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Structure of CPV17 polyhedrin determined by the improved analysis of serial femtosecond crystallographic data.

Ginn HM, Messerschmidt M, Ji X, Zhang H, Axford D, Gildea RJ, Winter G, Brewster AS, Hattne J, Wagner A, Grimes JM, Evans G, Sauter NK, Sutton G, Stuart DI - Nat Commun (2015)

Bottom Line: Here we use an XFEL to determine the room temperature atomic structure for the smallest cytoplasmic polyhedrosis virus polyhedra yet characterized, which we failed to solve at a synchrotron.These protein microcrystals, roughly a micron across, accrue within infected cells.Our improved methods should open up more challenging biological samples to XFEL analysis.

View Article: PubMed Central - PubMed

Affiliation: Division of Structural Biology, The Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford, Oxfordshire OX3 7BN, UK.

ABSTRACT
The X-ray free-electron laser (XFEL) allows the analysis of small weakly diffracting protein crystals, but has required very many crystals to obtain good data. Here we use an XFEL to determine the room temperature atomic structure for the smallest cytoplasmic polyhedrosis virus polyhedra yet characterized, which we failed to solve at a synchrotron. These protein microcrystals, roughly a micron across, accrue within infected cells. We use a new physical model for XFEL diffraction, which better estimates the experimental signal, delivering a high-resolution XFEL structure (1.75 Å), using fewer crystals than previously required for this resolution. The crystal lattice and protein core are conserved compared with a polyhedrin with less than 10% sequence identity. We explain how the conserved biological phenotype, the crystal lattice, is maintained in the face of extreme environmental challenge and massive evolutionary divergence. Our improved methods should open up more challenging biological samples to XFEL analysis.

No MeSH data available.


Related in: MedlinePlus

Structure of CPV17 polyhedrin.(a) Electron density around the ATP moiety contoured at 1.3 σ. (b) Cartoon of the CPV17 polyhedrin subunit coloured from blue to red, N terminus to C terminus that are denoted by spheres. Secondary structure elements are labelled and the ATP molecule shown as sticks. (c) Comparison of the CPV17 and CPV1 polyhedrin structures. The molecules were aligned with program SHP43. Both colour and tube thickness represent r.m.s. distance (r.m.s.d.) of equivalent C-alpha atoms (thin, blue: r.m.s.d.<1.0 Å, green, thicker: 1.0–2.5 Å, orange, thickest: >2.5 Å). Unaligned regions are coloured red and displayed with exaggerated thickness. Variable regions are labelled v1–v5 with the N- and C-terminal extensions of CPV1 drawn. (d) Comparison of variable regions between CPV17 (magenta) and CPV1 (cyan). (e) The interface between monomers (coloured differently) in the crystal is rich in tyrosines.
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f2: Structure of CPV17 polyhedrin.(a) Electron density around the ATP moiety contoured at 1.3 σ. (b) Cartoon of the CPV17 polyhedrin subunit coloured from blue to red, N terminus to C terminus that are denoted by spheres. Secondary structure elements are labelled and the ATP molecule shown as sticks. (c) Comparison of the CPV17 and CPV1 polyhedrin structures. The molecules were aligned with program SHP43. Both colour and tube thickness represent r.m.s. distance (r.m.s.d.) of equivalent C-alpha atoms (thin, blue: r.m.s.d.<1.0 Å, green, thicker: 1.0–2.5 Å, orange, thickest: >2.5 Å). Unaligned regions are coloured red and displayed with exaggerated thickness. Variable regions are labelled v1–v5 with the N- and C-terminal extensions of CPV1 drawn. (d) Comparison of variable regions between CPV17 (magenta) and CPV1 (cyan). (e) The interface between monomers (coloured differently) in the crystal is rich in tyrosines.

Mentions: The structure of CPV17 was solved by molecular replacement using the higher-resolution XFEL data. Manual rebuilding and refinement rapidly reduced Rwork/Rfree from 56.5%/54.1% to 12.2%/15.4% (Table 1). Beyond 2.0 Å resolution, at which point Rsplit exceeds 20%, the Rwork and Rfree increase significantly. However, the quality of the electron density meets that expected for a high-quality 1.75-Å resolution analysis (Supplementary Movie 1), for instance allowing the unambiguous identification of an unexpected bound adenosine triphosphate (ATP) (Fig. 2a). Using this room temperature structure the synchrotron 100 K structure was solved trivially and refined to 2.2 Å (Table 2).


Structure of CPV17 polyhedrin determined by the improved analysis of serial femtosecond crystallographic data.

Ginn HM, Messerschmidt M, Ji X, Zhang H, Axford D, Gildea RJ, Winter G, Brewster AS, Hattne J, Wagner A, Grimes JM, Evans G, Sauter NK, Sutton G, Stuart DI - Nat Commun (2015)

Structure of CPV17 polyhedrin.(a) Electron density around the ATP moiety contoured at 1.3 σ. (b) Cartoon of the CPV17 polyhedrin subunit coloured from blue to red, N terminus to C terminus that are denoted by spheres. Secondary structure elements are labelled and the ATP molecule shown as sticks. (c) Comparison of the CPV17 and CPV1 polyhedrin structures. The molecules were aligned with program SHP43. Both colour and tube thickness represent r.m.s. distance (r.m.s.d.) of equivalent C-alpha atoms (thin, blue: r.m.s.d.<1.0 Å, green, thicker: 1.0–2.5 Å, orange, thickest: >2.5 Å). Unaligned regions are coloured red and displayed with exaggerated thickness. Variable regions are labelled v1–v5 with the N- and C-terminal extensions of CPV1 drawn. (d) Comparison of variable regions between CPV17 (magenta) and CPV1 (cyan). (e) The interface between monomers (coloured differently) in the crystal is rich in tyrosines.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4403592&req=5

f2: Structure of CPV17 polyhedrin.(a) Electron density around the ATP moiety contoured at 1.3 σ. (b) Cartoon of the CPV17 polyhedrin subunit coloured from blue to red, N terminus to C terminus that are denoted by spheres. Secondary structure elements are labelled and the ATP molecule shown as sticks. (c) Comparison of the CPV17 and CPV1 polyhedrin structures. The molecules were aligned with program SHP43. Both colour and tube thickness represent r.m.s. distance (r.m.s.d.) of equivalent C-alpha atoms (thin, blue: r.m.s.d.<1.0 Å, green, thicker: 1.0–2.5 Å, orange, thickest: >2.5 Å). Unaligned regions are coloured red and displayed with exaggerated thickness. Variable regions are labelled v1–v5 with the N- and C-terminal extensions of CPV1 drawn. (d) Comparison of variable regions between CPV17 (magenta) and CPV1 (cyan). (e) The interface between monomers (coloured differently) in the crystal is rich in tyrosines.
Mentions: The structure of CPV17 was solved by molecular replacement using the higher-resolution XFEL data. Manual rebuilding and refinement rapidly reduced Rwork/Rfree from 56.5%/54.1% to 12.2%/15.4% (Table 1). Beyond 2.0 Å resolution, at which point Rsplit exceeds 20%, the Rwork and Rfree increase significantly. However, the quality of the electron density meets that expected for a high-quality 1.75-Å resolution analysis (Supplementary Movie 1), for instance allowing the unambiguous identification of an unexpected bound adenosine triphosphate (ATP) (Fig. 2a). Using this room temperature structure the synchrotron 100 K structure was solved trivially and refined to 2.2 Å (Table 2).

Bottom Line: Here we use an XFEL to determine the room temperature atomic structure for the smallest cytoplasmic polyhedrosis virus polyhedra yet characterized, which we failed to solve at a synchrotron.These protein microcrystals, roughly a micron across, accrue within infected cells.Our improved methods should open up more challenging biological samples to XFEL analysis.

View Article: PubMed Central - PubMed

Affiliation: Division of Structural Biology, The Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford, Oxfordshire OX3 7BN, UK.

ABSTRACT
The X-ray free-electron laser (XFEL) allows the analysis of small weakly diffracting protein crystals, but has required very many crystals to obtain good data. Here we use an XFEL to determine the room temperature atomic structure for the smallest cytoplasmic polyhedrosis virus polyhedra yet characterized, which we failed to solve at a synchrotron. These protein microcrystals, roughly a micron across, accrue within infected cells. We use a new physical model for XFEL diffraction, which better estimates the experimental signal, delivering a high-resolution XFEL structure (1.75 Å), using fewer crystals than previously required for this resolution. The crystal lattice and protein core are conserved compared with a polyhedrin with less than 10% sequence identity. We explain how the conserved biological phenotype, the crystal lattice, is maintained in the face of extreme environmental challenge and massive evolutionary divergence. Our improved methods should open up more challenging biological samples to XFEL analysis.

No MeSH data available.


Related in: MedlinePlus