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- EMDB-23661: Cryo-electron tomogram of a JCVI_Syn3A cell -

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Entry
Database: EMDB / ID: EMD-23661
TitleCryo-electron tomogram of a JCVI_Syn3A cell
Map datacryo-electron tomogram of a JCVI_Syn3A cell
Sample
  • Cell: Cryo-electron tomogram of whole JCVI-Syn3A synthetic mycoplasma.
Biological speciessynthetic bacterium JCVI-Syn3A (others)
Methodelectron tomography / cryo EM
AuthorsLam V / Villa E
Funding support United States, 4 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1DP2GM123494-01 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)5T32GM7240-40 United States
National Science Foundation (NSF, United States)1920374 United States
National Science Foundation (NSF, United States)1818344 United States
CitationJournal: Front Mol Biosci / Year: 2021
Title: Generating Chromosome Geometries in a Minimal Cell From Cryo-Electron Tomograms and Chromosome Conformation Capture Maps.
Authors: Benjamin R Gilbert / Zane R Thornburg / Vinson Lam / Fatema-Zahra M Rashid / John I Glass / Elizabeth Villa / Remus T Dame / Zaida Luthey-Schulten /
Abstract: JCVI-syn3A is a genetically minimal bacterial cell, consisting of 493 genes and only a single 543 kbp circular chromosome. Syn3A's genome and physical size are approximately one-tenth those of the ...JCVI-syn3A is a genetically minimal bacterial cell, consisting of 493 genes and only a single 543 kbp circular chromosome. Syn3A's genome and physical size are approximately one-tenth those of the model bacterial organism 's, and the corresponding reduction in complexity and scale provides a unique opportunity for whole-cell modeling. Previous work established genome-scale gene essentiality and proteomics data along with its essential metabolic network and a kinetic model of genetic information processing. In addition to that information, whole-cell, spatially-resolved kinetic models require cellular architecture, including spatial distributions of ribosomes and the circular chromosome's configuration. We reconstruct cellular architectures of Syn3A cells at the single-cell level directly from cryo-electron tomograms, including the ribosome distributions. We present a method of generating self-avoiding circular chromosome configurations in a lattice model with a resolution of 11.8 bp per monomer on a 4 nm cubic lattice. Realizations of the chromosome configurations are constrained by the ribosomes and geometry reconstructed from the tomograms and include DNA loops suggested by experimental chromosome conformation capture (3C) maps. Using ensembles of simulated chromosome configurations we predict chromosome contact maps for Syn3A cells at resolutions of 250 bp and greater and compare them to the experimental maps. Additionally, the spatial distributions of ribosomes and the DNA-crowding resulting from the individual chromosome configurations can be used to identify macromolecular structures formed from ribosomes and DNA, such as polysomes and expressomes.
History
DepositionMar 19, 2021-
Header (metadata) releaseAug 18, 2021-
Map releaseAug 18, 2021-
UpdateAug 18, 2021-
Current statusAug 18, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Solid view (volume rendering)
  • Imaged by UCSF Chimera
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  • Solid view (volume rendering)
  • Imaged by UCSF Chimera
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Supplemental images

emd_23661.png

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Map

FileDownload / File: emd_23661.map.gz / Format: CCP4 / Size: 77.3 MB / Type: IMAGE STORED AS SIGNED BYTE
Annotationcryo-electron tomogram of a JCVI_Syn3A cell
Voxel sizeX=Y=Z: 17.06 Å
Density
Minimum - Maximum-103.0 - 103.0
Average (Standard dev.)11.304837 (±7.4906077)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin15-16-18
Dimensions92896091
Spacing96092891
CellA: 16377.6 Å / B: 15831.68 Å / C: 1552.46 Å
α=β=γ: 90.0 °

CCP4 map header:

modeenvelope stored as signed bytes (from -128 lowest to 127 highest)
Å/pix. X/Y/Z17.0617.0617.06
M x/y/z96092891
origin x/y/z0.0000.0000.000
length x/y/z16377.60015831.6801552.460
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ500500500
MAP C/R/S123
start NC/NR/NS-1615-18
NC/NR/NS96092891
D min/max/mean-103.000103.00011.305

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Supplemental data

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Sample components

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Entire : Cryo-electron tomogram of whole JCVI-Syn3A synthetic mycoplasma.

EntireName: Cryo-electron tomogram of whole JCVI-Syn3A synthetic mycoplasma.
Components
  • Cell: Cryo-electron tomogram of whole JCVI-Syn3A synthetic mycoplasma.

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Supramolecule #1: Cryo-electron tomogram of whole JCVI-Syn3A synthetic mycoplasma.

SupramoleculeName: Cryo-electron tomogram of whole JCVI-Syn3A synthetic mycoplasma.
type: cell / ID: 1 / Parent: 0
Source (natural)Organism: synthetic bacterium JCVI-Syn3A (others)

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron tomography
Aggregation statecell

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Sample preparation

BufferpH: 7.5 / Details: nominal pH of SP4 medium
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.019 kPa / Details: current: 20 mA instrument: PELCO easiGlow
VitrificationCryogen name: ETHANE-PROPANE / Instrument: HOMEMADE PLUNGER
Detailsgrown in SP4 medium to mid-log phase
SectioningOther: NO SECTIONING

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 77.0 K
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3710 pixel / Digitization - Dimensions - Height: 3838 pixel / Digitization - Frames/image: 1-8 / Average exposure time: 1.98 sec. / Average electron dose: 1.72 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus min: 6.0 µm / Nominal magnification: 33000
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

Final reconstructionAlgorithm: BACK PROJECTION / Software - Name: IMOD (ver. 4.10.29) / Number images used: 61

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