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- EMDB-5892: CryoEM of C3PO assembled on ssRNA-ChemiC grids -

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Basic information

Entry
Database: EMDB / ID: EMD-5892
TitleCryoEM of C3PO assembled on ssRNA-ChemiC grids
Map dataC3PO on let7 ssRNA ChemiC grids
Sample
  • Sample: Complex 3 Promoter of RISC (C3PO) assembled on the let7 ssRNA presented by ChemiC grids
  • Protein or peptide: Component 3 Promoter of RISC
  • RNA: let7 ssRNA
KeywordsNuclease activity / RNA interference / ChemiC / C3PO / side port
Function / homology
Function and homology information


endoribonuclease complex / Small interfering RNA (siRNA) biogenesis / : / regulatory ncRNA-mediated post-transcriptional gene silencing / siRNA processing / male germ cell nucleus / single-stranded DNA binding / endonuclease activity / DNA recombination / sequence-specific DNA binding ...endoribonuclease complex / Small interfering RNA (siRNA) biogenesis / : / regulatory ncRNA-mediated post-transcriptional gene silencing / siRNA processing / male germ cell nucleus / single-stranded DNA binding / endonuclease activity / DNA recombination / sequence-specific DNA binding / Hydrolases; Acting on ester bonds / mRNA binding / endoplasmic reticulum / DNA binding / RNA binding / nucleoplasm / identical protein binding / nucleus / cytosol / cytoplasm
Similarity search - Function
Translin, N-terminal / Translin, C-terminal / Translin / Translin family / Translin family / Translin superfamily / Translin family
Similarity search - Domain/homology
Biological speciesHomo sapiens (human) / unidentified (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 15.0 Å
AuthorsLlaguno MC / Xu H / Shi L / Huang H / Zhang H / Liu Q / Jiang Q
CitationJournal: J Struct Biol / Year: 2014
Title: Chemically functionalized carbon films for single molecule imaging.
Authors: Marc C Llaguno / Hui Xu / Liang Shi / Nian Huang / Hong Zhang / Qinghua Liu / Qiu-Xing Jiang /
Abstract: Many biological complexes are naturally low in abundance and pose a significant challenge to their structural and functional studies. Here we describe a new method that utilizes strong oxidation and ...Many biological complexes are naturally low in abundance and pose a significant challenge to their structural and functional studies. Here we describe a new method that utilizes strong oxidation and chemical linkage to introduce a high density of bioactive ligands onto nanometer-thick carbon films and enable selective enrichment of individual macromolecular complexes at subnanogram levels. The introduced ligands are physically separated. Ni-NTA, Protein G and DNA/RNA oligonucleotides were covalently linked to the carbon surface. They embody negligible mass and their stability makes the functionalized films able to survive long-term storage and tolerate variations in pH, temperature, salts, detergents, and solvents. We demonstrated the application of the new method to the electron microscopic imaging of the substrate-bound C3PO, an RNA-processing enzyme important for the RNA interference pathway. On the ssRNA-linked carbon surface, the formation of C3PO oligomers at subnanomolar concentrations likely mimics their assembly onto ssRNA substrates presented by their native partners. Interestingly, the 3D reconstructions by negative stain EM reveal a side port in the C3PO/ssRNA complex, and the 15Å cryoEM map showed extra density right above the side port, which probably represents the ssRNA. These results suggest a new way for ssRNAs to interact with the active sites of the complex. Together our data demonstrate that the surface-engineered carbon films are suitable for selectively enriching low-abundance biological complexes at nanomolar level and for developing novel applications on a large number of surface-presented molecules.
History
DepositionJan 29, 2014-
Header (metadata) releaseMar 19, 2014-
Map releaseMar 19, 2014-
UpdateMar 19, 2014-
Current statusMar 19, 2014Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.068
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.068
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

400_5892.gif

Downloads & links

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Map

FileDownload / File: emd_5892.map.gz / Format: CCP4 / Size: 3.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationC3PO on let7 ssRNA ChemiC grids
Voxel sizeX=Y=Z: 2.33 Å
Density
Contour LevelBy AUTHOR: 0.068 / Movie #1: 0.068
Minimum - Maximum-0.41981968 - 1.28919888
Average (Standard dev.)0.00334152 (±0.06032538)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions969696
Spacing969696
CellA=B=C: 223.68 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.332.332.33
M x/y/z969696
origin x/y/z0.0000.0000.000
length x/y/z223.680223.680223.680
α/β/γ90.00090.00090.000
start NX/NY/NZ-95-75153
NX/NY/NZ200200200
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS969696
D min/max/mean-0.4201.2890.003

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Supplemental data

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Sample components

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Entire : Complex 3 Promoter of RISC (C3PO) assembled on the let7 ssRNA pre...

EntireName: Complex 3 Promoter of RISC (C3PO) assembled on the let7 ssRNA presented by ChemiC grids
Components
  • Sample: Complex 3 Promoter of RISC (C3PO) assembled on the let7 ssRNA presented by ChemiC grids
  • Protein or peptide: Component 3 Promoter of RISC
  • RNA: let7 ssRNA

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Supramolecule #1000: Complex 3 Promoter of RISC (C3PO) assembled on the let7 ssRNA pre...

SupramoleculeName: Complex 3 Promoter of RISC (C3PO) assembled on the let7 ssRNA presented by ChemiC grids
type: sample / ID: 1000 / Details: The sample was monodisperse. / Oligomeric state: One C3PO octamer binds to one let7 ssRNA / Number unique components: 2
Molecular weightExperimental: 240 KDa / Theoretical: 240 KDa / Method: Gel filtration and ultra-centrifugation

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Macromolecule #1: Component 3 Promoter of RISC

MacromoleculeName: Component 3 Promoter of RISC / type: protein_or_peptide / ID: 1 / Name.synonym: C3PO
Details: C3PO was incubated on let7 ssRNA presented on ChemiC films.
Number of copies: 1 / Oligomeric state: Octamer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Location in cell: cytoplasm
Molecular weightExperimental: 240 KDa / Theoretical: 240 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21(DE3) / Recombinant plasmid: pET Duet
SequenceUniProtKB: Translin / GO: GO: 0090305, RNA binding, nucleus / InterPro: Translin family

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Macromolecule #2: let7 ssRNA

MacromoleculeName: let7 ssRNA / type: rna / ID: 2 / Classification: OTHER / Structure: SINGLE STRANDED / Synthetic?: Yes
Source (natural)Organism: unidentified (others)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.01 mg/mL
BufferpH: 8 / Details: 150 mM NaCl, 10 mM Tris-HCl
GridDetails: 400 mesh copper grid with thin carbon with covalently bound let7 ssRNA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 77 K / Instrument: FEI VITROBOT MARK III / Method: blot for 7 seconds before plunging

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Electron microscopy

MicroscopeJEOL 2200FS
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 61198 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 60000
Specialist opticsEnergy filter - Name: JEOL Omega type / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 25.0 eV
Sample stageSpecimen holder: Liquid nitrogen cooled / Specimen holder model: GATAN LIQUID NITROGEN
TemperatureMin: 77 K / Max: 110 K / Average: 100 K
Alignment procedureLegacy - Astigmatism: Astigmatism was corrected at 100,000 times magnification.
Detailsweak exposure, 12 min developing, 5 min quick fixing
DateSep 4, 2012
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 14 µm / Number real images: 294 / Average electron dose: 20 e/Å2 / Details: film images selected in a diffractometer / Od range: 1.2 / Bits/pixel: 8

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Image processing

CTF correctionDetails: each particle
Final two d classificationNumber classes: 1081
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 15.0 Å / Resolution method: OTHER / Software - Name: IMAGIC, SPIDER, EMAN / Number images used: 38968
DetailsThe particles were selected manually using the EMAN boxer program.

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