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- EMDB-23533: D3-1.5C -

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Basic information

Entry
Database: EMDB / ID: EMD-23533
TitleD3-1.5C
Map data
Sample
  • Complex: D3-1.5C
    • Protein or peptide: D3-1.5C
Biological speciessynthetic construct (others)
Methodsingle particle reconstruction / negative staining / Resolution: 19.0 Å
AuthorsPark YJ / Ivan V / Baker D / Veesler D
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM120553 United States
CitationJournal: 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.
History
DepositionFeb 24, 2021-
Header (metadata) releaseJun 9, 2021-
Map releaseJun 9, 2021-
UpdateJul 28, 2021-
Current statusJul 28, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.15
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.15
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23533.png

Downloads & links

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Map

FileDownload / File: emd_23533.map.gz / Format: CCP4 / Size: 2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 6.4 Å
Density
Contour LevelBy AUTHOR: 0.15 / Movie #1: 0.15
Minimum - Maximum-0.008258775 - 0.7378947
Average (Standard dev.)0.0030631656 (±0.029194526)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions808080
Spacing808080
CellA=B=C: 512.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z6.46.46.4
M x/y/z808080
origin x/y/z0.0000.0000.000
length x/y/z512.000512.000512.000
α/β/γ90.00090.00090.000
start NX/NY/NZ1331310
NX/NY/NZ223226424
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS808080
D min/max/mean-0.0080.7380.003

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Supplemental data

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Additional map: #1

Fileemd_23533_additional_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_23533_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_23533_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : D3-1.5C

EntireName: D3-1.5C
Components
  • Complex: D3-1.5C
    • Protein or peptide: D3-1.5C

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Supramolecule #1: D3-1.5C

SupramoleculeName: D3-1.5C / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: synthetic construct (others)
Recombinant expressionOrganism: Escherichia coli K-12 (bacteria)

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Macromolecule #1: D3-1.5C

MacromoleculeName: D3-1.5C / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
SequenceString: MGSEKARIAV ENLEAALRLN RAAAEMQKSA AKIVADNASD EKALRYLRLT TKVLRMSVEL LRASLELAEK ALREEGSDDS AEKVRKEAEE ILKESTEILK EADQITEVAD LAFELANKAT DEELRKEISK CARLALELAS RSTNDELIKQ ILEVAKLAFE LASKATDEEL ...String:
MGSEKARIAV ENLEAALRLN RAAAEMQKSA AKIVADNASD EKALRYLRLT TKVLRMSVEL LRASLELAEK ALREEGSDDS AEKVRKEAEE ILKESTEILK EADQITEVAD LAFELANKAT DEELRKEISK CARLALELAS RSTNDELIKQ ILEVAKLAFE LASKATDEEL IKLILKCCQA AFERASRSTN DEEIKKILEV AKRAFETASK ATDEEEIKSI LLICAAALGN KDAVKSAIEN GADVNASDSD GRTPLHHAAE NGNAEVVALL IEKGADVNAK DSDGRTPLHH AAENGHDEVV LILLLKGADV NAKDSDGRTP LHHAAENGHK RVVLVLILAG ADVNTSDSDG RTPLDLAREH GNEEVVKALE KQGGWLEHHH HHH

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 8
StainingType: NEGATIVE / Material: UF

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Electron microscopy

MicroscopeFEI TECNAI 12
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Average electron dose: 50.0 e/Å2

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Image processing

Startup modelType of model: OTHER / Details: cryoSPARC ab initio
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING
Final reconstructionApplied symmetry - Point group: D3 (2x3 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 19.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 93635

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