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- EMDB-47327: Computationally Designed Bifaceted Protein Nanomaterial pD5-14 -

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Entry
Database: EMDB / ID: EMD-47327
TitleComputationally Designed Bifaceted Protein Nanomaterial pD5-14
Map dataMap sharpened using DeepEMhancer
Sample
  • Complex: Computationally Designed Bifaceted Protein Nanomaterial pD5-14
    • Protein or peptide: pD5-14 A component
    • Protein or peptide: pD5-14 B component
    • Protein or peptide: pD5-14 C component
    • Protein or peptide: pD5-14 D component
Keywordsnanomaterial / 4 component / bifaceted / penton / DE NOVO PROTEIN
Biological speciessynthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.3 Å
AuthorsCarr KD / Borst AJ / Weidle C
Funding support United States, Sweden, 4 items
OrganizationGrant numberCountry
Bill & Melinda Gates Foundation United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID) United States
Howard Hughes Medical Institute (HHMI) United States
Swedish Research Council Sweden
CitationJournal: bioRxiv / Year: 2024
Title: Computational design of bifaceted protein nanomaterials.
Authors: Sanela Rankovic / Kenneth D Carr / Justin Decarreau / Rebecca Skotheim / Ryan D Kibler / Sebastian Ols / Sangmin Lee / Jung-Ho Chun / Marti R Tooley / Justas Dauparas / Helen E Eisenach / ...Authors: Sanela Rankovic / Kenneth D Carr / Justin Decarreau / Rebecca Skotheim / Ryan D Kibler / Sebastian Ols / Sangmin Lee / Jung-Ho Chun / Marti R Tooley / Justas Dauparas / Helen E Eisenach / Matthias Glögl / Connor Weidle / Andrew J Borst / David Baker / Neil P King /
Abstract: Recent advances in computational methods have led to considerable progress in the design of self-assembling protein nanoparticles. However, nearly all nanoparticles designed to date exhibit strict ...Recent advances in computational methods have led to considerable progress in the design of self-assembling protein nanoparticles. However, nearly all nanoparticles designed to date exhibit strict point group symmetry, with each subunit occupying an identical, symmetrically related environment. This limits the structural diversity that can be achieved and precludes anisotropic functionalization. Here, we describe a general computational strategy for designing multi-component bifaceted protein nanomaterials with two distinctly addressable sides. The method centers on docking pseudosymmetric heterooligomeric building blocks in architectures with dihedral symmetry and designing an asymmetric protein-protein interface between them. We used this approach to obtain an initial 30-subunit assembly with pseudo-D5 symmetry, and then generated an additional 15 variants in which we controllably altered the size and morphology of the bifaceted nanoparticles by designing extensions to one of the subunits. Functionalization of the two distinct faces of the nanoparticles with protein minibinders enabled specific colocalization of two populations of polystyrene microparticles coated with target protein receptors. The ability to accurately design anisotropic protein nanomaterials with precisely tunable structures and functions could be broadly useful in applications that require colocalizing two or more distinct target moieties.
History
DepositionOct 16, 2024-
Header (metadata) releaseOct 30, 2024-
Map releaseOct 30, 2024-
UpdateNov 13, 2024-
Current statusNov 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_47327.png

Downloads & links

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Map

FileDownload / File: emd_47327.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap sharpened using DeepEMhancer
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.68 Å/pix.
x 400 pix.
= 672. Å		1.68 Å/pix.
x 400 pix.
= 672. Å		1.68 Å/pix.
x 400 pix.
= 672. Å

Surface

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Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.68 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.25
Minimum - Maximum-0.0030569197 - 1.4137489
Average (Standard dev.)0.00068716035 (±0.018876767)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 672.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Sharpened Map B-Factor: 239.9

Fileemd_47327_additional_1.map
AnnotationSharpened Map B-Factor: 239.9
Projections & Slices
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Additional map: unsharpened map

Fileemd_47327_additional_2.map
Annotationunsharpened map
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Half map: half map A

Fileemd_47327_half_map_1.map
Annotationhalf map A
Projections & Slices
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Half map: half map B

Fileemd_47327_half_map_2.map
Annotationhalf map B
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Sample components

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Entire : Computationally Designed Bifaceted Protein Nanomaterial pD5-14

EntireName: Computationally Designed Bifaceted Protein Nanomaterial pD5-14
Components
  • Complex: Computationally Designed Bifaceted Protein Nanomaterial pD5-14
    • Protein or peptide: pD5-14 A component
    • Protein or peptide: pD5-14 B component
    • Protein or peptide: pD5-14 C component
    • Protein or peptide: pD5-14 D component

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Supramolecule #1: Computationally Designed Bifaceted Protein Nanomaterial pD5-14

SupramoleculeName: Computationally Designed Bifaceted Protein Nanomaterial pD5-14
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Chains were expressed separately in E. coli. Batches of A, B, and C components or A, B, and D components were mixed, subsequently lysed and centrifuged to yield 2 distinct species of cyclic ...Details: Chains were expressed separately in E. coli. Batches of A, B, and C components or A, B, and D components were mixed, subsequently lysed and centrifuged to yield 2 distinct species of cyclic assemblies - (ABC)5 and (ABD)5. Following IMAC and SEC, (ABC)5 and (ABD)5 assemblies were mixed to form pseudo-D5 (ABC)5-(ABD)5 assembly pD5-14.
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 1.256 MDa

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Macromolecule #1: pD5-14 A component

MacromoleculeName: pD5-14 A component / type: protein_or_peptide / ID: 1 / Number of copies: 10 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 35.30207 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGSLELALKA LQILVNAAYV LAEIARDRGN EELLEKAARL AEEAARQAER IARQARKEGN LELALKALQI LVNAAYVLAE IARDRGNEE LLEYAARLAE EAARQAIEIW AQAMEEGNQQ LRTKAAHIIL RAAEVLLEIA RDRGNQELLE KAASLVDAVA A LQQAAAAI ...String:
MGSLELALKA LQILVNAAYV LAEIARDRGN EELLEKAARL AEEAARQAER IARQARKEGN LELALKALQI LVNAAYVLAE IARDRGNEE LLEYAARLAE EAARQAIEIW AQAMEEGNQQ LRTKAAHIIL RAAEVLLEIA RDRGNQELLE KAASLVDAVA A LQQAAAAI LEGDVEKAVR AAQEAVKAAK EAGDNDMLRA VAIAALRIAK EAEKQGNVEV AVKAARVAVE AAKQAGDNDV LR KVAEQAL RIAKEAEKQG NVEVAVKAAR VAVEAAKQAG DNDVLRKVAD QALEIAKAAL EQGDIDVAQK AMDVAVEALT QAG GSGGSH HHHHH

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Macromolecule #2: pD5-14 B component

MacromoleculeName: pD5-14 B component / type: protein_or_peptide / ID: 2 / Number of copies: 10 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 34.050781 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGSPRLVLRA LENMVRAAHT LAEIARDNGN EEWLERAARL AEEVARRAER LAREARKEGN LELALKALQI LVNAAYVLAE IARDRGNEE ELEYAARLAE EAARQAIEIA AQAMEEGNLE LALKALQIIV NAAYVLAEIA RDRGNEELLE KAASLAEAAA A LAEAIAAI ...String:
MGSPRLVLRA LENMVRAAHT LAEIARDNGN EEWLERAARL AEEVARRAER LAREARKEGN LELALKALQI LVNAAYVLAE IARDRGNEE ELEYAARLAE EAARQAIEIA AQAMEEGNLE LALKALQIIV NAAYVLAEIA RDRGNEELLE KAASLAEAAA A LAEAIAAI LEGDVEKAVR AAQEAVKAAK EAGDNDMLRA VAIAALRIAK EAEKQGNVEV AVKAARVAVE AAKQAGDNDV LR KVAEQAL RIAKEAEKQG NVEVAVKAAR VAVEAAKQAG DNDVLRKVAE QALEIAKKAA EQGDVGVMQK AMDVALRAAG QAG

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Macromolecule #3: pD5-14 C component

MacromoleculeName: pD5-14 C component / type: protein_or_peptide / ID: 3
Details: mScarlet fused to the N-terminus of the C component of pD5-14
Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 56.306781 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGSKGEAVIK EFMRFKVHME GSMNGHEFEI EGEGEGRPYE GTQTAKLKVT KGGPLPFSWD ILSPQFMYGS RAFIKHPADI PDYYKQSFP EGFKWERVMN FEDGGAVTVT QDTSLEDGTL IYKVKLRGTN FPPDGPVMQK KTMGWEASTE RLYPEDGVLK G DIKMALRL ...String:
MGSKGEAVIK EFMRFKVHME GSMNGHEFEI EGEGEGRPYE GTQTAKLKVT KGGPLPFSWD ILSPQFMYGS RAFIKHPADI PDYYKQSFP EGFKWERVMN FEDGGAVTVT QDTSLEDGTL IYKVKLRGTN FPPDGPVMQK KTMGWEASTE RLYPEDGVLK G DIKMALRL KDGGRYLADF KTTYKAKKPV QMPGAYNVDR KLDITSHNED YTVVEQYERS EGRHSTGGMD ELYKGSGSGP EL FLQDLRS LVEAARILAR LARQRGDEHA LERAARWAEQ AARQAERLAR QARKEGNLEL ALKALQILVN AAYVLAEIAR DRG NEELLE YAARLAEEAA RQAIEIAAQA MEEGNFELAL EALEIINEAA RVLARIAHHR GNQELLEKAA SLTHASAALS RAIA AILEG DVEKAVRAAQ EAVKAAKEAG DNDMLRAVAI AALRIAKEAE KQGNVEVAVK AARVAVEAAK QAGDNDVLRL VSERA LSIA ASSVKQGNYE VKEKAIRVAK EANKQAG

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Macromolecule #4: pD5-14 D component

MacromoleculeName: pD5-14 D component / type: protein_or_peptide / ID: 4
Details: mNeonGreen fused to the N-terminus of the D component of pD5-14
Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 56.573711 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGSKGEEDNM ASLPATHELH IFGSINGVDF DMVGQGTGNP NDGYEELNLK STKGDLQFSP WILVPHIGYG FHQYLPYPDG MSPFQAAMV DGSGYQVHRT MQFEDGASLT VNYRYTYEGS HIKGEAQVKG TGFPADGPVM TNSLTAADWC RSKKTYPNDK T IISTFKWS ...String:
MGSKGEEDNM ASLPATHELH IFGSINGVDF DMVGQGTGNP NDGYEELNLK STKGDLQFSP WILVPHIGYG FHQYLPYPDG MSPFQAAMV DGSGYQVHRT MQFEDGASLT VNYRYTYEGS HIKGEAQVKG TGFPADGPVM TNSLTAADWC RSKKTYPNDK T IISTFKWS YTTGNGKRYR STARTTYTFA KPMAANYLKN QPMYVFRKTE LKHSKTELNF KEWQKAFTDV MGMDELYKGS GS GPELFLQ DLRSLVEAAR ILARLARQRG DEHALERAAR WAEQAARQAE RLARQARKEG NLELALKALQ ILVNAAYVLA EIA RDRGNE ELLEYAARLA EEAARQAIEI AAQAMEEGNF ELALEALEII NEAARVLARI AHHRGNQELL EKAASLTHAS AALS RAIAA ILEGDVEKAV RAAQEAVKAA KEAGDNDMLR AVAIAALRIA KEAEKQGNVE VAVKAARVAV EAAKQAGDND VLKRV SETL LSIAAEATKQ GNSEVMEKAI RVSEEAEKQA G

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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

Concentration1.5 mg/mL
BufferpH: 8
GridModel: EMS Lacey Carbon / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: LACEY / Support film - Film thickness: 3
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number real images: 4871 / Average electron dose: 45.21 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 640897
Startup modelType of model: INSILICO MODEL / In silico model: ab-initio model / Details: ab-initio model
Final reconstructionNumber classes used: 18 / Applied symmetry - Point group: C5 (5 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 4.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.4.0) / Number images used: 209004
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.4.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.4.0)
Final 3D classificationNumber classes: 18 / Avg.num./class: 11611 / Software - Name: cryoSPARC (ver. 4.4.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

DetailsInitial fitting was performed in ChimeraX. Phenix, Namdinator, ISOLDE, and Coot were used for relaxation of the model to better fit the density.
RefinementSpace: REAL / Protocol: OTHER / Overall B value: 239.9 / Target criteria: Cross-correlation coefficient
Output model

PDB-9dze:
Computationally Designed Bifaceted Protein Nanomaterial pD5-14

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