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Yorodumi- EMDB-23199: Generation of ordered protein assemblies using rigid three-body fusion -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-23199 | |||||||||
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Title | Generation of ordered protein assemblies using rigid three-body fusion | |||||||||
Map data | raw map | |||||||||
Sample |
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Method | single particle reconstruction / cryo EM / Resolution: 4.8 Å | |||||||||
Authors | Yao Q / Vulovic I / Baker D / Jensen G | |||||||||
Funding support | United States, 1 items
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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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_23199.map.gz | 134.7 MB | EMDB map data format | |
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Header (meta data) | emd-23199-v30.xml emd-23199.xml | 18.6 KB 18.6 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_23199_fsc.xml | 14.4 KB | Display | FSC data file |
Images | emd_23199.png | 79 KB | ||
Masks | emd_23199_msk_1.map | 274.6 MB | Mask map | |
Others | emd_23199_additional_1.map.gz emd_23199_half_map_1.map.gz emd_23199_half_map_2.map.gz | 233.9 MB 254.6 MB 254.6 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-23199 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-23199 | HTTPS FTP |
-Validation report
Summary document | emd_23199_validation.pdf.gz | 497.4 KB | Display | EMDB validaton report |
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Full document | emd_23199_full_validation.pdf.gz | 496.9 KB | Display | |
Data in XML | emd_23199_validation.xml.gz | 23 KB | Display | |
Data in CIF | emd_23199_validation.cif.gz | 29.8 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-23199 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-23199 | HTTPS FTP |
-Related structure data
Related structure data | C: citing same article (ref.) |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_23199.map.gz / Format: CCP4 / Size: 274.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | raw map | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 0.834 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Mask #1
File | emd_23199_msk_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Additional map: sharpened map
File | emd_23199_additional_1.map | ||||||||||||
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Annotation | sharpened map | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half-map A
File | emd_23199_half_map_1.map | ||||||||||||
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Annotation | Half-map A | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half-map B
File | emd_23199_half_map_2.map | ||||||||||||
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Annotation | Half-map B | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : Cryo-EM characterization of DARPin-grafted scaffold D2-1.4H with GFP
Entire | Name: Cryo-EM characterization of DARPin-grafted scaffold D2-1.4H with GFP |
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Components |
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-Supramolecule #1: Cryo-EM characterization of DARPin-grafted scaffold D2-1.4H with GFP
Supramolecule | Name: Cryo-EM characterization of DARPin-grafted scaffold D2-1.4H with GFP type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1 |
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Molecular weight | Theoretical: 310 KDa |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 1.5 mg/mL | ||||||
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Buffer | pH: 7.5 / Component:
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Grid | Model: Quantifoil / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE | ||||||
Vitrification | Cryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK II |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 40.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 aperture diameter: 100.0 µm / Calibrated defocus max: 2.9 µm / Calibrated defocus min: 0.9 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.9 µm / Nominal defocus min: 0.9 µm |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |