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- EMDB-5951: Tomographic reconstruction of negatively-stained GALT tissue from... -

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Entry
Database: EMDB / ID: EMD-5951
TitleTomographic reconstruction of negatively-stained GALT tissue from an HIV-infected BLT (humanized) mouse. Shown is an HIV-1 budding profile that displays structures in the neck region that may represent portions of the host-encoded scission machinery (ESCRT).
Map datanegative-stain tomographic reconstruction of HIV-infected humanized mouse colon
Sample
  • Sample: negative-stain tomographic reconstruction of HIV-infected humanized mouse colon
  • Organelle or cellular component: HIV in Humanized Mouse GALT
KeywordsCellular electron tomography / HIV / GALT / negative-stain / ESCRT / colon / tissue / humanized mouse / BLT / gag
Biological speciesMus musculus (house mouse)
Methodelectron tomography / cryo EM / negative staining
AuthorsLadinsky MS / Kieffer C / Olson G / Deruaz M / Vrbanac V / Tager AM / Kwon DS / Bjorkman PJ
CitationJournal: PLoS Pathog / Year: 2014
Title: Electron tomography of HIV-1 infection in gut-associated lymphoid tissue.
Authors: Mark S Ladinsky / Collin Kieffer / Gregory Olson / Maud Deruaz / Vladimir Vrbanac / Andrew M Tager / Douglas S Kwon / Pamela J Bjorkman /
Abstract: Critical aspects of HIV-1 infection occur in mucosal tissues, particularly in the gut, which contains large numbers of HIV-1 target cells that are depleted early in infection. We used electron ...Critical aspects of HIV-1 infection occur in mucosal tissues, particularly in the gut, which contains large numbers of HIV-1 target cells that are depleted early in infection. We used electron tomography (ET) to image HIV-1 in gut-associated lymphoid tissue (GALT) of HIV-1-infected humanized mice, the first three-dimensional ultrastructural examination of HIV-1 infection in vivo. Human immune cells were successfully engrafted in the mice, and following infection with HIV-1, human T cells were reduced in GALT. Virions were found by ET at all stages of egress, including budding immature virions and free mature and immature viruses. Immuno-electron microscopy verified the virions were HIV-1 and showed CD4 sequestration in the endoplasmic reticulum of infected cells. Observation of HIV-1 in infected GALT tissue revealed that most HIV-1-infected cells, identified by immunolabeling and/or the presence of budding virions, were localized to intestinal crypts with pools of free virions concentrated in spaces between cells. Fewer infected cells were found in mucosal regions and the lamina propria. The preservation quality of reconstructed tissue volumes allowed details of budding virions, including structures interpreted as host-encoded scission machinery, to be resolved. Although HIV-1 virions released from infected cultured cells have been described as exclusively mature, we found pools of both immature and mature free virions within infected tissue. The pools could be classified as containing either mostly mature or mostly immature particles, and analyses of their proximities to the cell of origin supported a model of semi-synchronous waves of virion release. In addition to HIV-1 transmission by pools of free virus, we found evidence of transmission via virological synapses. Three-dimensional EM imaging of an active infection within tissue revealed important differences between cultured cell and tissue infection models and furthered the ultrastructural understanding of HIV-1 transmission within lymphoid tissue.
History
DepositionApr 21, 2014-
Header (metadata) releaseApr 30, 2014-
Map releaseApr 30, 2014-
UpdateApr 30, 2014-
Current statusApr 30, 2014Processing site: RCSB / Status: Released

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Structure visualization

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Structure viewerEM map:
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Supplemental images

400_5951.gif

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Map

FileDownload / File: emd_5951.map.gz / Format: CCP4 / Size: 141.5 MB / Type: IMAGE STORED AS SIGNED BYTE
Annotationnegative-stain tomographic reconstruction of HIV-infected humanized mouse colon
Voxel sizeX=Y=Z: 9.557 Å
Density
Minimum - Maximum-128.0 - 127.0
Average (Standard dev.)5.92945957 (±25.487979889999998)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-244014
Dimensions1279204858
Spacing1279204858
CellA: 19572.736 Å / B: 12223.403 Å / C: 554.306 Å
α=β=γ: 90.0 °

CCP4 map header:

modeenvelope stored as signed bytes (from -128 lowest to 127 highest)
Å/pix. X/Y/Z9.5579.5579.557
M x/y/z2048127958
origin x/y/z0.0000.0000.000
length x/y/z19572.73612223.403554.306
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ969680
MAP C/R/S123
start NC/NR/NS0-24414
NC/NR/NS2048127958
D min/max/mean-128.000127.0005.929

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Supplemental data

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Sample components

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Entire : negative-stain tomographic reconstruction of HIV-infected humaniz...

EntireName: negative-stain tomographic reconstruction of HIV-infected humanized mouse colon
Components
  • Sample: negative-stain tomographic reconstruction of HIV-infected humanized mouse colon
  • Organelle or cellular component: HIV in Humanized Mouse GALT

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Supramolecule #1000: negative-stain tomographic reconstruction of HIV-infected humaniz...

SupramoleculeName: negative-stain tomographic reconstruction of HIV-infected humanized mouse colon
type: sample / ID: 1000 / Details: Cellular Tomographic Reconstruction / Number unique components: 1

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Supramolecule #1: HIV in Humanized Mouse GALT

SupramoleculeName: HIV in Humanized Mouse GALT / type: organelle_or_cellular_component / ID: 1 / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Mus musculus (house mouse) / synonym: Mouse / Location in cell: Gut associated lymphoid tissue

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingelectron tomography
Aggregation statetissue

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Sample preparation

StainingType: NEGATIVE
Details: Negatively-stained (1% uranyl acetate), sucrose-impregnated cryosection of mouse tissue, imaged by electron tomography
VitrificationCryogen name: NITROGEN / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI 12
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus min: 0.5 µm / Nominal magnification: 6500
Sample stageSpecimen holder model: OTHER / Tilt series - Axis1 - Min angle: -60 ° / Tilt series - Axis1 - Max angle: 60 ° / Tilt series - Axis1 - Angle increment: 1 °
DateJul 13, 2012
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 1000 (2k x 2k)

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Image processing

Final reconstructionSoftware - Name: SerialEM, IMOD / Number images used: 242
DetailsTomogram acquired using SerialEM software package on a Tecai T-12 EM. Tomograms were analyzed using IMOD.

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