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- EMDB-8146: Methanococcus jannaschii box C/D sRNP -

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Basic information

Entry
Database: EMDB / ID: EMD-8146
TitleMethanococcus jannaschii box C/D sRNP
Map dataNone
Sample
  • Complex: Methanococcus janaschii box C/D sRNP
    • Protein or peptide: L7Ae
    • Protein or peptide: Nop5
    • Protein or peptide: Fibrillarin
    • RNA: Methanococcus jannaschii sR8 box C/D sRNA
Biological speciesMethanocaldococcus jannaschii (archaea)
Methodsingle particle reconstruction / cryo EM / Resolution: 9.0 Å
AuthorsYip WSV / Shigematsu H / Taylor DW / Baserga SJ
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM115710 United States
CitationJournal: Nucleic Acids Res / Year: 2016
Title: Box C/D sRNA stem ends act as stabilizing anchors for box C/D di-sRNPs.
Authors: W S Vincent Yip / Hideki Shigematsu / David W Taylor / Susan J Baserga /
Abstract: Ribosomal RNA (rRNA) modifications are essential for ribosome function in all cellular organisms. Box C/D small (nucleolar) ribonucleoproteins [s(no)RNPs] catalyze 2'-O-methylation, one rRNA ...Ribosomal RNA (rRNA) modifications are essential for ribosome function in all cellular organisms. Box C/D small (nucleolar) ribonucleoproteins [s(no)RNPs] catalyze 2'-O-methylation, one rRNA modification type in Eukarya and Archaea. Negatively stained electron microscopy (EM) models of archaeal box C/D sRNPs have demonstrated the dimeric sRNP (di-sRNP) architecture, which has been corroborated by nuclear magnetic resonance (NMR) studies. Due to limitations of the structural techniques, the orientation of the box C/D sRNAs has remained unclear. Here, we have used cryo-EM to elucidate the sRNA orientation in a M. jannaschii box C/D di-sRNP. The cryo-EM reconstruction suggests a parallel orientation of the two sRNAs. Biochemical and structural analyses of sRNPs assembled with mutant sRNAs indicate a potential interaction between the sRNA stem ends. Our results suggest that the parallel arrangement of the sRNAs juxtaposes their stem ends into close proximity to allow for a stabilizing interaction that helps maintain the di-sRNP architecture.
History
DepositionApr 13, 2016-
Header (metadata) releaseJul 6, 2016-
Map releaseJul 6, 2016-
UpdateJan 29, 2020-
Current statusJan 29, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0159
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0159
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8146.png

Downloads & links

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Map

FileDownload / File: emd_8146.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNone
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.25 Å/pix.
x 288 pix.
= 359.136 Å		1.25 Å/pix.
x 288 pix.
= 359.136 Å		1.25 Å/pix.
x 288 pix.
= 359.136 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.247 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0159 / Movie #1: 0.0159
Minimum - Maximum-0.023278335 - 0.058725648
Average (Standard dev.)0.00010509183 (±0.002525227)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-144-144-144
Dimensions288288288
Spacing288288288
CellA=B=C: 359.136 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.2471.2471.247
M x/y/z288288288
origin x/y/z0.0000.0000.000
length x/y/z359.136359.136359.136
α/β/γ90.00090.00090.000
start NX/NY/NZ-38-19-20
NX/NY/NZ858082
MAP C/R/S123
start NC/NR/NS-144-144-144
NC/NR/NS288288288
D min/max/mean-0.0230.0590.000

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Supplemental data

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Sample components

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Entire : Methanococcus janaschii box C/D sRNP

EntireName: Methanococcus janaschii box C/D sRNP
Components
  • Complex: Methanococcus janaschii box C/D sRNP
    • Protein or peptide: L7Ae
    • Protein or peptide: Nop5
    • Protein or peptide: Fibrillarin
    • RNA: Methanococcus jannaschii sR8 box C/D sRNA

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Supramolecule #1: Methanococcus janaschii box C/D sRNP

SupramoleculeName: Methanococcus janaschii box C/D sRNP / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Methanocaldococcus jannaschii (archaea)
Molecular weightTheoretical: 366 KDa

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Macromolecule #1: L7Ae

MacromoleculeName: L7Ae / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Methanocaldococcus jannaschii (archaea)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MAVYVKFKVP EEIQKELLDA VAKAQKIKKG ANEVTKAVER GIAKLVIIAE DVKPEEVVAH LPYLCEEKGI PYAYVASKQD LGKAAGLEVA ASSVAIINEG DAEELKVLIE KVNVLKQ

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Macromolecule #2: Nop5

MacromoleculeName: Nop5 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Methanocaldococcus jannaschii (archaea)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MIYVTFTPYG AFGVKDNKEV SGLEDIEYKK LFNEEEIPDI MFKLKTQPNK IADELKEEWG DEIKLETLST EPFNIGEFLR NNLFKVGKEL GYFNNYDEFR KKMHYWSTEL TKKVIKSYAQ QKDKIIIQVA EAISDLDKTL NLLSERLREW YSLYFPELDH LVNKHEVYAN ...String:
MIYVTFTPYG AFGVKDNKEV SGLEDIEYKK LFNEEEIPDI MFKLKTQPNK IADELKEEWG DEIKLETLST EPFNIGEFLR NNLFKVGKEL GYFNNYDEFR KKMHYWSTEL TKKVIKSYAQ QKDKIIIQVA EAISDLDKTL NLLSERLREW YSLYFPELDH LVNKHEVYAN LITKLGKRKN FTKSQLKKIL PSKLAGKIAE AAKNSMGGEL EDYDLDVIVK FAEEINHLYE KRKELYNYLE KLMNEEAPNI TKLAGVSLGA RLIGLAGGLE KLAKMPASTI QVLGAEKALF AHLRMGVEPP KHGIIYNHPL IQGSPHWQRG KIARALACKL AIAARADYVG DYIADELLEK LNKRVEEIRR KYPKPPK

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Macromolecule #3: Fibrillarin

MacromoleculeName: Fibrillarin / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Methanocaldococcus jannaschii (archaea)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MEDIKIKEIF ENIYEVDLGD GLKRIATKSI VKGKKVYDEK IIKIGDEEYR IWNPNKSKLA AAIIKGLKVM PIKRDSKILY LGASAGTTPS HVADIADKGI VYAIEYAPRI MRELLDACAE RENIIPILGD ANKPQEYANI VEKVDVIYED VAQPNQAEIL IKNAKWFLKK ...String:
MEDIKIKEIF ENIYEVDLGD GLKRIATKSI VKGKKVYDEK IIKIGDEEYR IWNPNKSKLA AAIIKGLKVM PIKRDSKILY LGASAGTTPS HVADIADKGI VYAIEYAPRI MRELLDACAE RENIIPILGD ANKPQEYANI VEKVDVIYED VAQPNQAEIL IKNAKWFLKK GGYGMIAIKA RSIDVTKDPK EIFKEQKEIL EAGGFKIVDE VDIEPFEKDH VMFVGIWEGK

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Macromolecule #4: Methanococcus jannaschii sR8 box C/D sRNA

MacromoleculeName: Methanococcus jannaschii sR8 box C/D sRNA / type: rna / ID: 4
SequenceString:
AAAUCGCCAA UGAUGACGAU UGGCUUUGCU GAGUCUGUGA UGAACCGUAU GAGCACUGAG GCGAUUU

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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

BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHEPES
300.0 mMNaClSodium chloridesodium chloride
1.5 mMMgCl2magnesium chloride
GridModel: Electron Microscopy Sciences / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 281 K / Instrument: FEI VITROBOT MARK III
Details: Blotting time 5 seconds; Blot offset -1 mm; plunged into liquid ethane (FEI VITROBOT MARK III)..

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 2.5 µm / Nominal magnification: 29000
Sample stageCooling holder cryogen: NITROGEN
Image recording#0 - Image recording ID: 1 / #0 - Film or detector model: GATAN K2 SUMMIT (4k x 4k) / #0 - Detector mode: COUNTING / #0 - Number grids imaged: 1 / #0 - Number real images: 833 / #0 - Average exposure time: 4.25 sec. / #0 - Average electron dose: 30.0 e/Å2 / #0 - Details: Session 1 / #1 - Image recording ID: 2 / #1 - Film or detector model: GATAN K2 SUMMIT (4k x 4k) / #1 - Detector mode: COUNTING / #1 - Number grids imaged: 1 / #1 - Number real images: 281 / #1 - Average exposure time: 7.25 sec. / #1 - Average electron dose: 30.0 e/Å2 / #1 - Details: Session 2 / #2 - Image recording ID: 3 / #2 - Film or detector model: GATAN K2 SUMMIT (4k x 4k) / #2 - Detector mode: COUNTING / #2 - Number grids imaged: 1 / #2 - Number real images: 546 / #2 - Average exposure time: 6.25 sec. / #2 - Average electron dose: 30.0 e/Å2 / #2 - Details: Session 3
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 164282
CTF correctionSoftware:
Namedetails
CTFFIND3CTFFIND3 was used for CTF estimation.
RELION (ver. 1.3)CTF was corrected in Relion using the parameters from CTFFIND3.

Details: CTFFIND3 in Relion was used for CTF estimation
Startup modelType of model: EMDB MAP
EMDB ID:

1636


Details: A Methanococcus jannaschii negatively-stained EM map low-passed to 60 Angstrom resolution was used as the initial model for 3D reconstruction.
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: Reion (ver. 1.3) / Software - details: Relion 3D auto-refine
Details: Refinement was performed using Relion 3D auto-refine. Initial angular sampling was 7.5 degrees; initial offset range was 5 pixels; initial offset step was 1 pixel.
Final 3D classificationNumber classes: 3 / Avg.num./class: 24000 / Software - Name: RELION (ver. 1.3) / Software - details: Relion 3D Classification
Details: 3D classification was performed on "shiny" particles that have beam-induced motions corrected by lm-bfgs alignparts.
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.3) / Software - details: Relion 3D auto-refine
Details: Refinement was performed using Relion 3D auto-refine. Angular sampling, offset range, and offset step sizes were automatically adjusted in Relion.
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 9.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.3) / Software - details: 3D auto-refine
Details: 3D refinement was performed on "shiny" particles that have beam-induced motions corrected by lm-bfgs alignparts.
Number images used: 32771
Image recording ID1

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Atomic model buiding 1

Initial model
PDB IDChain

3nvi


2nnw

chain_id: A, residue_range: 8-126

2nnw

chain_id: B
DetailsTo perform rigid-body docking, two copies of the co-crystal structure from P. furiosus [PDB ID 3NVI, each containing two copies of L7Ae, kink-turn RNA, and Nop5 lacking the NTD (amino acids 127-373)] were docked as rigid bodies into the center of the EM volume (using "Fit in Map" in Chimera) based on prior knowledge of the location of the Nop5 coiled-coil domain. Subsequently, four copies of P. furiosus Nop5 N-terminal domain (amino acids 8-126) and fibrillarin from PDB ID 2NNW were docked into the four corners of the volume using "Fit to Segments" in Chimera without changing the relative orientation between the two proteins. To refine the docking, a simultaneous multi-fragment docking refinement was performed using Collage in Situs 2.8.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: Correlation

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