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- EMDB-23771: 3D reconstruction generated using the locations and orientations ... -

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Basic information

Entry
Database: EMDB / ID: EMD-23771
Title3D reconstruction generated using the locations and orientations of 5,080 50S subunits detected in 220 images using 2DTM with a M. pneumoniae 50S template
Map data20-Angstrom filtered reconstruction
Sample
  • Complex: Mycoplasma pneumoniae 70S ribosome
Biological speciesMycoplasma pneumoniae (Filterable agent of primary atypical pneumonia)
Methodsingle particle reconstruction / cryo EM / Resolution: 20.0 Å
AuthorsLucas BA / Himes BA / Xue L / Grant T / Mahamid J / Grigorieff N
Funding support Germany, 1 items
OrganizationGrant numberCountry
European Research Council (ERC)760067 Germany
CitationJournal: Elife / Year: 2021
Title: Locating macromolecular assemblies in cells by 2D template matching with cisTEM.
Authors: Bronwyn A Lucas / Benjamin A Himes / Liang Xue / Timothy Grant / Julia Mahamid / Nikolaus Grigorieff /
Abstract: For a more complete understanding of molecular mechanisms, it is important to study macromolecules and their assemblies in the broader context of the cell. This context can be visualized at nanometer ...For a more complete understanding of molecular mechanisms, it is important to study macromolecules and their assemblies in the broader context of the cell. This context can be visualized at nanometer resolution in three dimensions (3D) using electron cryo-tomography, which requires tilt series to be recorded and computationally aligned, currently limiting throughput. Additionally, the high-resolution signal preserved in the raw tomograms is currently limited by a number of technical difficulties, leading to an increased false-positive detection rate when using 3D template matching to find molecular complexes in tomograms. We have recently described a 2D template matching approach that addresses these issues by including high-resolution signal preserved in single-tilt images. A current limitation of this approach is the high computational cost that limits throughput. We describe here a GPU-accelerated implementation of 2D template matching in the image processing software TEM that allows for easy scaling and improves the accessibility of this approach. We apply 2D template matching to identify ribosomes in images of frozen-hydrated cells with high precision and sensitivity, demonstrating that this is a versatile tool for in situ visual proteomics and in situ structure determination. We benchmark the results with 3D template matching of tomograms acquired on identical sample locations and identify strengths and weaknesses of both techniques, which offer complementary information about target localization and identity.
History
DepositionApr 5, 2021-
Header (metadata) releaseJun 23, 2021-
Map releaseJun 23, 2021-
UpdateJun 23, 2021-
Current statusJun 23, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 1
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23771.png

Downloads & links

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Map

FileDownload / File: emd_23771.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation20-Angstrom filtered reconstruction
Voxel sizeX=Y=Z: 1.5 Å
Density
Contour LevelBy AUTHOR: 1.0 / Movie #1: 1
Minimum - Maximum-2.0888515 - 5.559764
Average (Standard dev.)0.049143806 (±0.54958534)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 384.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.51.51.5
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z384.000384.000384.000
α/β/γ90.00090.00090.000
start NX/NY/NZ192139186
NX/NY/NZ211274246
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-2.0895.5600.049

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Supplemental data

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Additional map: Unfiltered reconstruction

Fileemd_23771_additional_1.map
AnnotationUnfiltered reconstruction
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1

Fileemd_23771_half_map_1.map
AnnotationHalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2

Fileemd_23771_half_map_2.map
AnnotationHalf map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Mycoplasma pneumoniae 70S ribosome

EntireName: Mycoplasma pneumoniae 70S ribosome
Components
  • Complex: Mycoplasma pneumoniae 70S ribosome

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Supramolecule #1: Mycoplasma pneumoniae 70S ribosome

SupramoleculeName: Mycoplasma pneumoniae 70S ribosome / type: complex / ID: 1 / Parent: 0
Details: 3D reconstruction generated using the locations and orientations of 5,080 50S subunits detected in 220 images using 2DTM with a M. pneumoniae 50S template
Source (natural)Organism: Mycoplasma pneumoniae (Filterable agent of primary atypical pneumonia)
Molecular weightTheoretical: 2.5 MDa

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation statecell

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Sample preparation

BufferpH: 7
GridModel: Quantifoil / Material: GOLD / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV
Details: Grids were washed with PBS buffer containing 10 nm protein A-conjugated gold beads (Aurion), blotted from the back side for 2 s and plunged into mixed liquid ethane/propane at liquid N2 ...Details: Grids were washed with PBS buffer containing 10 nm protein A-conjugated gold beads (Aurion), blotted from the back side for 2 s and plunged into mixed liquid ethane/propane at liquid N2 temperature with a manual plunger..
DetailsWhole Mycoplasma pneumoniae cells deposited on a cryo-EM grid

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Electron microscopy

MicroscopeTFS KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 215000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 10 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Digitization - Sampling interval: 5.0 µm / Digitization - Frames/image: 1-20 / Number real images: 220 / Average exposure time: 2.0 sec. / Average electron dose: 32.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionDetails: Particles were selected based on 50S targets detected by 2D template matching
CTF correctionSoftware - Name: cisTEM (ver. 2.0) / Software - details: See github repository
Startup modelType of model: NONE / Details: Orientations are based on template matching
Initial angle assignmentType: NOT APPLICABLE / Software - Name: cisTEM (ver. 2.0) / Software - details: See github repository
Final angle assignmentType: PROJECTION MATCHING
Projection matching processing - Number reference projections: 2500000
Projection matching processing - Merit function: SNR
Projection matching processing - Angular sampling: 1.5 degrees
Software - Name: cisTEM (ver. 2.0) / Software - details: See github repository
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 20.0 Å / Resolution method: OTHER / Software - Name: cisTEM (ver. 2.0) / Software - details: See github repository
Details: Resolution not determined since the FSC has template bias
Number images used: 5080
DetailsThe reconstruction was calculated using standard procedures implemented in cisTEM. See https://github.com/timothygrant80/cisTEM
FSC plot (resolution estimation)

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