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Yorodumi- EMDB-2978: Time-resolved Cryo Electron Microscopy of ribosome subunit association -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-2978 | |||||||||
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Title | Time-resolved Cryo Electron Microscopy of ribosome subunit association | |||||||||
Map data | Reconstruction of E. Coli naked 70S ribosome in rotated (RT) conformation | |||||||||
Sample |
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Keywords | time-resolved / cryo-EM / mixing-spraying / ribosome subunit association / structural dynamics | |||||||||
Function / homology | Function and homology information Cajal-Retzius cell differentiation / positive regulation of L-glutamate import across plasma membrane / amyloid precursor protein biosynthetic process / positive regulation of coagulation / protein catabolic process at postsynapse / negative regulation of core promoter binding / gamma-secretase complex / aspartic endopeptidase activity, intramembrane cleaving / short-term synaptic potentiation / positive regulation of amyloid precursor protein biosynthetic process ...Cajal-Retzius cell differentiation / positive regulation of L-glutamate import across plasma membrane / amyloid precursor protein biosynthetic process / positive regulation of coagulation / protein catabolic process at postsynapse / negative regulation of core promoter binding / gamma-secretase complex / aspartic endopeptidase activity, intramembrane cleaving / short-term synaptic potentiation / positive regulation of amyloid precursor protein biosynthetic process / Noncanonical activation of NOTCH3 / positive regulation of endopeptidase activity / choline transport / Notch receptor processing / central nervous system myelination / sequestering of calcium ion / membrane protein intracellular domain proteolysis / synaptic vesicle targeting / negative regulation of axonogenesis / regulation of resting membrane potential / T cell activation involved in immune response / skin morphogenesis / NOTCH4 Activation and Transmission of Signal to the Nucleus / growth factor receptor binding / neural retina development / dorsal/ventral neural tube patterning / regulation of synaptic vesicle cycle / L-glutamate import across plasma membrane / myeloid dendritic cell differentiation / Regulated proteolysis of p75NTR / amyloid precursor protein metabolic process / regulation of phosphorylation / locomotion / brain morphogenesis / glutamate receptor signaling pathway / endoplasmic reticulum calcium ion homeostasis / nuclear outer membrane / smooth endoplasmic reticulum calcium ion homeostasis / astrocyte activation involved in immune response / regulation of canonical Wnt signaling pathway / aggresome / regulation of long-term synaptic potentiation / embryonic limb morphogenesis / skeletal system morphogenesis / positive regulation of amyloid fibril formation / cell fate specification / regulation of postsynapse organization / positive regulation of dendritic spine development / ciliary rootlet / myeloid cell homeostasis / azurophil granule membrane / dopamine receptor signaling pathway / Hydrolases; Acting on peptide bonds (peptidases); Aspartic endopeptidases / adult behavior / positive regulation of receptor recycling / mitochondrial transport / positive regulation of catalytic activity / regulation of neuron projection development / heart looping / blood vessel development / amyloid precursor protein catabolic process / cerebral cortex cell migration / smooth endoplasmic reticulum / protein glycosylation / amyloid-beta formation / negative regulation of apoptotic signaling pathway / autophagosome assembly / membrane protein ectodomain proteolysis / EPH-ephrin mediated repulsion of cells / neuron development / rough endoplasmic reticulum / Nuclear signaling by ERBB4 / hematopoietic progenitor cell differentiation / somitogenesis / amyloid-beta metabolic process / T cell proliferation / negative regulation of ubiquitin-dependent protein catabolic process / Notch signaling pathway / regulation of synaptic transmission, glutamatergic / viral release from host cell by cytolysis / NOTCH2 Activation and Transmission of Signal to the Nucleus / cellular response to calcium ion / neuron projection maintenance / NRIF signals cell death from the nucleus / Activated NOTCH1 Transmits Signal to the Nucleus / Degradation of the extracellular matrix / cerebellum development / positive regulation of glycolytic process / peptidoglycan catabolic process / post-embryonic development / dendritic shaft / thymus development / negative regulation of protein phosphorylation / epithelial cell proliferation / PDZ domain binding / NOTCH3 Activation and Transmission of Signal to the Nucleus / astrocyte activation / apoptotic signaling pathway / synapse organization / neuron migration Similarity search - Function | |||||||||
Biological species | Escherichia coli (E. coli) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 11.6 Å | |||||||||
Authors | Chen B / Kaledhonkar S / Sun M / Shen B / Lu Z / Barnard D / Lu T / Gonzalez Jr R / Frank J | |||||||||
Citation | Journal: Structure / Year: 2015 Title: Structural dynamics of ribosome subunit association studied by mixing-spraying time-resolved cryogenic electron microscopy. Authors: Bo Chen / Sandip Kaledhonkar / Ming Sun / Bingxin Shen / Zonghuan Lu / David Barnard / Toh-Ming Lu / Ruben L Gonzalez / Joachim Frank / Abstract: Ribosomal subunit association is a key checkpoint in translation initiation but its structural dynamics are poorly understood. Here, we used a recently developed mixing-spraying, time-resolved, ...Ribosomal subunit association is a key checkpoint in translation initiation but its structural dynamics are poorly understood. Here, we used a recently developed mixing-spraying, time-resolved, cryogenic electron microscopy (cryo-EM) method to study ribosomal subunit association in the sub-second time range. We have improved this method and increased the cryo-EM data yield by tenfold. Pre-equilibrium states of the association reaction were captured by reacting the mixture of ribosomal subunits for 60 ms and 140 ms. We also identified three distinct ribosome conformations in the associated ribosomes. The observed proportions of these conformations are the same in these two time points, suggesting that ribosomes equilibrate among the three conformations within less than 60 ms upon formation. Our results demonstrate that the mixing-spraying method can capture multiple states of macromolecules during a sub-second reaction. Other fast processes, such as translation initiation, decoding, and ribosome recycling, are amenable to study with this method. | |||||||||
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_2978.map.gz | 14.3 MB | EMDB map data format | |
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Header (meta data) | emd-2978-v30.xml emd-2978.xml | 9.2 KB 9.2 KB | Display Display | EMDB header |
Images | emd_2978.png | 281.5 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-2978 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-2978 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_2978.map.gz / Format: CCP4 / Size: 15.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Reconstruction of E. Coli naked 70S ribosome in rotated (RT) conformation | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 2.2451 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 : E. Coli 70S Ribosome
Entire | Name: E. Coli 70S Ribosome |
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Components |
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-Supramolecule #1000: E. Coli 70S Ribosome
Supramolecule | Name: E. Coli 70S Ribosome / type: sample / ID: 1000 / Number unique components: 1 |
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-Supramolecule #1: 70S ribosome
Supramolecule | Name: 70S ribosome / type: complex / ID: 1 / Recombinant expression: No / Ribosome-details: ribosome-prokaryote: LSU 50S, SSU 30S |
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Source (natural) | Organism: Escherichia coli (E. coli) / Strain: MRE600 |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Buffer | pH: 7.6 Details: 25 mM Tris-HCl, 60 mM NH4Cl, 5 mM 2-mercaptoethanol, 3.5 mM MgCl2 |
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Grid | Details: Quantifoil R2/2 300 mesh copper grid with thin carbon sipport |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 80 % / Chamber temperature: 80 K / Instrument: OTHER Details: Equal volume of 1.2 microM 30S and 0.6 microM 50S (final concentration after mixing) were injected into the mixing-spraying device each at flow rate of 3 microL/s. The computer-controlled ...Details: Equal volume of 1.2 microM 30S and 0.6 microM 50S (final concentration after mixing) were injected into the mixing-spraying device each at flow rate of 3 microL/s. The computer-controlled plunging device was purchased from Dr. Howard White (Eastern Virginia Medical School, VA). Timed resolved state: Vitrified after spraying |
-Electron microscopy
Microscope | FEI TECNAI F20 |
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Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Calibrated magnification: 66318 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 50000 |
Sample stage | Specimen holder: CT 3500 / Specimen holder model: GATAN LIQUID NITROGEN |
Temperature | Average: 80 K |
Details | Low dose, Data was collected over two years time |
Date | Sep 13, 2013 |
Image recording | Category: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 3402 / Average electron dose: 17 e/Å2 |
Experimental equipment | Model: Tecnai F20 / Image courtesy: FEI Company |
-Image processing
CTF correction | Details: each Micrograph |
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Final reconstruction | Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 11.6 Å / Resolution method: OTHER / Software - Name: Arachnid, RELION, SPIDER / Number images used: 11129 |
Details | The partciles were selected with Autopicker (Langlois et al., 2014), and 3D classification and reconstruction with RELION |