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

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Basic information

Entry
Database: PDB / ID: 5kp9
TitleStructure of Nanoparticle Released from Enveloped Protein Nanoparticle
ComponentsEPN-01*
KeywordsSTRUCTURAL PROTEIN / protein design / icosahedral assemblies / cell transduction / enveloped viruses / virus assembly / enveloped protein / nanoparticle
Function / homology
Function and homology information


viral budding via host ESCRT complex / host multivesicular body / viral nucleocapsid / lyase activity / host cell nucleus / structural molecule activity / host cell plasma membrane / virion membrane / RNA binding / zinc ion binding ...viral budding via host ESCRT complex / host multivesicular body / viral nucleocapsid / lyase activity / host cell nucleus / structural molecule activity / host cell plasma membrane / virion membrane / RNA binding / zinc ion binding / membrane / identical protein binding
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) / Retroviral nucleocapsid Gag protein p24, C-terminal domain / Gag protein p24 C-terminal domain / Matrix protein, lentiviral and alpha-retroviral, N-terminal ...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) / Retroviral nucleocapsid Gag protein p24, C-terminal domain / Gag protein p24 C-terminal domain / Matrix protein, lentiviral and alpha-retroviral, N-terminal / Retrovirus capsid, C-terminal / Retroviral matrix protein / 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
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 5.7 Å
AuthorsVotteler, J. / Ogohara, C. / Yi, S. / Hsia, Y. / Natterman, U. / Belnap, D.M. / King, N.P. / Sundquist, W.I.
Funding support Germany, United States, 5items
OrganizationGrant numberCountry
German Research Foundation (DFG)Fellowship VO 1836/1-1 Germany
Bill & Melinda Gates FoundationOPP1118840 United States
Defense Advanced Research Projects AgencyW911NF-14-1-0162 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI51174 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM082545 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, 2016Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 7, 2016Provider: repository / Type: Initial release
Revision 1.1Jan 11, 2017Group: Database references
Revision 1.2Sep 13, 2017Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.3Sep 27, 2017Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.4Jul 18, 2018Group: Data collection / Experimental preparation / Category: em_sample_support / em_software / Item: _em_sample_support.grid_type / _em_software.name
Revision 1.5Nov 27, 2019Group: Author supporting evidence / Category: pdbx_audit_support
Item: _pdbx_audit_support.funding_organization / _pdbx_audit_support.grant_number
Revision 1.6Dec 11, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization

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Structure visualization

Movie
  • Biological unit as complete icosahedral assembly
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  • Biological unit as icosahedral pentamer
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  • Biological unit as icosahedral 23 hexamer
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  • Deposited structure unit
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  • Superimposition on EM map
  • EMDB-8278
  • Imaged by UCSF Chimera
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  • Superimposition on EM map
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Structure viewerMolecule:
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Assembly

Deposited unit
B: EPN-01*


Theoretical massNumber of molelcules
Total (without water)30,3051
Polymers30,3051
Non-polymers00
Water0
1
B: EPN-01*
x 60


Theoretical massNumber of molelcules
Total (without water)1,818,29560
Polymers1,818,29560
Non-polymers00
Water0
TypeNameSymmetry operationNumber
point symmetry operation60
2


  • Idetical with deposited unit in distinct coordinate
  • icosahedral asymmetric unit
TypeNameSymmetry operationNumber
point symmetry operation1
3
B: EPN-01*
x 5


  • icosahedral pentamer
  • 152 kDa, 5 polymers
Theoretical massNumber of molelcules
Total (without water)151,5255
Polymers151,5255
Non-polymers00
Water0
TypeNameSymmetry operationNumber
point symmetry operation5
4
B: EPN-01*
x 6


  • icosahedral 23 hexamer
  • 182 kDa, 6 polymers
Theoretical massNumber of molelcules
Total (without water)181,8306
Polymers181,8306
Non-polymers00
Water0
TypeNameSymmetry operationNumber
point symmetry operation6
5


  • Idetical with deposited unit in distinct coordinate
  • icosahedral asymmetric unit, std point frame
TypeNameSymmetry operationNumber
transform to point frame1
SymmetryPoint symmetry: (Schoenflies symbol: I (icosahedral))

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Components

#1: Protein EPN-01* / Enveloped protein nanoparticle


Mass: 30304.920 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermotoga maritima (bacteria), (gene. exp.) Human immunodeficiency virus type 1 group M subtype B (isolate BH10)
Gene: TM_0066, gag / Strain: isolate BH10 / Cell line (production host): HEK293T / Production host: Homo sapiens (human) / References: UniProt: Q9WXS1, UniProt: P03347

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: EPN-01* / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Thermotoga maritima (bacteria)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293T / Plasmid: EPN-01*
Buffer solutionpH: 7.5 / Details: Phosphate-buffered saline
SpecimenConc.: 0.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: EPN nanoparticles released from vesicles by detergent treatment (0.75% CHAPS)
Specimen supportGrid material: COPPER / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK II / Cryogen name: ETHANE / Humidity: 80 % / Chamber temperature: 277 K / Details: 11 second blot, 0 mm offset

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI F20
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Calibrated magnification: 41911 X / Calibrated defocus min: 700 nm / Calibrated defocus max: 3300 nm / Cs: 2 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN
Specimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Image recordingElectron dose: 2 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Details: Electron dose at specimen was not recorded.
Image scansSampling size: 2.5 µm / Width: 7420 / Height: 7676 / Movie frames/image: 60

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Processing

EM software
IDNameCategory
1Scipionparticle selection
2Xmippparticle selection
3SerialEMimage acquisition
5CTFFINDCTF correction
6ScipionCTF correction
9UCSF Chimeramodel fitting
11RELIONinitial Euler assignment
12Scipioninitial Euler assignment
13RELIONfinal Euler assignment
14Scipionfinal Euler assignment
15RELIONclassification
16Scipionclassification
17RELION3D reconstruction
18Scipion3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 9177
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionResolution: 5.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 8573 / Algorithm: FOURIER SPACE / Num. of class averages: 10 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT

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