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- EMDB-2978: Time-resolved Cryo Electron Microscopy of ribosome subunit association -

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Basic information

Entry
Database: EMDB / ID: EMD-2978
TitleTime-resolved Cryo Electron Microscopy of ribosome subunit association
Map dataReconstruction of E. Coli naked 70S ribosome in rotated (RT) conformation
Sample
  • Sample: E. Coli 70S Ribosome
  • Complex: 70S ribosomeRibosome
Keywordstime-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 / adult behavior / Hydrolases; Acting on peptide bonds (peptidases); Aspartic endopeptidases / positive regulation of receptor recycling / mitochondrial transport / positive regulation of catalytic activity / heart looping / blood vessel development / regulation of neuron projection 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 / Nuclear signaling by ERBB4 / hematopoietic progenitor cell differentiation / rough endoplasmic reticulum / 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 / thymus development / dendritic shaft / negative regulation of protein phosphorylation / epithelial cell proliferation / PDZ domain binding / NOTCH3 Activation and Transmission of Signal to the Nucleus / apoptotic signaling pathway / astrocyte activation / synapse organization / neuron migration
Similarity search - Function
Peptidase A22A, presenilin 1 / Peptidase A22A, presenilin / Presenilin, C-terminal / Presenilin / Nicastrin / Nicastrin, small lobe / Nicastrin / Nicastrin small lobe / Presenilin/signal peptide peptidase / Presenilin, signal peptide peptidase, family ...Peptidase A22A, presenilin 1 / Peptidase A22A, presenilin / Presenilin, C-terminal / Presenilin / Nicastrin / Nicastrin, small lobe / Nicastrin / Nicastrin small lobe / Presenilin/signal peptide peptidase / Presenilin, signal peptide peptidase, family / Endolysin T4 type / T4-type lysozyme / Glycoside hydrolase, family 24 / Lysozyme domain superfamily / Phage lysozyme / Lysozyme-like domain superfamily
Similarity search - Domain/homology
Endolysin / Endolysin / Presenilin-1 / Nicastrin
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 11.6 Å
AuthorsChen B / Kaledhonkar S / Sun M / Shen B / Lu Z / Barnard D / Lu T / Gonzalez Jr R / Frank J
CitationJournal: 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
DepositionApr 7, 2015-
Header (metadata) releaseJun 17, 2015-
Map releaseJun 17, 2015-
UpdateJul 1, 2015-
Current statusJul 1, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2978.png

Downloads & links

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Map

FileDownload / File: emd_2978.map.gz / Format: CCP4 / Size: 15.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of E. Coli naked 70S ribosome in rotated (RT) conformation
Voxel sizeX=Y=Z: 2.2451 Å
Density
Contour LevelBy AUTHOR: 0.04 / Movie #1: 0.04
Minimum - Maximum-0.09098771 - 0.1893954
Average (Standard dev.)0.00143521 (±0.02883799)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin111
Dimensions160160160
Spacing160160160
CellA=B=C: 359.216 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.24512.24512.2451
M x/y/z160160160
origin x/y/z0.0000.0000.000
length x/y/z359.216359.216359.216
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS111
NC/NR/NS160160160
D min/max/mean-0.0910.1890.001

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Supplemental data

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Sample components

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Entire : E. Coli 70S Ribosome

EntireName: E. Coli 70S Ribosome
Components
  • Sample: E. Coli 70S Ribosome
  • Complex: 70S ribosomeRibosome

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Supramolecule #1000: E. Coli 70S Ribosome

SupramoleculeName: E. Coli 70S Ribosome / type: sample / ID: 1000 / Number unique components: 1

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Supramolecule #1: 70S ribosome

SupramoleculeName: 70S ribosome / type: complex / ID: 1 / Recombinant expression: No / Ribosome-details: ribosome-prokaryote: LSU 50S, SSU 30S
Source (natural)Organism: Escherichia coli (E. coli) / Strain: MRE600

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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: 7.6
Details: 25 mM Tris-HCl, 60 mM NH4Cl, 5 mM 2-mercaptoethanol, 3.5 mM MgCl2
GridDetails: Quantifoil R2/2 300 mesh copper grid with thin carbon sipport
VitrificationCryogen 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

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated 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 stageSpecimen holder: CT 3500 / Specimen holder model: GATAN LIQUID NITROGEN
TemperatureAverage: 80 K
DetailsLow dose, Data was collected over two years time
DateSep 13, 2013
Image recordingCategory: 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

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Image processing

CTF correctionDetails: each Micrograph
Final reconstructionApplied 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
DetailsThe partciles were selected with Autopicker (Langlois et al., 2014), and 3D classification and reconstruction with RELION

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