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- EMDB-42917: De novo designed KWOCA 18 nanoparticle - Assembly in D2 symmetry -

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Basic information

Entry
Database: EMDB / ID: EMD-42917
TitleDe novo designed KWOCA 18 nanoparticle - Assembly in D2 symmetry
Map dataMain map
Sample
  • Complex: Designed nanoparticle KWOCA 18 - D2 assembly
Keywordsprotein design / nanoparticles / cryptic sites / degreaser / soluble expression / DE NOVO PROTEIN
Biological speciessynthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 8.0 Å
AuthorsAntanasijevic A / Ward AB
Funding support United States, 1 items
OrganizationGrant numberCountry
International AIDS Vaccine InitiativeOPP1196345 United States
CitationJournal: Nat Struct Mol Biol / Year: 2025
Title: Local structural flexibility drives oligomorphism in computationally designed protein assemblies.
Authors: Alena Khmelinskaia / Neville P Bethel / Farzad Fatehi / Bhoomika Basu Mallik / Aleksandar Antanasijevic / Andrew J Borst / Szu-Hsueh Lai / Ho Yeung Chim / Jing Yang 'John' Wang / Marcos C ...Authors: Alena Khmelinskaia / Neville P Bethel / Farzad Fatehi / Bhoomika Basu Mallik / Aleksandar Antanasijevic / Andrew J Borst / Szu-Hsueh Lai / Ho Yeung Chim / Jing Yang 'John' Wang / Marcos C Miranda / Andrew M Watkins / Cassandra Ogohara / Shane Caldwell / Mengyu Wu / Albert J R Heck / David Veesler / Andrew B Ward / David Baker / Reidun Twarock / Neil P King /
Abstract: Many naturally occurring protein assemblies have dynamic structures that allow them to perform specialized functions. Although computational methods for designing novel self-assembling proteins have ...Many naturally occurring protein assemblies have dynamic structures that allow them to perform specialized functions. Although computational methods for designing novel self-assembling proteins have advanced substantially over the past decade, they primarily focus on designing static structures. Here we characterize three distinct computationally designed protein assemblies that exhibit unanticipated structural diversity arising from flexibility in their subunits. Cryo-EM single-particle reconstructions and native mass spectrometry reveal two distinct architectures for two assemblies, while six cryo-EM reconstructions for the third likely represent a subset of its solution-phase structures. Structural modeling and molecular dynamics simulations indicate that constrained flexibility within the subunits of each assembly promotes a defined range of architectures rather than nonspecific aggregation. Redesigning the flexible region in one building block rescues the intended monomorphic assembly. These findings highlight structural flexibility as a powerful design principle, enabling exploration of new structural and functional spaces in protein assembly design.
History
DepositionNov 23, 2023-
Header (metadata) releaseDec 11, 2024-
Map releaseDec 11, 2024-
UpdateJul 2, 2025-
Current statusJul 2, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_42917.png

Downloads & links

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Map

FileDownload / File: emd_42917.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMain map
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
1.15 Å/pix.
x 320 pix.
= 368. Å		1.15 Å/pix.
x 320 pix.
= 368. Å		1.15 Å/pix.
x 320 pix.
= 368. Å

Surface

Projections

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.15 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.004
Minimum - Maximum-0.0036207465 - 0.01009946
Average (Standard dev.)0.000029458308 (±0.0008471048)
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_42917_msk_1.map
Projections & Slices
AxesZYX

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Half map: Half-map 1

Fileemd_42917_half_map_1.map
AnnotationHalf-map 1
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Half map: Half-map 2

Fileemd_42917_half_map_2.map
AnnotationHalf-map 2
Projections & Slices
AxesZYX

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Histogram

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Sample components

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Entire : Designed nanoparticle KWOCA 18 - D2 assembly

EntireName: Designed nanoparticle KWOCA 18 - D2 assembly
Components
  • Complex: Designed nanoparticle KWOCA 18 - D2 assembly

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Supramolecule #1: Designed nanoparticle KWOCA 18 - D2 assembly

SupramoleculeName: Designed nanoparticle KWOCA 18 - D2 assembly / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: Self-assembling nanoparticle expressed in mammalian cells
Source (natural)Organism: synthetic construct (others)

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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

Concentration4.93 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
25.0 mMTris-HClTris-HCl
150.0 mMNaClSodium chloride
2.0 %GlycerolGlycerol

Details: TBS + 2% glycerol
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK IV / Details: Blotting time varied between 4 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
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: 1803 / Average exposure time: 9.8 sec. / Average electron dose: 50.1 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / 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
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

DetailsFrames were aligned using MotionCorr and GCTF was applied for estimation of CTF parameters.
Particle selectionNumber selected: 399059 / Details: CryoSPARC template picker
CTF correctionSoftware: (Name: RELION (ver. 3.0), cryoSPARC (ver. 2.9)) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: OTHER
Details: Map obtained from Ab initio reconstruction in cryoSPARC with application of symmetry
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: D2 (2x2 fold dihedral) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 8.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0)
Details: 3D auto-refinement in relion, D2 symmetry applied. Solvent mask around the nanoparticle
Number images used: 45922
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final 3D classificationSoftware - Name: RELION (ver. 3.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: RIGID BODY FIT

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