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- EMDB-5420: Pyrococcus abyssi methylation-guide sRNP -

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Basic information

Entry
Database: EMDB / ID: EMD-5420
TitlePyrococcus abyssi methylation-guide sRNP
Map dataProjection matching single particle reconstruction of Pyrococcus abyssi box C/D sRNP, 25 Angstrom resolution
Sample
  • Sample: Pyrococcus abyssi methylation-guide sRNP
  • Protein or peptide: ribonucleoprotein
Keywordsarchaea / box C/D sRNP / single particle EM / rRNA modification / RNA-protein complex / non-coding RNA
Biological speciesPyrococcus abyssi (archaea)
Methodsingle particle reconstruction / negative staining / Resolution: 25.0 Å
AuthorsBower-Phipps KR / Taylor DW / Wang HW / Baserga SJ
CitationJournal: RNA / Year: 2012
Title: The box C/D sRNP dimeric architecture is conserved across domain Archaea.
Authors: Kathleen R Bower-Phipps / David W Taylor / Hong-Wei Wang / Susan J Baserga /
Abstract: Box C/D small (nucleolar) ribonucleoproteins [s(no)RNPs] catalyze RNA-guided 2'-O-ribose methylation in two of the three domains of life. Recent structural studies have led to a controversy over ...Box C/D small (nucleolar) ribonucleoproteins [s(no)RNPs] catalyze RNA-guided 2'-O-ribose methylation in two of the three domains of life. Recent structural studies have led to a controversy over whether box C/D sRNPs functionally assemble as monomeric or dimeric macromolecules. The archaeal box C/D sRNP from Methanococcus jannaschii (Mj) has been shown by glycerol gradient sedimentation, gel filtration chromatography, native gel analysis, and single-particle electron microscopy (EM) to adopt a di-sRNP architecture, containing four copies of each box C/D core protein and two copies of the Mj sR8 sRNA. Subsequently, investigators used a two-stranded artificial guide sRNA, CD45, to assemble a box C/D sRNP from Sulfolobus solfataricus with a short RNA methylation substrate, yielding a crystal structure of a mono-sRNP. To more closely examine box C/D sRNP architecture, we investigate the role of the omnipresent sRNA loop as a structural determinant of sRNP assembly. We show through sRNA mutagenesis, native gel electrophoresis, and single-particle EM that a di-sRNP is the near exclusive architecture obtained when reconstituting box C/D sRNPs with natural or artificial sRNAs containing an internal loop. Our results span three distantly related archaeal species--Sulfolobus solfataricus, Pyrococcus abyssi, and Archaeoglobus fulgidus--indicating that the di-sRNP architecture is broadly conserved across the entire archaeal domain.
History
DepositionMay 8, 2012-
Header (metadata) releaseJun 5, 2012-
Map releaseJul 9, 2012-
UpdateJul 9, 2012-
Current statusJul 9, 2012Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 3
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5420_1.png

Downloads & links

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Map

FileDownload / File: emd_5420.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationProjection matching single particle reconstruction of Pyrococcus abyssi box C/D sRNP, 25 Angstrom resolution
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.7 Å/pix.
x 128 pix.
= 345.6 Å		2.7 Å/pix.
x 128 pix.
= 345.6 Å		2.7 Å/pix.
x 128 pix.
= 345.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.7 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 3.0 / Movie #1: 3
Minimum - Maximum-7.35317993 - 17.610933299999999
Average (Standard dev.)0.0 (±0.99999976)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 345.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.72.72.7
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z345.600345.600345.600
α/β/γ90.00090.00090.000
start NX/NY/NZ-62-62-62
NX/NY/NZ125125125
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-7.35317.611-0.000

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Supplemental data

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Sample components

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Entire : Pyrococcus abyssi methylation-guide sRNP

EntireName: Pyrococcus abyssi methylation-guide sRNP
Components
  • Sample: Pyrococcus abyssi methylation-guide sRNP
  • Protein or peptide: ribonucleoprotein

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Supramolecule #1000: Pyrococcus abyssi methylation-guide sRNP

SupramoleculeName: Pyrococcus abyssi methylation-guide sRNP / type: sample / ID: 1000
Oligomeric state: Four copies of each box C/D core protein could be docked in this map.
Number unique components: 1
Molecular weightTheoretical: 368 KDa

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Macromolecule #1: ribonucleoprotein

MacromoleculeName: ribonucleoprotein / type: protein_or_peptide / ID: 1 / Name.synonym: box C/D sRNP / Recombinant expression: Yes / Database: NCBI
Source (natural)Organism: Pyrococcus abyssi (archaea)
Molecular weightTheoretical: 368 KDa

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5 / Details: 20mM HEPES, 500mM NaCl, 1.5mM MgCl2
StainingType: NEGATIVE / Details: 2% uranyl formate
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI 12
Detailslow-dose conditions
DateSep 8, 2011
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Average electron dose: 20 e/Å2
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal magnification: 42000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC

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Image processing

Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 25.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER / Number images used: 9248

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Atomic model buiding 1

Initial modelPDB ID:

3nmu

SoftwareName: Chimera, Situs
RefinementSpace: REAL

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