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- EMDB-1806: Cryo-electron tomography derived density map of a conserved retro... -

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Entry
Database: EMDB / ID: EMD-1806
TitleCryo-electron tomography derived density map of a conserved retroviral RNA packaging element from Moloney Murine Leukemia Virus.
Map dataThe final average of 38 subvolumes of two sets of two stem loop structures (CD2) of MoMuLV.
Sample
  • Sample: MLV Tandem Hairpin RNA
  • RNA: MLV Tandem Hairpin RNA
Keywordscryo-ET / tomography / retroviral RNA / MoMuLV / Moloney Murine Leukemia Virus / double hairpin
Biological speciesMoloney murine leukemia virus
Methodsubtomogram averaging / cryo EM
AuthorsMiyazaki Y / Irobalieva RN / Tolbert B / Smalls-Mantey A / Iyalla K / Loeliger K / DSouza V / Khant H / Schmid MF / Garcia E ...Miyazaki Y / Irobalieva RN / Tolbert B / Smalls-Mantey A / Iyalla K / Loeliger K / DSouza V / Khant H / Schmid MF / Garcia E / Telesnitsky A / Chiu W / Summers MF
CitationJournal: J Mol Biol / Year: 2010
Title: Structure of a conserved retroviral RNA packaging element by NMR spectroscopy and cryo-electron tomography.
Authors: Yasuyuki Miyazaki / Rossitza N Irobalieva / Blanton S Tolbert / Adjoa Smalls-Mantey / Kilali Iyalla / Kelsey Loeliger / Victoria D'Souza / Htet Khant / Michael F Schmid / Eric L Garcia / ...Authors: Yasuyuki Miyazaki / Rossitza N Irobalieva / Blanton S Tolbert / Adjoa Smalls-Mantey / Kilali Iyalla / Kelsey Loeliger / Victoria D'Souza / Htet Khant / Michael F Schmid / Eric L Garcia / Alice Telesnitsky / Wah Chiu / Michael F Summers /
Abstract: The 5'-untranslated regions of all gammaretroviruses contain a conserved "double-hairpin motif" (Ψ(CD)) that is required for genome packaging. Both hairpins (SL-C and SL-D) contain GACG tetraloops ...The 5'-untranslated regions of all gammaretroviruses contain a conserved "double-hairpin motif" (Ψ(CD)) that is required for genome packaging. Both hairpins (SL-C and SL-D) contain GACG tetraloops that, in isolated RNAs, are capable of forming "kissing" interactions stabilized by two intermolecular G-C base pairs. We have determined the three-dimensional structure of the double hairpin from the Moloney murine leukemia virus ([Ψ(CD)](2), 132 nt, 42.8 kDa) using a (2)H-edited NMR-spectroscopy-based approach. This approach enabled the detection of (1)H-(1)H dipolar interactions that were not observed in previous studies of isolated SL-C and SL-D hairpin RNAs using traditional (1)H-(1)H correlated and (1)H-(13)C-edited NMR methods. The hairpins participate in intermolecular cross-kissing interactions (SL-C to SL-D' and SLC' to SL-D) and stack in an end-to-end manner (SL-C to SL-D and SL-C' to SL-D') that gives rise to an elongated overall shape (ca 95 Å×45 Å×25 Å). The global structure was confirmed by cryo-electron tomography (cryo-ET), making [Ψ(CD)](2) simultaneously the smallest RNA to be structurally characterized to date by cryo-ET and among the largest to be determined by NMR. Our findings suggest that, in addition to promoting dimerization, [Ψ(CD)](2) functions as a scaffold that helps initiate virus assembly by exposing a cluster of conserved UCUG elements for binding to the cognate nucleocapsid domains of assembling viral Gag proteins.
History
DepositionOct 18, 2010-
Header (metadata) releaseJan 14, 2011-
Map releaseJan 14, 2011-
UpdateJan 14, 2011-
Current statusJan 14, 2011Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 8
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 8
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1806.png

Downloads & links

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Map

FileDownload / File: emd_1806.map.gz / Format: CCP4 / Size: 1001 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThe final average of 38 subvolumes of two sets of two stem loop structures (CD2) of MoMuLV.
Voxel sizeX=Y=Z: 6.487 Å
Density
Contour LevelBy AUTHOR: 8.0 / Movie #1: 8
Minimum - Maximum-8.041969999999999 - 25.148700000000002
Average (Standard dev.)0.00000000256321 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-35-31-32
Dimensions646464
Spacing646464
CellA=B=C: 415.168 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z6.4876.4876.487
M x/y/z646464
origin x/y/z0.0000.0000.000
length x/y/z415.168415.168415.168
α/β/γ90.00090.00090.000
start NX/NY/NZ-150-150-149
NX/NY/NZ300300300
MAP C/R/S123
start NC/NR/NS-31-35-32
NC/NR/NS646464
D min/max/mean-8.04225.1490.000

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Supplemental data

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Sample components

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Entire : MLV Tandem Hairpin RNA

EntireName: MLV Tandem Hairpin RNA
Components
  • Sample: MLV Tandem Hairpin RNA
  • RNA: MLV Tandem Hairpin RNA

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Supramolecule #1000: MLV Tandem Hairpin RNA

SupramoleculeName: MLV Tandem Hairpin RNA / type: sample / ID: 1000
Details: RNA synthesized by in vitro transcription and purified by polyacrylamide gel electrophoresis. Sequence of the RNA confirmed by NMR.
Oligomeric state: Homodimer / Number unique components: 1
Molecular weightTheoretical: 42.8 KDa / Method: Not determined but identity confirmed by NMR

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Macromolecule #1: MLV Tandem Hairpin RNA

MacromoleculeName: MLV Tandem Hairpin RNA / type: rna / ID: 1 / Name.synonym: MLV Tandem Hairpin RNA / Classification: OTHER / Structure: SINGLE STRANDED / Synthetic?: No
Source (natural)Organism: Moloney murine leukemia virus / synonym: Moloney Murine Leukemia Virus
Molecular weightTheoretical: 42.8 KDa
SequenceString:
GGCGGACCCG UGGUGGAACA GACGUGUUCG GAACACCCGG CCGCAACCCU GGGAGACGUC CCAGGG

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging

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Sample preparation

Concentration1.03 mg/mL
BufferpH: 7
Details: Tris buffer, pH 7.0, containing 140 mM KCl and 2 mM MgCl2
GridDetails: 200 mesh gold grid
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK III / Details: Vitrification instrument: Vitrobot Mark III / Method: 1 blot, 1 second

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Electron microscopy

MicroscopeJEOL 2200FSC
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 23123 / Illumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal magnification: 20000
Specialist opticsEnergy filter - Name: In column Omega-type filter
Sample stageSpecimen holder: Gatan 70 degree holder / Specimen holder model: GATAN LIQUID NITROGEN / Tilt series - Axis1 - Min angle: -60 ° / Tilt series - Axis1 - Max angle: 60 °
TemperatureMin: 98 K / Max: 98 K / Average: 98 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 100,000 times magnification
Image recordingCategory: CCD / Film or detector model: GENERIC GATAN (4k x 4k) / Average electron dose: 85 e/Å2

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Image processing

Final reconstructionAlgorithm: OTHER / Software - Name: IMOD / Details: Final map was an average of 38 subvolumes.
DetailsTomogram was reconstructed using IMOD. Average number of tilts used in the 3D reconstructions: 60. Average tomographic tilt angle increment: 2.

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Atomic model buiding 1

Initial modelPDB ID:

2l1f

SoftwareName: Chimera, X-plor
DetailsProtocol: Distance geometry refinement with the Cyana software package using NMR-derived restraints. The cryo-ET data were not employed as refinement restraints. The determined NMR structure (2LIF) was fitted into the cryo-ET density map using Chimera and X-plor.
RefinementSpace: REAL
Target criteria: Lowest target functions, equals sum of the square of the distance and angle restraint violations

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