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- EMDB-6533: Electron cryo-microscopy of human TAP transporter inhibited by th... -

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Basic information

Entry
Database: EMDB / ID: EMD-6533
TitleElectron cryo-microscopy of human TAP transporter inhibited by the ICP47 viral inhibitor from herpes simplex virus type 1
Map dataReconstruction of human TAP transporter inhibited by herpes simplex virus type 1 ICP47 with Factor correction
Sample
  • Sample: TAP1/TAP2 heterodimer bound to herpes simplex virus type 1 inhibitor, ICP47
  • Protein or peptide: TAP1
  • Protein or peptide: TAP2
  • Protein or peptide: ICP47
Keywordsviral invasion / ATP-Binding / Cassette (ABC) transporter / antigen processing / major histocompatibility complex class 1 / inward-facing
Function / homology
Function and homology information


MHC protein binding / ABC-type peptide antigen transporter activity / TAP complex / ABC-type peptide transporter activity / antigen processing and presentation of exogenous peptide antigen via MHC class I, TAP-dependent / symbiont-mediated suppression of host adaptive immune response / symbiont-mediated suppression of host antigen processing and presentation / TAP2 binding / MHC class I protein binding / defense response ...MHC protein binding / ABC-type peptide antigen transporter activity / TAP complex / ABC-type peptide transporter activity / antigen processing and presentation of exogenous peptide antigen via MHC class I, TAP-dependent / symbiont-mediated suppression of host adaptive immune response / symbiont-mediated suppression of host antigen processing and presentation / TAP2 binding / MHC class I protein binding / defense response / antigen processing and presentation of endogenous peptide antigen via MHC class I / host cell cytoplasm / adaptive immune response / nuclear speck / host cell nucleus / endoplasmic reticulum / ATP binding / cytoplasm
Similarity search - Function
Antigen peptide transporter 1 / Antigen peptide transporter 1 / Antigen peptide transporter 2 / Antigen peptide transporter 2 / Herpesvirus ICP47 / Herpesvirus ICP47 / Herpesvirus US12 family / ABC transporter Tap-like / Type 1 protein exporter / ABC transporter transmembrane region ...Antigen peptide transporter 1 / Antigen peptide transporter 1 / Antigen peptide transporter 2 / Antigen peptide transporter 2 / Herpesvirus ICP47 / Herpesvirus ICP47 / Herpesvirus US12 family / ABC transporter Tap-like / Type 1 protein exporter / ABC transporter transmembrane region / ABC transporter type 1, transmembrane domain / ABC transporter integral membrane type-1 fused domain profile. / ABC transporter type 1, transmembrane domain superfamily / ABC transporter-like, conserved site / ABC transporters family signature. / ABC transporter / ABC transporter-like, ATP-binding domain / ATP-binding cassette, ABC transporter-type domain profile. / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ICP47 protein / TAP2 / TAP1
Similarity search - Component
Biological speciesHomo sapiens (human) / Human herpesvirus 1 (Herpes simplex virus type 1)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.5 Å
AuthorsOldham ML / Hite RK / Steffen AM / Damko E / Li Z / Walz T / Chen J
CitationJournal: Nature / Year: 2016
Title: A mechanism of viral immune evasion revealed by cryo-EM analysis of the TAP transporter.
Authors: Michael L Oldham / Richard K Hite / Alanna M Steffen / Ermelinda Damko / Zongli Li / Thomas Walz / Jue Chen /
Abstract: Cellular immunity against viral infection and tumour cells depends on antigen presentation by major histocompatibility complex class I (MHC I) molecules. Intracellular antigenic peptides are ...Cellular immunity against viral infection and tumour cells depends on antigen presentation by major histocompatibility complex class I (MHC I) molecules. Intracellular antigenic peptides are transported into the endoplasmic reticulum by the transporter associated with antigen processing (TAP) and then loaded onto the nascent MHC I molecules, which are exported to the cell surface and present peptides to the immune system. Cytotoxic T lymphocytes recognize non-self peptides and program the infected or malignant cells for apoptosis. Defects in TAP account for immunodeficiency and tumour development. To escape immune surveillance, some viruses have evolved strategies either to downregulate TAP expression or directly inhibit TAP activity. So far, neither the architecture of TAP nor the mechanism of viral inhibition has been elucidated at the structural level. Here we describe the cryo-electron microscopy structure of human TAP in complex with its inhibitor ICP47, a small protein produced by the herpes simplex virus I. Here we show that the 12 transmembrane helices and 2 cytosolic nucleotide-binding domains of the transporter adopt an inward-facing conformation with the two nucleotide-binding domains separated. The viral inhibitor ICP47 forms a long helical hairpin, which plugs the translocation pathway of TAP from the cytoplasmic side. Association of ICP47 precludes substrate binding and prevents nucleotide-binding domain closure necessary for ATP hydrolysis. This work illustrates a striking example of immune evasion by persistent viruses. By blocking viral antigens from entering the endoplasmic reticulum, herpes simplex virus is hidden from cytotoxic T lymphocytes, which may contribute to establishing a lifelong infection in the host.
History
DepositionNov 20, 2015-
Header (metadata) releaseJan 20, 2016-
Map releaseJan 20, 2016-
UpdateFeb 3, 2016-
Current statusFeb 3, 2016Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

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Map

FileDownload / File: emd_6533.map.gz / Format: CCP4 / Size: 62.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of human TAP transporter inhibited by herpes simplex virus type 1 ICP47 with Factor correction
Voxel sizeX=Y=Z: 1.35 Å
Density
Contour LevelBy EMDB: 0.05 / Movie #1: 0.05
Minimum - Maximum-0.04499828 - 0.15952149
Average (Standard dev.)0.0001944 (±0.00494422)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 345.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.351.351.35
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z345.600345.600345.600
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0450.1600.000

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Supplemental data

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Sample components

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Entire : TAP1/TAP2 heterodimer bound to herpes simplex virus type 1 inhibi...

EntireName: TAP1/TAP2 heterodimer bound to herpes simplex virus type 1 inhibitor, ICP47
Components
  • Sample: TAP1/TAP2 heterodimer bound to herpes simplex virus type 1 inhibitor, ICP47
  • Protein or peptide: TAP1
  • Protein or peptide: TAP2
  • Protein or peptide: ICP47

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Supramolecule #1000: TAP1/TAP2 heterodimer bound to herpes simplex virus type 1 inhibi...

SupramoleculeName: TAP1/TAP2 heterodimer bound to herpes simplex virus type 1 inhibitor, ICP47
type: sample / ID: 1000
Oligomeric state: One heterodimer of TAP1 and TAP2 bound to one monomer of ICP47
Number unique components: 3
Molecular weightTheoretical: 166.4 KDa

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Macromolecule #1: TAP1

MacromoleculeName: TAP1 / type: protein_or_peptide / ID: 1
Name.synonym: transporter associated with antigen processing 1, ABCB2
Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Organelle: Endoplasmic reticulum / Location in cell: membrane
Molecular weightTheoretical: 80.964 KDa
Recombinant expressionOrganism: Komagataella pastoris (fungus) / Recombinant strain: SMD1163 His+ / Recombinant plasmid: modified pPICZ A
SequenceUniProtKB: TAP1 / InterPro: Antigen peptide transporter 1

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Macromolecule #2: TAP2

MacromoleculeName: TAP2 / type: protein_or_peptide / ID: 2
Name.synonym: transporter associated with antigen processing 2, ABCB3
Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Organelle: Endoplasmic reticulum / Location in cell: membrane
Molecular weightTheoretical: 75.6638 KDa
Recombinant expressionOrganism: Komagataella pastoris (fungus) / Recombinant strain: SMD1163 His+ / Recombinant plasmid: modified pPICZ A
SequenceUniProtKB: TAP2 / InterPro: Antigen peptide transporter 2

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Macromolecule #3: ICP47

MacromoleculeName: ICP47 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Human herpesvirus 1 (Herpes simplex virus type 1) / Organelle: Endoplasmic reticulum / Location in cell: membrane
Molecular weightTheoretical: 9.806 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21(DE3) RIL / Recombinant plasmid: pMCSG20
SequenceUniProtKB: ICP47 protein / InterPro: Herpesvirus ICP47

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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

Concentration2 mg/mL
BufferpH: 7.4
Details: 20 mM HEPES sodium, 150 mM NaCl, 2 mM TCEP, 1 mM n-Dodecyl-beta-D-Maltopyranoside (DDM), 1 mM Octaethylene Glycol Monododecyl Ether (C12E8)
GridDetails: C-flat grids (CF-1.2/1.3-4C), 1.2 micron hole, 1.3 micron space, 400 mesh copper TEM grid, glow-discharged in partial air vacuum
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 120 K / Instrument: FEI VITROBOT MARK IV / Method: Blot for 4 seconds before plunging.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 81000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 0.01 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 1.6 µm / Nominal magnification: 81000
Specialist opticsEnergy filter - Name: Gatan / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV
Sample stageSpecimen holder: liquid nitrogen-cooled / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
TemperatureAverage: 120 K
Alignment procedureLegacy - Astigmatism: FEI Image corrector
DetailsBRIGHT FIELD illumination
DateAug 3, 2015
Image recordingCategory: CCD / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Digitization - Sampling interval: 5 µm / Number real images: 5163 / Average electron dose: 88 e/Å2
Details: 40 sub-frames were recorded for each image in super-resolution counting mode.
Bits/pixel: 32
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: CTFFIND4; Each image
Final two d classificationNumber classes: 104
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 6.5 Å / Resolution method: OTHER / Software - Name: SPARX, RELION, FREALIGN
Details: Manually picked particles with BOXER and RELION. 2D and 3D classification in RELION. Final map generated with FREALIGN.
Number images used: 139293
DetailsThe particles were selected using automatic selection in RELION.

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