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- EMDB-12093: Higher-order structures of the foot-and-mouth disease virus RNA-d... -

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Basic information

Entry
Database: EMDB / ID: EMD-12093
TitleHigher-order structures of the foot-and-mouth disease virus RNA-dependent RNA polymerase required for dynamic inter-molecular interactions involved in viral genome replication
Map dataHelical reconstruction of fibril formed of foot and mouth disease virus RNA dependent RNA polymerase 3Dpol
Sample
  • Complex: Helical fibril of foot and mouth disease virus RNA dependent RNA polymerase (3Dpol)
Function / homology
Function and homology information


modulation by virus of host chromatin organization / ribonucleoside triphosphate phosphatase activity / T=pseudo3 icosahedral viral capsid / host cell cytoplasmic vesicle membrane / cytoplasmic vesicle membrane / : / regulation of translation / protein complex oligomerization / monoatomic ion channel activity / clathrin-dependent endocytosis of virus by host cell ...modulation by virus of host chromatin organization / ribonucleoside triphosphate phosphatase activity / T=pseudo3 icosahedral viral capsid / host cell cytoplasmic vesicle membrane / cytoplasmic vesicle membrane / : / regulation of translation / protein complex oligomerization / monoatomic ion channel activity / clathrin-dependent endocytosis of virus by host cell / RNA helicase activity / viral protein processing / viral RNA genome replication / cysteine-type endopeptidase activity / RNA-dependent RNA polymerase activity / DNA-templated transcription / virion attachment to host cell / structural molecule activity / proteolysis / RNA binding / ATP binding
Similarity search - Function
Aphthovirus leader protease (L(pro)) domain profile. / Peptidase C28, foot-and-mouth virus L-proteinase / Foot-and-mouth virus L-proteinase / Foot-and-mouth disease virus VP1 coat / Capsid protein VP4, Picornavirus / Viral protein VP4 subunit / Capsid protein VP4 superfamily, Picornavirus / Helicase/polymerase/peptidase polyprotein, Calicivirus-type / Picornavirales 3C/3C-like protease domain / Picornavirales 3C/3C-like protease domain profile. ...Aphthovirus leader protease (L(pro)) domain profile. / Peptidase C28, foot-and-mouth virus L-proteinase / Foot-and-mouth virus L-proteinase / Foot-and-mouth disease virus VP1 coat / Capsid protein VP4, Picornavirus / Viral protein VP4 subunit / Capsid protein VP4 superfamily, Picornavirus / Helicase/polymerase/peptidase polyprotein, Calicivirus-type / Picornavirales 3C/3C-like protease domain / Picornavirales 3C/3C-like protease domain profile. / Peptidase C3A/C3B, picornaviral / 3C cysteine protease (picornain 3C) / Picornavirus capsid / picornavirus capsid protein / Helicase, superfamily 3, single-stranded RNA virus / Superfamily 3 helicase of positive ssRNA viruses domain profile. / Helicase, superfamily 3, single-stranded DNA/RNA virus / RNA helicase / Papain-like cysteine peptidase superfamily / Picornavirus/Calicivirus coat protein / Viral coat protein subunit / RNA-directed RNA polymerase, C-terminal domain / Viral RNA-dependent RNA polymerase / Reverse transcriptase/Diguanylate cyclase domain / RNA-directed RNA polymerase, catalytic domain / RdRp of positive ssRNA viruses catalytic domain profile. / Peptidase S1, PA clan, chymotrypsin-like fold / Peptidase S1, PA clan / DNA/RNA polymerase superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Biological speciesFoot-and-mouth disease virus
Methodhelical reconstruction / cryo EM / Resolution: 7.3 Å
AuthorsLoundras EA / Streetley J / Herod MJ / Thompson R / Harris M / Bhella D / Stonehouse NJ
Funding support United Kingdom, 7 items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MC_UU_12014/7 United Kingdom
Medical Research Council (MRC, United Kingdom)MR/M000451/1 United Kingdom
Biotechnology and Biological Sciences Research Council (BBSRC)BB/F01614X/1 United Kingdom
Biotechnology and Biological Sciences Research Council (BBSRC)BB/K003801/1 United Kingdom
Biotechnology and Biological Sciences Research Council (BBSRC)BB/P001459/1 United Kingdom
Wellcome Trust108466/Z/15/Z United Kingdom
Wellcome Trust096670/Z/11/Z United Kingdom
CitationJournal: Commun Biol / Year: 2022
Title: Higher-order structures of the foot-and-mouth disease virus RNA-dependent RNA polymerase required for genome replication.
Authors: Eleni-Anna Loundras / James Streetley / Morgan R Herod / Rebecca Thompson / Mark Harris / David Bhella / Nicola J Stonehouse /
Abstract: Replication of many positive-sense RNA viruses occurs within intracellular membrane-associated compartments. These are thought to provide a favourable environment for replication to occur, ...Replication of many positive-sense RNA viruses occurs within intracellular membrane-associated compartments. These are thought to provide a favourable environment for replication to occur, concentrating essential viral structural and nonstructural components, as well as protecting these components from host-cell pathogen recognition and innate immune responses. However, the details of the molecular interactions and dynamics within these structures is very limited. One of the key components of the replication machinery is the RNA-dependent RNA polymerase, RdRp. This enzyme has been shown to form higher-order fibrils in vitro. Here, using the RdRp from foot-and-mouth disease virus (termed 3D), we report fibril structures, solved at ~7-9 Å resolution by cryo-EM, revealing multiple conformations of a flexible assembly. Fitting high-resolution coordinates led to the definition of potential intermolecular interactions. We employed mutagenesis using a sub-genomic replicon system to probe the importance of these interactions for replication. We use these data to propose models for the role of higher-order 3D complexes as a dynamic scaffold within which RNA replication can occur.
History
DepositionDec 14, 2020-
Header (metadata) releaseNov 24, 2021-
Map releaseNov 24, 2021-
UpdateFeb 2, 2022-
Current statusFeb 2, 2022Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12093.png

Downloads & links

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Map

FileDownload / File: emd_12093.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationHelical reconstruction of fibril formed of foot and mouth disease virus RNA dependent RNA polymerase 3Dpol
Voxel sizeX=Y=Z: 2.13 Å
Density
Contour LevelBy AUTHOR: 0.05 / Movie #1: 0.05
Minimum - Maximum-0.082748726 - 0.22391324
Average (Standard dev.)0.00092561665 (±0.011186446)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 426.00003 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.132.132.13
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z426.000426.000426.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-0.0830.2240.001

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Supplemental data

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Sample components

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Entire : Helical fibril of foot and mouth disease virus RNA dependent RNA ...

EntireName: Helical fibril of foot and mouth disease virus RNA dependent RNA polymerase (3Dpol)
Components
  • Complex: Helical fibril of foot and mouth disease virus RNA dependent RNA polymerase (3Dpol)

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Supramolecule #1: Helical fibril of foot and mouth disease virus RNA dependent RNA ...

SupramoleculeName: Helical fibril of foot and mouth disease virus RNA dependent RNA polymerase (3Dpol)
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Foot-and-mouth disease virus
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pET-28a

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

BufferpH: 7
GridModel: C-flat-1.2/1.3 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: Specimen was loaded onto C-flat (1.2/1.3), 400 mesh holey carbon support films bearing a continuous carbon layer. Allowed to adsorb for one minute before blotting for 6-seconds and plunge freezing..
DetailsFibrils were formed by mixing FMDV 3DPol with a 13mer of poly-adenosine RNA, oligo-d(T) primer and UTP in the presence of 5mM glutaraldehyde.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.8000000000000003 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 75000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: INTEGRATING / Number grids imaged: 2 / Number real images: 5417 / Average exposure time: 2.0 sec. / Average electron dose: 108.0 e/Å2
Details: The dose rate is 60 e/pix/s, giving a dose of 54 e/a2/second. We are taking a 2 second exposure, with 79 frames in total, giving 1.4 electrons/frame
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Segment selectionNumber selected: 266263 / Software - Name: RELION (ver. 3.0)
CTF correctionSoftware - Name: Gctf
Details: Defocus estimation was performed using GCTF and corrected in RELION
Startup modelType of model: NONE
Details: Initial model generated from 2D classes using SPIDER
Final angle assignmentType: NOT APPLICABLE / Software - Name: RELION (ver. 3.1)
Final reconstructionNumber classes used: 1
Applied symmetry - Helical parameters - Δz: 26.9991 Å
Applied symmetry - Helical parameters - Δ&Phi: 39.003 °
Applied symmetry - Helical parameters - Axial symmetry: C2 (2 fold cyclic)
Resolution.type: BY AUTHOR / Resolution: 7.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1)
Details: Maps had 2-fold symmetry that was not incorporated into the reconstruction strategy. Half-maps were subjected to real-space averaging using UCSF chimera to align and average volumes ...Details: Maps had 2-fold symmetry that was not incorporated into the reconstruction strategy. Half-maps were subjected to real-space averaging using UCSF chimera to align and average volumes following rotation about the x-axis.
Number images used: 38886
DetailsImages were motion corrected using MotionCor2
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

2ec0

RefinementProtocol: RIGID BODY FIT

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