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- EMDB-8278: Structure of Nanoparticle Released from Enveloped Protein Nanoparticle -

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Basic information

Entry
Database: EMDB / ID: EMD-8278
TitleStructure of Nanoparticle Released from Enveloped Protein Nanoparticle
Map dataIcosahedrally-symmetrized, average reconstruction of nanoparticle released from EPN
Sample
  • Complex: EPN-01*
    • Protein or peptide: EPN-01*
Function / homology
Function and homology information


viral budding via host ESCRT complex / host multivesicular body / viral nucleocapsid / lyase activity / viral translational frameshifting / host cell nucleus / host cell plasma membrane / structural molecule activity / virion membrane / RNA binding ...viral budding via host ESCRT complex / host multivesicular body / viral nucleocapsid / lyase activity / viral translational frameshifting / host cell nucleus / host cell plasma membrane / structural molecule activity / virion membrane / RNA binding / zinc ion binding / identical protein binding / membrane
Similarity search - Function
KDPG/KHG aldolase / KDPG and KHG aldolase / Gag protein p6 / Gag protein p6 / : / gag protein p24 N-terminal domain / Immunodeficiency lentiviral matrix, N-terminal / gag gene protein p17 (matrix protein) / Matrix protein, lentiviral and alpha-retroviral, N-terminal / Retroviral nucleocapsid Gag protein p24, C-terminal domain ...KDPG/KHG aldolase / KDPG and KHG aldolase / Gag protein p6 / Gag protein p6 / : / gag protein p24 N-terminal domain / Immunodeficiency lentiviral matrix, N-terminal / gag gene protein p17 (matrix protein) / Matrix protein, lentiviral and alpha-retroviral, N-terminal / Retroviral nucleocapsid Gag protein p24, C-terminal domain / Gag protein p24 C-terminal domain / Retroviral matrix protein / Retrovirus capsid, C-terminal / Retrovirus capsid, N-terminal / zinc finger / Zinc knuckle / Zinc finger, CCHC-type superfamily / Zinc finger, CCHC-type / Zinc finger CCHC-type profile. / Aldolase-type TIM barrel
Similarity search - Domain/homology
Gag polyprotein / 2-dehydro-3-deoxyphosphogluconate aldolase/4-hydroxy-2-oxoglutarate aldolase
Similarity search - Component
Biological speciesThermotoga maritima (bacteria) / Human immunodeficiency virus type 1 group M subtype B (isolate BH10)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.7 Å
AuthorsVotteler J / Ogohara C / Yi S / Hsia Y / Natterman U / Belnap DM / King NP / Sundquist WI
Funding support Germany, United States, 5 items
OrganizationGrant numberCountry
German Research Foundation (DFG)Fellowship VO 1836/1-1 Germany
Defense Advanced Research Projects AgencyW911NF-14-1-0162 United States
Bill & Melinda Gates FoundationOPP1118840 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM082545 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI51174 United States
CitationJournal: Nature / Year: 2016
Title: Designed proteins induce the formation of nanocage-containing extracellular vesicles.
Authors: Jörg Votteler / Cassandra Ogohara / Sue Yi / Yang Hsia / Una Nattermann / David M Belnap / Neil P King / Wesley I Sundquist /
Abstract: Complex biological processes are often performed by self-organizing nanostructures comprising multiple classes of macromolecules, such as ribosomes (proteins and RNA) or enveloped viruses (proteins, ...Complex biological processes are often performed by self-organizing nanostructures comprising multiple classes of macromolecules, such as ribosomes (proteins and RNA) or enveloped viruses (proteins, nucleic acids and lipids). Approaches have been developed for designing self-assembling structures consisting of either nucleic acids or proteins, but strategies for engineering hybrid biological materials are only beginning to emerge. Here we describe the design of self-assembling protein nanocages that direct their own release from human cells inside small vesicles in a manner that resembles some viruses. We refer to these hybrid biomaterials as 'enveloped protein nanocages' (EPNs). Robust EPN biogenesis requires protein sequence elements that encode three distinct functions: membrane binding, self-assembly, and recruitment of the endosomal sorting complexes required for transport (ESCRT) machinery. A variety of synthetic proteins with these functional elements induce EPN biogenesis, highlighting the modularity and generality of the design strategy. Biochemical analyses and cryo-electron microscopy reveal that one design, EPN-01, comprises small (~100 nm) vesicles containing multiple protein nanocages that closely match the structure of the designed 60-subunit self-assembling scaffold. EPNs that incorporate the vesicular stomatitis viral glycoprotein can fuse with target cells and deliver their contents, thereby transferring cargoes from one cell to another. These results show how proteins can be programmed to direct the formation of hybrid biological materials that perform complex tasks, and establish EPNs as a class of designed, modular, genetically-encoded nanomaterials that can transfer molecules between cells.
History
DepositionJul 2, 2016-
Header (metadata) releaseAug 10, 2016-
Map releaseDec 7, 2016-
UpdateDec 11, 2019-
Current statusDec 11, 2019Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0263
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.0263
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5kp9
  • Surface level: 0.0263
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-5kp9
  • Surface level: 0.035
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8278.png

Downloads & links

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Map

FileDownload / File: emd_8278.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationIcosahedrally-symmetrized, average reconstruction of nanoparticle released from EPN
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.19 Å/pix.
x 300 pix.
= 357.9 Å		1.19 Å/pix.
x 300 pix.
= 357.9 Å		1.19 Å/pix.
x 300 pix.
= 357.9 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.193 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0263 / Movie #1: 0.0263
Minimum - Maximum-0.021441469 - 0.07595806
Average (Standard dev.)0.0013056165 (±0.005869076)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-150-150-150
Dimensions300300300
Spacing300300300
CellA=B=C: 357.9 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.1931.1931.193
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z357.900357.900357.900
α/β/γ90.00090.00090.000
start NX/NY/NZ-64-64-64
NX/NY/NZ128128128
MAP C/R/S123
start NC/NR/NS-150-150-150
NC/NR/NS300300300
D min/max/mean-0.0210.0760.001

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Supplemental data

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Sample components

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Entire : EPN-01*

EntireName: EPN-01*
Components
  • Complex: EPN-01*
    • Protein or peptide: EPN-01*

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Supramolecule #1: EPN-01*

SupramoleculeName: EPN-01* / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Thermotoga maritima (bacteria)
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293T / Recombinant plasmid: EPN-01*

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Macromolecule #1: EPN-01*

MacromoleculeName: EPN-01* / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Human immunodeficiency virus type 1 group M subtype B (isolate BH10)
Strain: isolate BH10
Molecular weightTheoretical: 30.30492 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MGARASGSKS GSGSDSGSKM EELFKKHKIV AVLRANSVEE AKKKALAVFL GGVHLIEITF TVPDADTVIK ELSFLKEMGA IIGAGTVTS VEQCRKAVES GAEFIVSPHL DEEISQFCKE KGVFYMPGVM TPTELVKAMK LGHTILKLFP GEVVGPQFVK A MKGPFPNV ...String:
MGARASGSKS GSGSDSGSKM EELFKKHKIV AVLRANSVEE AKKKALAVFL GGVHLIEITF TVPDADTVIK ELSFLKEMGA IIGAGTVTS VEQCRKAVES GAEFIVSPHL DEEISQFCKE KGVFYMPGVM TPTELVKAMK LGHTILKLFP GEVVGPQFVK A MKGPFPNV KFVPTGGVNL DNVCEWFKAG VLAVGVGSAL VKGTPVEVAE KAKAFVEKIR GCTEQKLISE EDLQSRPEPT AP PEESFRS GVETTTPPQK QEPIDKELYP LTSLRSLFGN DPSSQ

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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

Concentration0.2 mg/mL
BufferpH: 7.5 / Details: Phosphate-buffered saline
GridModel: Quantifoil R2/2 / Material: COPPER / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 80 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK II / Details: 11 second blot, 0 mm offset.
DetailsEPN nanoparticles released from vesicles by detergent treatment (0.75% CHAPS)

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Electron microscopy

MicroscopeFEI TECNAI F20
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 7420 pixel / Digitization - Dimensions - Height: 7676 pixel / Digitization - Sampling interval: 2.5 µm / Average electron dose: 2.0 e/Å2 / Details: Electron dose at specimen was not recorded.
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 3.3 µm / Calibrated defocus min: 0.7 µm / Calibrated magnification: 41911 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm
Sample stageSpecimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 9177
CTF correctionSoftware: (Name: CTFFIND, Scipion)
Startup modelType of model: OTHER
Details: Map generated by Xmipp RANSAC protocol, icosahedral symmetry applied
Final reconstructionNumber classes used: 10 / Applied symmetry - Point group: I (icosahedral) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 5.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software: (Name: RELION, Scipion) / Number images used: 8573
Initial angle assignmentType: PROJECTION MATCHING / Software: (Name: RELION, Scipion)
Final angle assignmentType: PROJECTION MATCHING / Software: (Name: RELION, Scipion)
Final 3D classificationNumber classes: 20 / Software: (Name: RELION, Scipion)

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Atomic model buiding 1

RefinementProtocol: RIGID BODY FIT
Output model

PDB-5kp9:
Structure of Nanoparticle Released from Enveloped Protein Nanoparticle

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