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- EMDB-28929: Improving the secretion of designed protein assemblies through ne... -

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Basic information

Entry
Database: EMDB / ID: EMD-28929
TitleImproving the secretion of designed protein assemblies through negative design of cryptic transmembrane domains - KWOCA51
Map dataUnsharpened Map
Sample
  • Complex: KWOCA51
    • Protein or peptide: KWOCA51
Keywordstetradron / KWOCA / secretable / DE NOVO PROTEIN / design / degreaser / cage / vaccine
Biological speciessynthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.87 Å
AuthorsBorst AJ / King NP
Funding support United States, 1 items
OrganizationGrant numberCountry
Bill & Melinda Gates Foundation 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 21, 2022-
Header (metadata) releaseMar 22, 2023-
Map releaseMar 22, 2023-
UpdateJan 17, 2024-
Current statusJan 17, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_28929.png

Downloads & links

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Map

FileDownload / File: emd_28929.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationUnsharpened Map
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.84 Å/pix.
x 360 pix.
= 302.4 Å		0.84 Å/pix.
x 360 pix.
= 302.4 Å		0.84 Å/pix.
x 360 pix.
= 302.4 Å

Surface

Projections

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.84 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.161
Minimum - Maximum-0.30373022 - 0.5903399
Average (Standard dev.)-0.0023801876 (±0.02469611)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 302.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Sharpened Map

Fileemd_28929_additional_1.map
AnnotationSharpened Map
Projections & Slices
AxesZYX

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Half map: Half Map A

Fileemd_28929_half_map_1.map
AnnotationHalf Map A
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Half map: Half Map B

Fileemd_28929_half_map_2.map
AnnotationHalf Map B
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Sample components

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Entire : KWOCA51

EntireName: KWOCA51
Components
  • Complex: KWOCA51
    • Protein or peptide: KWOCA51

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Supramolecule #1: KWOCA51

SupramoleculeName: KWOCA51 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: synthetic construct (others)

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Macromolecule #1: KWOCA51

MacromoleculeName: KWOCA51 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
SequenceString: METDTLLLWV LLLWVPGSTG DGSHHHHHHG GSEQKLISEE DLSGGGSWSG SSDEEEAREW AERAEEAAKE ALEQAKREGD EIARLCAKML EILAEEARRK KDSEEAEAVY WAARAVLAAL EALEQAKREG DEDARRCAEE LLRLACSAAA RQDSEQARAV YEAARAVLAA ...String:
METDTLLLWV LLLWVPGSTG DGSHHHHHHG GSEQKLISEE DLSGGGSWSG SSDEEEAREW AERAEEAAKE ALEQAKREGD EIARLCAKML EILAEEARRK KDSEEAEAVY WAARAVLAAL EALEQAKREG DEDARRCAEE LLRLACSAAA RQDSEQARAV YEAARAVLAA LRALEAAKRA GMEEARKEAE ELLRRACEAA RKQDPELARA VRDKAELLKA LADLFKALKE LKKSLDELER SLEELEKNPS EDALVENNRL NVENNKIIVE VLRIIAEVLR INARAV

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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

Concentration3.0 mg/mL
BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 65.476 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: -1.8 µm / Nominal defocus min: -0.7000000000000001 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER / Details: Ab initio 3D - cryosparc
Final reconstructionResolution.type: BY AUTHOR / Resolution: 5.87 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 397630
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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