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Title: In cellulo structure determination of a novel cypovirus polyhedrin

Abstract

The crystal structure of a previously unsolved type of cypovirus polyhedrin has been determined from data collected directly from frozen live insect cells. This work demonstrates that with the use of a microfocus synchrotron beam the structure of a novel viral polyhedrin could be successfully determined from microcrystals within cells, removing the preparatory step of sample isolation and maintaining a favourable biological environment. The data obtained are of high quality, comparable to that obtained from isolated crystals, and enabled a facile structure determination. A small but significant difference is observed between the unit-cell parameters and the mosaic spread of in cellulo and isolated crystals, suggesting that even these robust crystals are adversely affected by removal from the cell.

Authors:
 [1];  [2];  [1];  [3];  [2];  [3]
  1. Diamond Light Source Ltd, Harwell Oxford, Didcot OX11 0DE (United Kingdom)
  2. University of Oxford, Oxford OX3 7BN (United Kingdom)
  3. (United Kingdom)
Publication Date:
OSTI Identifier:
22347799
Resource Type:
Journal Article
Resource Relation:
Journal Name: Acta Crystallographica. Section D: Biological Crystallography; Journal Volume: 70; Journal Issue: Pt 5; Other Information: PMCID: PMC4014125; PMID: 24816111; PUBLISHER-ID: wa5065; OAI: oai:pubmedcentral.nih.gov:4014125; Copyright (c) Axford et al. 2014; This is an open-access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited.; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
Denmark
Language:
English
Subject:
75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND SUPERFLUIDITY; BEAMS; CRYSTAL STRUCTURE; CRYSTALS; ENVIRONMENT; IRON; SYNCHROTRONS

Citation Formats

Axford, Danny, Ji, Xiaoyun, Stuart, David I., University of Oxford, Oxford OX3 7BN, Sutton, Geoff, E-mail: geoff@strubi.ox.ac.uk, and Diamond Light Source Ltd, Harwell Oxford, Didcot OX11 0DE. In cellulo structure determination of a novel cypovirus polyhedrin. Denmark: N. p., 2014. Web. doi:10.1107/S1399004714004714.
Axford, Danny, Ji, Xiaoyun, Stuart, David I., University of Oxford, Oxford OX3 7BN, Sutton, Geoff, E-mail: geoff@strubi.ox.ac.uk, & Diamond Light Source Ltd, Harwell Oxford, Didcot OX11 0DE. In cellulo structure determination of a novel cypovirus polyhedrin. Denmark. doi:10.1107/S1399004714004714.
Axford, Danny, Ji, Xiaoyun, Stuart, David I., University of Oxford, Oxford OX3 7BN, Sutton, Geoff, E-mail: geoff@strubi.ox.ac.uk, and Diamond Light Source Ltd, Harwell Oxford, Didcot OX11 0DE. Thu . "In cellulo structure determination of a novel cypovirus polyhedrin". Denmark. doi:10.1107/S1399004714004714.
@article{osti_22347799,
title = {In cellulo structure determination of a novel cypovirus polyhedrin},
author = {Axford, Danny and Ji, Xiaoyun and Stuart, David I. and University of Oxford, Oxford OX3 7BN and Sutton, Geoff, E-mail: geoff@strubi.ox.ac.uk and Diamond Light Source Ltd, Harwell Oxford, Didcot OX11 0DE},
abstractNote = {The crystal structure of a previously unsolved type of cypovirus polyhedrin has been determined from data collected directly from frozen live insect cells. This work demonstrates that with the use of a microfocus synchrotron beam the structure of a novel viral polyhedrin could be successfully determined from microcrystals within cells, removing the preparatory step of sample isolation and maintaining a favourable biological environment. The data obtained are of high quality, comparable to that obtained from isolated crystals, and enabled a facile structure determination. A small but significant difference is observed between the unit-cell parameters and the mosaic spread of in cellulo and isolated crystals, suggesting that even these robust crystals are adversely affected by removal from the cell.},
doi = {10.1107/S1399004714004714},
journal = {Acta Crystallographica. Section D: Biological Crystallography},
number = Pt 5,
volume = 70,
place = {Denmark},
year = {Thu May 01 00:00:00 EDT 2014},
month = {Thu May 01 00:00:00 EDT 2014}
}
  • 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 proteinmore » 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.« less
  • The segments 10 (S10) of the 11 double stranded RNA genomes from Antheraea mylitta cytoplasmic polyhedrosis virus (AmCPV) encoding a novel polyhedrin polypeptide was converted to cDNA, cloned, and sequenced. Three cDNA clones consisting of 1502 (AmCPV10-1), 1120 (AmCPV10-2), and 1415 (AmCPV10-3) nucleotides encoding polyhedrin of 254, 339, and 319 amino acids with molecular masses of 29, 39, and 37 kDa, respectively, were obtained, and verified by Northern analysis. These clones showed 70-94% sequence identity among them but none with any sequences in databases. The expression of AmCPV10-1 cDNA encoded polyhedrin in Sf-9 cells was detected by immunoblot analysis andmore » formation of polyhedra by electron microscopy, as observed in AmCPV-infected gut cells, but no expression of AmCPV10-2 or AmCPV10-3 cDNA was detected, indicating that during AmCPV replication, along with functional S10 RNA, some defective variant forms of S10 RNAs are packaged in virion particles.« less
  • Polyhedrin (polh) and p10 are the two hyper-expressed very late genes of nucleopolyhedroviruses. Alpha amanitin resistant transcription from Spodoptera litura nucleopolyhedrovirus (SpltNPV-I) polyhedrin promoter was observed with virus infected nuclear extract of NIV-HA-197 cells but not with that from uninfected nuclear extract. Anti-protein kinase-1 (pk1) antibody inhibited the transcription and the inhibition reversed on addition of pk1, however, pk1 mutant protein, K50M having no phosphorylation activity did not overcome the transcription inhibition. Chromatin immuno-precipitation assays with viral anti-pk1 antibody showed the interaction of pk1 with the polh while electrophoretic mobility shift assays indicated the strong binding affinity (K {sub d}more » {approx} 5.5 x 10{sup -11}) of purified pk1 with the polh promoter. These results suggested that the viral coded pk1 acts as a transcription factor in transcribing baculovirus very late genes.« less
  • A new mixed valence molybdate, La{sub 7}Mo{sub 7}O{sub 30}, first prepared by high energy ball milling, has been successfully synthesized by controlled hydrogen reduction of La{sub 2}Mo{sub 2}O{sub 9}. Its original crystal structure was determined from X-ray and neutron powder diffraction (space group R{sub 3}; a = b = 17.0051(2) {angstrom}, c = 6.8607(1) {angstrom}; Z = 3; reliability factors: R{sub p} = 0.081, R{sub wp} = 0.091, {chi}{sup 2} = 3.1, R{sub Bragg} = 0.049, R{sub F} = 0.033). It consists in the hexagonal stacking of individual cylinders of perovskite-type arrangement. These cylinders are built up from perovskite cagesmore » sharing corners in trans-position along their diagonal axis. Two different mixed-valence molybdenum sites coexist, with more (Mo{sup +5.75}) or less (Mo{sup +4.5}) distorted octahedral environments. Lanthanum atoms are located within the perovskite cages and around them, very close to their regular positions in the perovskite structure. Lanthanum and molybdenum atoms thus form two rows of almost perfect cubes, shifted from each other by c/2. An electron microscopy study revealed the defect-free cationic and octahedral arrangements in the (a,b) plane.« less
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