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- EMDB-23199: Generation of ordered protein assemblies using rigid three-body fusion -

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Entry
Database: EMDB / ID: EMD-23199
TitleGeneration of ordered protein assemblies using rigid three-body fusion
Map dataraw map
Sample
  • Complex: Cryo-EM characterization of DARPin-grafted scaffold D2-1.4H with GFP
Methodsingle particle reconstruction / cryo EM / Resolution: 4.8 Å
AuthorsYao Q / Vulovic I / Baker D / Jensen G
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM120553 United States
Citation
Journal: Proc Natl Acad Sci U S A / Year: 2021
Title: Generation of ordered protein assemblies using rigid three-body fusion.
Authors: Ivan Vulovic / Qing Yao / Young-Jun Park / Alexis Courbet / Andrew Norris / Florian Busch / Aniruddha Sahasrabuddhe / Hannes Merten / Danny D Sahtoe / George Ueda / Jorge A Fallas / Sara J ...Authors: Ivan Vulovic / Qing Yao / Young-Jun Park / Alexis Courbet / Andrew Norris / Florian Busch / Aniruddha Sahasrabuddhe / Hannes Merten / Danny D Sahtoe / George Ueda / Jorge A Fallas / Sara J Weaver / Yang Hsia / Robert A Langan / Andreas Plückthun / Vicki H Wysocki / David Veesler / Grant J Jensen / David Baker /
Abstract: Protein nanomaterial design is an emerging discipline with applications in medicine and beyond. A long-standing design approach uses genetic fusion to join protein homo-oligomer subunits via α- ...Protein nanomaterial design is an emerging discipline with applications in medicine and beyond. A long-standing design approach uses genetic fusion to join protein homo-oligomer subunits via α-helical linkers to form more complex symmetric assemblies, but this method is hampered by linker flexibility and a dearth of geometric solutions. Here, we describe a general computational method for rigidly fusing homo-oligomer and spacer building blocks to generate user-defined architectures that generates far more geometric solutions than previous approaches. The fusion junctions are then optimized using Rosetta to minimize flexibility. We apply this method to design and test 92 dihedral symmetric protein assemblies using a set of designed homodimers and repeat protein building blocks. Experimental validation by native mass spectrometry, small-angle X-ray scattering, and negative-stain single-particle electron microscopy confirms the assembly states for 11 designs. Most of these assemblies are constructed from designed ankyrin repeat proteins (DARPins), held in place on one end by α-helical fusion and on the other by a designed homodimer interface, and we explored their use for cryogenic electron microscopy (cryo-EM) structure determination by incorporating DARPin variants selected to bind targets of interest. Although the target resolution was limited by preferred orientation effects and small scaffold size, we found that the dual anchoring strategy reduced the flexibility of the target-DARPIN complex with respect to the overall assembly, suggesting that multipoint anchoring of binding domains could contribute to cryo-EM structure determination of small proteins.
#1: Journal: Biorxiv / Year: 2020
Title: Generation of ordered protein assemblies using rigid three-body fusion
Authors: Vulovic I / Yao Q / Park Y-J / Courbet A / Norris A / Busch F / Sahasrabuddhe A / Merten H / Sahtoe DD / Ueda G / Fallas JA / Weaver SJ / Hsia Y / Langan RA / Pluckthun A / Wysocki VH / ...Authors: Vulovic I / Yao Q / Park Y-J / Courbet A / Norris A / Busch F / Sahasrabuddhe A / Merten H / Sahtoe DD / Ueda G / Fallas JA / Weaver SJ / Hsia Y / Langan RA / Pluckthun A / Wysocki VH / Veesler D / Jensen GJ / Baker D
History
DepositionDec 23, 2020-
Header (metadata) releaseJun 9, 2021-
Map releaseJun 9, 2021-
UpdateSep 8, 2021-
Current statusSep 8, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.206
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.206
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23199.png

Downloads & links

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Map

FileDownload / File: emd_23199.map.gz / Format: CCP4 / Size: 274.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationraw map
Voxel sizeX=Y=Z: 0.834 Å
Density
Contour LevelBy AUTHOR: 0.206 / Movie #1: 0.206
Minimum - Maximum-0.19682191 - 1.0528694
Average (Standard dev.)0.00043094324 (±0.035225492)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions416416416
Spacing416416416
CellA=B=C: 346.944 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8340.8340.834
M x/y/z416416416
origin x/y/z0.0000.0000.000
length x/y/z346.944346.944346.944
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ300300300
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS416416416
D min/max/mean-0.1971.0530.000

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Supplemental data

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Mask #1

Fileemd_23199_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: sharpened map

Fileemd_23199_additional_1.map
Annotationsharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map A

Fileemd_23199_half_map_1.map
AnnotationHalf-map A
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map B

Fileemd_23199_half_map_2.map
AnnotationHalf-map B
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Sample components

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Entire : Cryo-EM characterization of DARPin-grafted scaffold D2-1.4H with GFP

EntireName: Cryo-EM characterization of DARPin-grafted scaffold D2-1.4H with GFP
Components
  • Complex: Cryo-EM characterization of DARPin-grafted scaffold D2-1.4H with GFP

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Supramolecule #1: Cryo-EM characterization of DARPin-grafted scaffold D2-1.4H with GFP

SupramoleculeName: Cryo-EM characterization of DARPin-grafted scaffold D2-1.4H with GFP
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Molecular weightTheoretical: 310 KDa

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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: 7.5
Component:
ConcentrationFormula
50.0 mMTris
150.0 mMNaClSodium chloride
GridModel: Quantifoil / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK II

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated defocus max: 2.9 µm / Calibrated defocus min: 0.9 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.9 µm / Nominal defocus min: 0.9 µm
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.11)
Final reconstructionAlgorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 4.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.11) / Number images used: 138348
FSC plot (resolution estimation)

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