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- EMDB-8803: 80S ribsome from Oryctolagus cuniculus, class II - with non-rotat... -

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Basic information

Entry
Database: EMDB / ID: EMD-8803
Title80S ribsome from Oryctolagus cuniculus, class II - with non-rotated/non-swiveled 40S with P site tRNA and ternary complex, derived from EMPIAR-10064 using eClarity
Map dataRabbit 80S ribosome derived from EMPIAR-10064 using emClarity. Non-rotated/non-swiveled 40S with P site tRNA and sampling ternary complex.
Sample
  • Complex: 80s ribsome from Oryctolagus cuniculus
Biological speciesOryctolagus cuniculus (rabbit)
Methodsubtomogram averaging / cryo EM / Resolution: 9.9 Å
AuthorsHimes BA / Zhang P
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM085043 United States
CitationJournal: Nat Methods / Year: 2018
Title: emClarity: software for high-resolution cryo-electron tomography and subtomogram averaging.
Authors: Benjamin A Himes / Peijun Zhang /
Abstract: Macromolecular complexes are intrinsically flexible and often challenging to purify for structure determination by single-particle cryo-electron microscopy (cryo-EM). Such complexes can be studied by ...Macromolecular complexes are intrinsically flexible and often challenging to purify for structure determination by single-particle cryo-electron microscopy (cryo-EM). Such complexes can be studied by cryo-electron tomography (cryo-ET) combined with subtomogram alignment and classification, which in exceptional cases achieves subnanometer resolution, yielding insight into structure-function relationships. However, it remains challenging to apply this approach to specimens that exhibit conformational or compositional heterogeneity or are present in low abundance. To address this, we developed emClarity ( https://github.com/bHimes/emClarity/wiki ), a GPU-accelerated image-processing package featuring an iterative tomographic tilt-series refinement algorithm that uses subtomograms as fiducial markers and a 3D-sampling-function-compensated, multi-scale principal component analysis classification method. We demonstrate that our approach offers substantial improvement in the resolution of maps and in the separation of different functional states of macromolecular complexes compared with current state-of-the-art software.
History
DepositionJun 30, 2017-
Header (metadata) releaseOct 10, 2018-
Map releaseOct 10, 2018-
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: 2
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 2
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8803.png

Downloads & links

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Map

FileDownload / File: emd_8803.map.gz / Format: CCP4 / Size: 144.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRabbit 80S ribosome derived from EMPIAR-10064 using emClarity. Non-rotated/non-swiveled 40S with P site tRNA and sampling ternary complex.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.62 Å/pix.
x 336 pix.
= 880.32 Å		2.62 Å/pix.
x 336 pix.
= 880.32 Å		2.62 Å/pix.
x 336 pix.
= 880.32 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.62 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 2 / Movie #1: 2
Minimum - Maximum-9.342286 - 14.31954
Average (Standard dev.)0.010950878 (±0.30197886)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions336336336
Spacing336336336
CellA=B=C: 880.31995 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.622.622.62
M x/y/z336336336
origin x/y/z0.0000.0000.000
length x/y/z880.320880.320880.320
α/β/γ90.00090.00090.000
start NX/NY/NZ-38-19-20
NX/NY/NZ858082
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS336336336
D min/max/mean-9.34214.3200.011

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Supplemental data

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Sample components

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Entire : 80s ribsome from Oryctolagus cuniculus

EntireName: 80s ribsome from Oryctolagus cuniculus
Components
  • Complex: 80s ribsome from Oryctolagus cuniculus

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Supramolecule #1: 80s ribsome from Oryctolagus cuniculus

SupramoleculeName: 80s ribsome from Oryctolagus cuniculus / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Oryctolagus cuniculus (rabbit) / Tissue: Reticulocyte

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation stateparticle

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Sample preparation

BufferpH: 7.6
Component:
ConcentrationNameFormula
20.0 mMHEPES
50.0 mMpotassium chlorideKCl
2.0 mMmagnesium chlorideMgCl2
GridModel: Quantifoil R2/1 / Material: COPPER / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING
VitrificationCryogen name: ETHANE / Chamber humidity: 60 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
DetailsData collection 20 --> -60, 22 --> 60 degrees
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: INTEGRATING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Average electron dose: 1.5 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 0.0037 µm / Nominal defocus min: 0.0024 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 9.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: emClarity (ver. 1.0) / Software - details: adapted single-particle wiener filter
Details: Independent beyond 3.6 nm as determined by the FSC at the time of dividing the half-sets
Number subtomograms used: 922
ExtractionNumber tomograms: 4 / Number images used: 3090
CTF correctionSoftware - Name: emClarity (ver. 1.0)
Software - details: ncluded astigmatism and per-tilt CTF determination
Details: Phases corrected on the projections by multiplying by the CTF in tiles compensating for the defocus gradient in tilted images.
Final 3D classificationNumber classes: 5 / Avg.num./class: 500 / Software - Name: emClarity (ver. 1.0)
Details: 3,090 total subtomograms were assigned to 5 separate classes using multi-scale PCA with full-3dCTF compensated estimators. 1.0,1.6,2.4,5.4 nm were the length-scales used for the msPCA.
Final angle assignmentType: OTHER / Software - Name: emClarity (ver. 1.0)
Details: 3D refinement with two half-sets fully separated after template matching, at which point the FSC 0.5 was 3.6 nanometers. Reference and subtomograms filtered dynamically according to the FSC ...Details: 3D refinement with two half-sets fully separated after template matching, at which point the FSC 0.5 was 3.6 nanometers. Reference and subtomograms filtered dynamically according to the FSC at each cycle using the FOM approach.
FSC plot (resolution estimation)

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