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- EMDB-28862: KWOCA 4 nanoparticle -

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Basic information

Entry
Database: EMDB / ID: EMD-28862
TitleKWOCA 4 nanoparticle
Map dataKWOCA 4 nanoparticle - The main cryoEM map used for data interpretation
Sample
  • Complex: Designed octahedral nanoparticle KWOCA 4
    • Protein or peptide: KWOCA 4
Biological speciessynthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.57 Å
AuthorsAntanasijevic A / Ward AB
Funding support United States, 1 items
OrganizationGrant numberCountry
International AIDS Vaccine InitiativeOPP1196345 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2023
Title: Improving the secretion of designed protein assemblies through negative design of cryptic transmembrane domains.
Authors: Jing Yang John Wang / Alena Khmelinskaia / William Sheffler / Marcos C Miranda / Aleksandar Antanasijevic / Andrew J Borst / Susana V Torres / Chelsea Shu / Yang Hsia / Una Nattermann / ...Authors: Jing Yang John Wang / Alena Khmelinskaia / William Sheffler / Marcos C Miranda / Aleksandar Antanasijevic / Andrew J Borst / Susana V Torres / Chelsea Shu / Yang Hsia / Una Nattermann / Daniel Ellis / Carl Walkey / Maggie Ahlrichs / Sidney Chan / Alex Kang / Hannah Nguyen / Claire Sydeman / Banumathi Sankaran / Mengyu Wu / Asim K Bera / Lauren Carter / Brooke Fiala / Michael Murphy / David Baker / Andrew B Ward / Neil P King /
Abstract: Computationally designed protein nanoparticles have recently emerged as a promising platform for the development of new vaccines and biologics. For many applications, secretion of designed ...Computationally designed protein nanoparticles have recently emerged as a promising platform for the development of new vaccines and biologics. For many applications, secretion of designed nanoparticles from eukaryotic cells would be advantageous, but in practice, they often secrete poorly. Here we show that designed hydrophobic interfaces that drive nanoparticle assembly are often predicted to form cryptic transmembrane domains, suggesting that interaction with the membrane insertion machinery could limit efficient secretion. We develop a general computational protocol, the Degreaser, to design away cryptic transmembrane domains without sacrificing protein stability. The retroactive application of the Degreaser to previously designed nanoparticle components and nanoparticles considerably improves secretion, and modular integration of the Degreaser into design pipelines results in new nanoparticles that secrete as robustly as naturally occurring protein assemblies. Both the Degreaser protocol and the nanoparticles we describe may be broadly useful in biotechnological applications.
History
DepositionNov 12, 2022-
Header (metadata) releaseMar 29, 2023-
Map releaseMar 29, 2023-
UpdateMar 29, 2023-
Current statusMar 29, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_28862.png

Downloads & links

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Map

FileDownload / File: emd_28862.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationKWOCA 4 nanoparticle - The main cryoEM map used for data interpretation
Voxel sizeX=Y=Z: 1.15 Å
Density
Contour LevelBy AUTHOR: 0.005
Minimum - Maximum-0.0077859103 - 0.024581805
Average (Standard dev.)0.000105992316 (±0.001161157)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 368.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_28862_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: KWOCA 4 nanoparticle - Half Map 1

Fileemd_28862_half_map_1.map
AnnotationKWOCA 4 nanoparticle - Half Map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: KWOCA 4 nanoparticle - Half Map 2

Fileemd_28862_half_map_2.map
AnnotationKWOCA 4 nanoparticle - Half Map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Designed octahedral nanoparticle KWOCA 4

EntireName: Designed octahedral nanoparticle KWOCA 4
Components
  • Complex: Designed octahedral nanoparticle KWOCA 4
    • Protein or peptide: KWOCA 4

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Supramolecule #1: Designed octahedral nanoparticle KWOCA 4

SupramoleculeName: Designed octahedral nanoparticle KWOCA 4 / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: all
Details: Self-assembling nanoparticle expressed in mammalian cells
Source (natural)Organism: synthetic construct (others)

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Macromolecule #1: KWOCA 4

MacromoleculeName: KWOCA 4 / type: protein_or_peptide / ID: 1 / Details: Sequence for expression in mammalian cells / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: METDTLLLWV LLLWVPGSTG DGSHHHHHHG GSEQKLISEE DLSGGGSWSG STEVEKKARE VAKEAVELAS LLRSETAIRV AQAILEAAEA AKRAAEQGKT EVAKLALKVL EEAIELAKEK RSEEALKVVL EIARAALAAA QAAEEGFTDV AKMALEVLER AIELAKDDRS ...String:
METDTLLLWV LLLWVPGSTG DGSHHHHHHG GSEQKLISEE DLSGGGSWSG STEVEKKARE VAKEAVELAS LLRSETAIRV AQAILEAAEA AKRAAEQGKT EVAKLALKVL EEAIELAKEK RSEEALKVVL EIARAALAAA QAAEEGFTDV AKMALEVLER AIELAKDDRS EEALKEVLEI ARAALAAAQL AKKGRDDEAR KILMKLRIRI TLRKLEESLR ELRRILEELK EMLERLEKNP DKDVIVKVLK VIVKAIEASV ENQRISAENQ KALAELA

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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

Concentration3.58 mg/mL
BufferpH: 8
Component:
ConcentrationNameFormula
20.0 mMTris-HClTris
150.0 mMSodium chlorideNaClSodium chloride
5.0 %GlycerolGlycerol

Details: TBS + 5% glycerol
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 10 sec. / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK IV / Details: Blotting time varied between 3 and 7 seconds..
DetailsSelf-assembling nanoparticle was expressed in mammalian cells and purified using affinity chromatography and size-exclusion chromatography.

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 36000
Sample stageSpecimen holder model: OTHER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-49 / Number grids imaged: 1 / Number real images: 1341 / Average exposure time: 9.8 sec. / Average electron dose: 50.1 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 130000 / Details: CryoSPARC template picker
Startup modelType of model: OTHER
Details: Map obtained from Ab initio reconstruction in cryoSPARC with application of symmetry
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final 3D classificationSoftware - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: O (octahedral) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 6.57 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 17416
DetailsFrames were aligned using MotionCorr and GCTF was applied for estimation of CTF parameters.
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

4zk7

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