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- EMDB-23538: DARPin HAS local refinement -

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Basic information

Entry
Database: EMDB / ID: EMD-23538
TitleDARPin HAS local refinement
Map data
Sample
  • Complex: DARPin D2-21.8.HSA-C9.v2
    • Protein or peptide: DARPin 21.8.HSA-C9.v2
Biological speciessynthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.91 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 14, 2021-
Current statusJul 14, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.6
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.6
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23538.png

Downloads & links

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Map

FileDownload / File: emd_23538.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.05 Å/pix.
x 400 pix.
= 420. Å		1.05 Å/pix.
x 400 pix.
= 420. Å		1.05 Å/pix.
x 400 pix.
= 420. Å

Surface

Projections

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.05 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.4 / Movie #1: 0.6
Minimum - Maximum-1.1563004 - 3.1776876
Average (Standard dev.)0.0005753041 (±0.027377019)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 419.99997 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.051.051.05
M x/y/z400400400
origin x/y/z0.0000.0000.000
length x/y/z420.000420.000420.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS400400400
D min/max/mean-1.1563.1780.001

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Supplemental data

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Additional map: #1

Fileemd_23538_additional_1.map
Projections & Slices
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Half map: #1

Fileemd_23538_half_map_1.map
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Half map: #2

Fileemd_23538_half_map_2.map
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Sample components

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Entire : DARPin D2-21.8.HSA-C9.v2

EntireName: DARPin D2-21.8.HSA-C9.v2
Components
  • Complex: DARPin D2-21.8.HSA-C9.v2
    • Protein or peptide: DARPin 21.8.HSA-C9.v2

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Supramolecule #1: DARPin D2-21.8.HSA-C9.v2

SupramoleculeName: DARPin D2-21.8.HSA-C9.v2 / 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: DARPin 21.8.HSA-C9.v2

MacromoleculeName: DARPin 21.8.HSA-C9.v2 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
SequenceString: MHHHHHHSEK ARIAVENLEA ALRLNRAAAE MQKSAIKIMR DNSSDEKAFR YLLLTTKVLK MSVELLRASL ELAEKALREE GSDDSAEKVR KEAEEILKES TEILKRAELE TLKAAVRVAA EAAARNATDE EERKRIEEEL KKAEERANRS TNEEEIKKIL EEALARFLLE ...String:
MHHHHHHSEK ARIAVENLEA ALRLNRAAAE MQKSAIKIMR DNSSDEKAFR YLLLTTKVLK MSVELLRASL ELAEKALREE GSDDSAEKVR KEAEEILKES TEILKRAELE TLKAAVRVAA EAAARNATDE EERKRIEEEL KKAEERANRS TNEEEIKKIL EEALARFLLE AAWKGAKEAV KLALEAGADV NAADYFGHTP LHLAARNGHA KVVLLLLEQG ADPNADDFAG STPLHLAARA GHAVVVALLL MHGADPNAVD SNGFTPLHLA AQKGHEEVVI LLLAMGADPN AQDKFGKTPF DLAIDNGNEE VVKVLEDHG

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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

BufferpH: 7
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 70.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER / Details: cryoSPARC ab initio
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.91 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 164745
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING

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