+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-8146 | |||||||||
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Title | Methanococcus jannaschii box C/D sRNP | |||||||||
Map data | None | |||||||||
Sample |
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Biological species | Methanocaldococcus jannaschii (archaea) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 9.0 Å | |||||||||
Authors | Yip WSV / Shigematsu H / Taylor DW / Baserga SJ | |||||||||
Funding support | United States, 1 items
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Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_8146.map.gz | 85.2 MB | EMDB map data format | |
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Header (meta data) | emd-8146-v30.xml emd-8146.xml | 20.1 KB 20.1 KB | Display Display | EMDB header |
Images | emd_8146.png | 54.8 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-8146 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-8146 | HTTPS FTP |
-Validation report
Summary document | emd_8146_validation.pdf.gz | 78.7 KB | Display | EMDB validaton report |
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Full document | emd_8146_full_validation.pdf.gz | 77.9 KB | Display | |
Data in XML | emd_8146_validation.xml.gz | 493 B | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-8146 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-8146 | HTTPS FTP |
-Related structure data
Similar structure data |
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-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_8146.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | None | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.247 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : Methanococcus janaschii box C/D sRNP
Entire | Name: Methanococcus janaschii box C/D sRNP |
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Components |
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-Supramolecule #1: Methanococcus janaschii box C/D sRNP
Supramolecule | Name: Methanococcus janaschii box C/D sRNP / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all |
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Source (natural) | Organism: Methanocaldococcus jannaschii (archaea) |
Molecular weight | Theoretical: 366 KDa |
-Macromolecule #1: L7Ae
Macromolecule | Name: L7Ae / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Methanocaldococcus jannaschii (archaea) |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: MAVYVKFKVP EEIQKELLDA VAKAQKIKKG ANEVTKAVER GIAKLVIIAE DVKPEEVVAH LPYLCEEKGI PYAYVASKQD LGKAAGLEVA ASSVAIINEG DAEELKVLIE KVNVLKQ |
-Macromolecule #2: Nop5
Macromolecule | Name: Nop5 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO |
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Source (natural) | Organism: Methanocaldococcus jannaschii (archaea) |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: 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 |
-Macromolecule #3: Fibrillarin
Macromolecule | Name: Fibrillarin / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO |
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Source (natural) | Organism: Methanocaldococcus jannaschii (archaea) |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: 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 |
-Macromolecule #4: Methanococcus jannaschii sR8 box C/D sRNA
Macromolecule | Name: Methanococcus jannaschii sR8 box C/D sRNA / type: rna / ID: 4 |
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Sequence | String: AAAUCGCCAA UGAUGACGAU UGGCUUUGCU GAGUCUGUGA UGAACCGUAU GAGCACUGAG GCGAUUU |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Buffer | pH: 7.5 Component:
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Grid | Model: Electron Microscopy Sciences / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Atmosphere: OTHER | ||||||||||||
Vitrification | Cryogen 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).. |
-Electron microscopy
Microscope | FEI TECNAI F20 |
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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 |
Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 2.5 µm / Nominal magnification: 29000 |
Sample stage | Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Tecnai F20 / Image courtesy: FEI Company |
+Image processing
-Atomic model buiding 1
Initial model |
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Details | To 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. | ||||||||
Refinement | Space: REAL / Protocol: RIGID BODY FIT / Target criteria: Correlation |