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- EMDB-24659: E. coli bL17-limitation ribosome assembly intermediate #33 -

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Basic information

Entry
Database: EMDB / ID: EMD-24659
TitleE. coli bL17-limitation ribosome assembly intermediate #33
Map dataE. coli bL17-limitation ribosome assembly intermediate #33 (C-class) sharpened map
Sample
  • Complex: bL17-depleted large ribosomal subunit assembly intermediate #33
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 9.8 Å
AuthorsRabuck-Gibbons JN / Lyumkis D / Williamson JR
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)DP5-OD021396 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)U54 AI150472 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35-GM136412 United States
CitationJournal: Structure / Year: 2022
Title: Quantitative mining of compositional heterogeneity in cryo-EM datasets of ribosome assembly intermediates.
Authors: Jessica N Rabuck-Gibbons / Dmitry Lyumkis / James R Williamson /
Abstract: Single-particle cryoelectron microscopy (cryo-EM) offers a unique opportunity to characterize macromolecular structural heterogeneity by virtue of its ability to place distinct particle populations ...Single-particle cryoelectron microscopy (cryo-EM) offers a unique opportunity to characterize macromolecular structural heterogeneity by virtue of its ability to place distinct particle populations into different groups through computational classification. However, there is a dearth of tools for surveying the heterogeneity landscape, quantitatively analyzing heterogeneous particle populations after classification, deciding how many unique classes are represented by the data, and accurately cross-comparing reconstructions. Here, we develop a workflow that contains discovery and analysis modules to quantitatively mine cryo-EM data for sets of structures with maximal diversity. This workflow was applied to a dataset of E. coli 50S ribosome assembly intermediates, which are characterized by significant structural heterogeneity. We identified more detailed branchpoints in the assembly process and characterized the interactions of an assembly factor with immature intermediates. While the tools described here were developed for ribosome assembly, they should be broadly applicable to the analysis of other heterogeneous cryo-EM datasets.
History
DepositionAug 9, 2021-
Header (metadata) releaseDec 29, 2021-
Map releaseDec 29, 2021-
UpdateApr 20, 2022-
Current statusApr 20, 2022Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 5
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_24659.png

Downloads & links

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Map

FileDownload / File: emd_24659.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationE. coli bL17-limitation ribosome assembly intermediate #33 (C-class) sharpened map
Projections & slices

Image control

Size
Brightness
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.31 Å/pix.
x 320 pix.
= 419.2 Å		1.31 Å/pix.
x 320 pix.
= 419.2 Å		1.31 Å/pix.
x 320 pix.
= 419.2 Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.31 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 5.8 / Movie #1: 5
Minimum - Maximum-5.9287133 - 16.102722
Average (Standard dev.)-0.43324733 (±1.0287404)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 419.19998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.311.311.31
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z419.200419.200419.200
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ448448448
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-5.92916.103-0.433

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Supplemental data

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Additional map: E. coli bL17-limitation ribosome assembly intermediate #33 (C-class)...

Fileemd_24659_additional_1.map
AnnotationE. coli bL17-limitation ribosome assembly intermediate #33 (C-class) binarized map
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Additional map: E. coli bL17-limitation ribosome assembly intermediate #33 (C-class)...

Fileemd_24659_additional_2.map
AnnotationE. coli bL17-limitation ribosome assembly intermediate #33 (C-class) unsharpened map
Projections & Slices
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Additional map: E. coli bL17-limitation ribosome assembly intermediate #33 (C-class)...

Fileemd_24659_additional_3.map
AnnotationE. coli bL17-limitation ribosome assembly intermediate #33 (C-class) binned, unmasked map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

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Half map: E. coli bL17-limitation ribosome assembly intermediate #33 (C-class)...

Fileemd_24659_half_map_1.map
AnnotationE. coli bL17-limitation ribosome assembly intermediate #33 (C-class) odd halfmap
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: E. coli bL17-limitation ribosome assembly intermediate #33 (C-class)...

Fileemd_24659_half_map_2.map
AnnotationE. coli bL17-limitation ribosome assembly intermediate #33 (C-class) even halfmap
Projections & Slices
AxesZYX

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Sample components

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Entire : bL17-depleted large ribosomal subunit assembly intermediate #33

EntireName: bL17-depleted large ribosomal subunit assembly intermediate #33
Components
  • Complex: bL17-depleted large ribosomal subunit assembly intermediate #33

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Supramolecule #1: bL17-depleted large ribosomal subunit assembly intermediate #33

SupramoleculeName: bL17-depleted large ribosomal subunit assembly intermediate #33
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Escherichia coli (E. coli) / Strain: NCM3722
Molecular weightTheoretical: 1.5 MDa

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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: 7.5
Component:
ConcentrationNameFormula
20.0 mMTris-HCl
100.0 mMammonium chlorideNH4Cl
10.0 mMmagnesium chlorideMgCl2
0.5 mMEDTA
6.0 mMb-mercaptoethanol
GridModel: Quantifoil R1.2/1.3 / Support film - Material: GOLD / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Instrument: HOMEMADE PLUNGER
Details: 3 ul of this sample was added to 3 a plasma cleaned (Gatan, Solarus) 1.2mm hole, 1.3mm spacing holey gold grids (Russo and Passmore, 2014). Grids were manually frozen in liquid ethane..
Detailsdepleted ribosome assembly intermediate purified by sucrose gradient

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Electron microscopy

MicroscopeFEI TITAN KRIOS
DetailsIn order to account for highly preferred orientation of the specimen, data were acquired using a tilt of -20 degrees.
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Frames/image: 1-50 / Number grids imaged: 1 / Number real images: 833 / Average exposure time: 10.0 sec. / Average electron dose: 35.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal magnification: 22500
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: CTFFIND
Final reconstructionResolution.type: BY AUTHOR / Resolution: 9.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: FREALIGN (ver. X) / Number images used: 2783
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: FREALIGN (ver. X) / Details: FrealignX
Final 3D classificationSoftware - Name: FREALIGN (ver. X)
FSC plot (resolution estimation)

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