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- EMDB-25894: Single-molecule 3D density map of HIV cellular entry by liquid-ph... -

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Basic information

Entry
Database: EMDB / ID: EMD-25894
TitleSingle-molecule 3D density map of HIV cellular entry by liquid-phase electron tomography (particle #3)
Map data
Sample
  • Organelle or cellular component: A HIV totally enter cell membrane
    • Organelle or cellular component: cell membrane
    • Virus: Human immunodeficiency virus
KeywordsHIV cellular entry / CELL INVASION
Biological speciesHomo sapiens (human) / Human immunodeficiency virus
Methodelectron tomography / Resolution: 65.0 Å
AuthorsKong L / Ren G
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)R01HL115153 United States
National Institutes of Health/National Institute of Mental Health (NIH/NIMH)R01MH077303 United States
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK)R01DK042667 United States
CitationJournal: Nat Commun / Year: 2023
Title: Facile hermetic TEM grid preparation for molecular imaging of hydrated biological samples at room temperature.
Authors: Lingli Kong / Jianfang Liu / Meng Zhang / Zhuoyang Lu / Han Xue / Amy Ren / Jiankang Liu / Jinping Li / Wai Li Ling / Gang Ren /
Abstract: Although structures of vitrified supramolecular complexes have been determined at near-atomic resolution, elucidating in situ molecular structure in living cells remains a challenge. Here, we report ...Although structures of vitrified supramolecular complexes have been determined at near-atomic resolution, elucidating in situ molecular structure in living cells remains a challenge. Here, we report a straightforward liquid cell technique, originally developed for real-time visualization of dynamics at a liquid-gas interface using transmission electron microscopy, to image wet biological samples. Due to the scattering effects from the liquid phase, the micrographs display an amplitude contrast comparable to that observed in negatively stained samples. We succeed in resolving subunits within the protein complex GroEL imaged in a buffer solution at room temperature. Additionally, we capture various stages of virus cell entry, a process for which only sparse structural data exists due to their transient nature. To scrutinize the morphological details further, we used individual particle electron tomography for 3D reconstruction of each virus. These findings showcase this approach potential as an efficient, cost-effective complement to other microscopy technique in addressing biological questions at the molecular level.
History
DepositionJan 11, 2022-
Header (metadata) releaseAug 16, 2023-
Map releaseAug 16, 2023-
UpdateSep 27, 2023-
Current statusSep 27, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_25894.png

Downloads & links

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Map

FileDownload / File: emd_25894.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 21.44 Å
Density
Minimum - Maximum-0.4023509 - 1.4776587
Average (Standard dev.)0.028734766 (±0.10721366)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-64-64-64
Dimensions128128128
Spacing128128128
CellA=B=C: 2744.32 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : A HIV totally enter cell membrane

EntireName: A HIV totally enter cell membrane
Components
  • Organelle or cellular component: A HIV totally enter cell membrane
    • Organelle or cellular component: cell membrane
    • Virus: Human immunodeficiency virus

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Supramolecule #1: A HIV totally enter cell membrane

SupramoleculeName: A HIV totally enter cell membrane / type: organelle_or_cellular_component / ID: 1 / Parent: 0
Details: In the penetrating stage of viral entry, in which a virus half embeds into the cell membrane.

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Supramolecule #3: cell membrane

SupramoleculeName: cell membrane / type: organelle_or_cellular_component / ID: 3 / Parent: 1
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #2: Human immunodeficiency virus

SupramoleculeName: Human immunodeficiency virus / type: virus / ID: 2 / Parent: 1 / NCBI-ID: 12721 / Sci species name: Human immunodeficiency virus / Virus type: VIRION / Virus isolate: OTHER / Virus enveloped: Yes / Virus empty: No
Host (natural)Organism: Homo sapiens (human)

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Experimental details

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Structure determination

Processingelectron tomography
Aggregation statecell

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Sample preparation

BufferpH: 7.4
Component:
ConcentrationName
100.0 %Gibco minimum essential media
10.0 %FBS

Details: HeLa cells were grown in Gibco minimum essential media (MEM, containing 10 percent FBS) at 37 degree with 5 percent CO2, and virus-infected cells were prepared by incubating HeLa cells with ...Details: HeLa cells were grown in Gibco minimum essential media (MEM, containing 10 percent FBS) at 37 degree with 5 percent CO2, and virus-infected cells were prepared by incubating HeLa cells with virus at a ratio of 50 to 1 for 12 hours.
Sugar embeddingMaterial: sanwiched by two formvar films
Details: sample solution (native liquid state, label-free, without any staining) was deposited and then sandwiched between two layers of 300 mesh TEM Formvar-coated copper grids at room temperature.
GridModel: Homemade / Material: COPPER / Support film - Material: FORMVAR / Support film - topology: CONTINUOUS
DetailsHeLa cells were grown in Gibco minimum essential media and virus-infected cells were prepared by incubating HeLa cells with virus at a ratio of 50:1 for 12 hours.
Cryo protectantLiquid-phase
SectioningOther: NO SECTIONING

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Electron microscopy

MicroscopeZEISS LIBRA120PLUS
Specialist opticsEnergy filter - Name: In-column Omega Filter / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 97 / Average electron dose: 1.0 e/Å2
Details: The total illumination electron doses were ~30 electron per angstrom square.
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsCalibrated defocus max: 11.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.4 mm / Nominal defocus max: 11.0 µm / Nominal defocus min: 3.0 µm

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Image processing

DetailsThe tilt series of the targeted particles, including HeLa cells, viruses and nanoparticles, was reconstructed by IPET software (PLoS ONE, 2012, 7(1): e30249). In brief, a circular mask with a Gaussian edge was applied to each image, followed by 3D reconstruction via an iteration refinement process with a series of soft-boundary masks and filters. To display the objects with positive or negative contrast, a superimposed 3D density map was generated by combining the positive contour map of the final IPET 3D density maps with its negative contour map by using Chimera software. The resolution of the final 3D map was estimated based on the intra-Fourier shell correlation (FSC). Briefly, the aligned images were then split into two groups based on having an odd- or even-numbered tilting index so that two 3D reconstructions were generated to compute the FSC curves, and the frequency at which the FSC curve fell to a value of 0.5 was used to represent the resolution.
Final reconstructionAlgorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 65.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: IPET (ver. 1.0) / Software - details: PLoS ONE, 2012, 7(1): e30249 / Number images used: 97
FSC plot (resolution estimation)

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