganesh ; Basu, Shibom ; Liu, Haiguang ; Zuo, Jian Min ; Spence, John. / Solving protein nanocrystals by cryo-EM diffraction : Multiple scattering artifacts. In: Ultramicroscopy. 2015 ; Vol. 148. pp. 87-93.
    @article{71368cd7dbcc476dbd746a4133239cdb,
    title = "Solving protein nanocrystals by cryo-EM diffraction: Multiple scattering artifacts",
    abstract = "The maximum thickness permissible within the single-scattering approximation for the determination of the structure of perfectly ordered protein microcrystals by transmission electron diffraction is estimated for tetragonal hen-egg lysozyme protein crystals using several approaches. Multislice simulations are performed for many diffraction conditions and beam energies to determine the validity domain of the required single-scattering approximation and hence the limit on crystal thickness. The effects of erroneous experimental structure factor amplitudes on the charge density map for lysozyme are noted and their threshold limits calculated. The maximum thickness of lysozyme permissible under the single-scattering approximation is also estimated using R-factor analysis. Successful reconstruction of density maps is found to result mainly from the use of the phase information provided by modeling based on the protein data base through molecular replacement (MR), which dominates the effect of poor quality electron diffraction data at thicknesses larger than about 200 {\AA}. For perfectly ordered protein nanocrystals, a maximum thickness of about 1000 {\AA} is predicted at 200 keV if MR can be used, using R-factor analysis performed over a subset of the simulated diffracted beams. The effects of crystal bending, mosaicity (which has recently been directly imaged by cryo-EM) and secondary scattering are discussed. Structure-independent tests for single-scattering and new microfluidic methods for growing and sorting nanocrystals by size are reviewed.",
    keywords = "Charge density map, Electron diffraction, Lysozyme, Multiple scattering, Protein crystallography, R-factor",
    author = "Ganesh Subramanian and Shibom Basu and Haiguang Liu and Zuo, {Jian Min} and John Spence",
    year = "2015",
    month = "1",
    day = "1",
    doi = "10.1016/j.ultramic.2014.08.013",
    language = "English (US)",
    volume = "148",
    pages = "87--93",
    journal = "Ultramicroscopy",
    issn = "0304-3991",
    publisher = "Elsevier",

    }

    TY - JOUR

    T1 - Solving protein nanocrystals by cryo-EM diffraction

    T2 - Multiple scattering artifacts

    AU - Subramanian, Ganesh

    AU - Basu, Shibom

    AU - Liu, Haiguang

    AU - Zuo, Jian Min

    AU - Spence, John

    PY - 2015/1/1

    Y1 - 2015/1/1

    N2 - The maximum thickness permissible within the single-scattering approximation for the determination of the structure of perfectly ordered protein microcrystals by transmission electron diffraction is estimated for tetragonal hen-egg lysozyme protein crystals using several approaches. Multislice simulations are performed for many diffraction conditions and beam energies to determine the validity domain of the required single-scattering approximation and hence the limit on crystal thickness. The effects of erroneous experimental structure factor amplitudes on the charge density map for lysozyme are noted and their threshold limits calculated. The maximum thickness of lysozyme permissible under the single-scattering approximation is also estimated using R-factor analysis. Successful reconstruction of density maps is found to result mainly from the use of the phase information provided by modeling based on the protein data base through molecular replacement (MR), which dominates the effect of poor quality electron diffraction data at thicknesses larger than about 200 Å. For perfectly ordered protein nanocrystals, a maximum thickness of about 1000 Å is predicted at 200 keV if MR can be used, using R-factor analysis performed over a subset of the simulated diffracted beams. The effects of crystal bending, mosaicity (which has recently been directly imaged by cryo-EM) and secondary scattering are discussed. Structure-independent tests for single-scattering and new microfluidic methods for growing and sorting nanocrystals by size are reviewed.

    AB - The maximum thickness permissible within the single-scattering approximation for the determination of the structure of perfectly ordered protein microcrystals by transmission electron diffraction is estimated for tetragonal hen-egg lysozyme protein crystals using several approaches. Multislice simulations are performed for many diffraction conditions and beam energies to determine the validity domain of the required single-scattering approximation and hence the limit on crystal thickness. The effects of erroneous experimental structure factor amplitudes on the charge density map for lysozyme are noted and their threshold limits calculated. The maximum thickness of lysozyme permissible under the single-scattering approximation is also estimated using R-factor analysis. Successful reconstruction of density maps is found to result mainly from the use of the phase information provided by modeling based on the protein data base through molecular replacement (MR), which dominates the effect of poor quality electron diffraction data at thicknesses larger than about 200 Å. For perfectly ordered protein nanocrystals, a maximum thickness of about 1000 Å is predicted at 200 keV if MR can be used, using R-factor analysis performed over a subset of the simulated diffracted beams. The effects of crystal bending, mosaicity (which has recently been directly imaged by cryo-EM) and secondary scattering are discussed. Structure-independent tests for single-scattering and new microfluidic methods for growing and sorting nanocrystals by size are reviewed.

    KW - Charge density map

    KW - Electron diffraction

    KW - Lysozyme

    KW - Multiple scattering

    KW - Protein crystallography

    KW - R-factor

    UR - http://www.scopus.com/inward/record.url?scp=84908434486&partnerID=8YFLogxK

    UR - http://www.scopus.com/inward/citedby.url?scp=84908434486&partnerID=8YFLogxK

    U2 - 10.1016/j.ultramic.2014.08.013

    DO - 10.1016/j.ultramic.2014.08.013

    M3 - Article

    VL - 148

    SP - 87

    EP - 93

    JO - Ultramicroscopy

    JF - Ultramicroscopy

    SN - 0304-3991

    ER -