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Basic information

Entry
Database: PDB / ID: 6nht
TitleSingle particle reconstruction of the symmetric core an engineered protein scaffold
Components
  • DARP14 - Subunit A with DARPin
  • DARP14 - Subunit B
KeywordsBIOSYNTHETIC PROTEIN / protein engineering / symmetric scaffold / small protein cryo-EM / display platform
Function / homology5-carboxymethyl-2-hydroxymuconate isomerase / Tautomerase/MIF superfamily / 5-carboxymethyl-2-hydroxymuconate isomerase / 5-carboxymethyl-2-hydroxymuconate delta-isomerase activity / aromatic compound catabolic process / Uncharacterized protein
Function and homology information
Specimen sourcePyrococcus horikoshii (archaea)
Pseudomonas aeruginosa (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsLiu, Y. / Huynh, D. / Yeates, T.O.
Funding supportUnited States , 6件
OrganizationGrant numberCountry
Department of Energy (United States)DE-FC02-02ER63421United States
National Institutes of Health/National Human Genome Research InstituteS10RR23057United States
National Institutes of Health/National Human Genome Research InstituteS10OD018111United States
National Institutes of Health/National Human Genome Research InstituteU24GM11679United States
National Science Foundation (United States)DBI-1338135United States
National Science Foundation (United States)DMR-1548924United States
CitationJournal: Nat Commun / Year: 2019
Title: A 3.8 Å resolution cryo-EM structure of a small protein bound to an imaging scaffold.
Authors: Yuxi Liu / Duc T Huynh / Todd O Yeates /
Abstract: Proteins smaller than about 50 kDa are currently too small to be imaged at high resolution by cryo-electron microscopy (cryo-EM), leaving most protein molecules in the cell beyond the reach of this ...Proteins smaller than about 50 kDa are currently too small to be imaged at high resolution by cryo-electron microscopy (cryo-EM), leaving most protein molecules in the cell beyond the reach of this powerful structural technique. Here we use a designed protein scaffold to bind and symmetrically display 12 copies of a small 26 kDa protein, green fluorescent protein (GFP). We show that the bound cargo protein is held rigidly enough to visualize it at a resolution of 3.8 Å by cryo-EM, where specific structural features of the protein are visible. The designed scaffold is modular and can be modified through modest changes in its amino acid sequence to bind and display diverse proteins for imaging, thus providing a general method to break through the lower size limitation in cryo-EM.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Dec 23, 2018 / Release: May 8, 2019Array

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Structure visualization

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  • Deposited structure unit
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  • Superimposition on EM map
  • EMDB-9373
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
A: DARP14 - Subunit A with DARPin
B: DARP14 - Subunit A with DARPin
C: DARP14 - Subunit A with DARPin
D: DARP14 - Subunit A with DARPin
E: DARP14 - Subunit B
F: DARP14 - Subunit B
G: DARP14 - Subunit B
H: DARP14 - Subunit B
I: DARP14 - Subunit A with DARPin
J: DARP14 - Subunit A with DARPin
K: DARP14 - Subunit A with DARPin
L: DARP14 - Subunit A with DARPin
M: DARP14 - Subunit B
N: DARP14 - Subunit B
O: DARP14 - Subunit B
P: DARP14 - Subunit B
Q: DARP14 - Subunit A with DARPin
R: DARP14 - Subunit A with DARPin
S: DARP14 - Subunit A with DARPin
T: DARP14 - Subunit A with DARPin
U: DARP14 - Subunit B
V: DARP14 - Subunit B
W: DARP14 - Subunit B
X: DARP14 - Subunit B


Theoretical massNumber of molelcules
Total (without water)588,76924
Polymers588,76924
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, native gel electrophoresis, microscopy, Negative stain EM
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TypeNameSymmetry operationNumber
identity operation1_5551
Buried area66320 Å2
ΔGint-335 kcal/mol
Surface area137510 Å2

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Components

#1: Protein/peptide
DARP14 - Subunit A with DARPin


Mass: 34717.820 Da / Num. of mol.: 12
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus horikoshii (strain ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3) (archaea)
Strain: ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3
Production host: Escherichia coli (E. coli)
#2: Protein/peptide
DARP14 - Subunit B


Mass: 14346.274 Da / Num. of mol.: 12
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1) (bacteria)
Strain: ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1
Gene: PA1966 / Production host: Escherichia coli (E. coli) / References: UniProt: Q9I2D8

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component

Type: COMPLEX

IDNameEntity IDParent-IDSource
1Subunit A with DARPin Subunit B1, 20MULTIPLE SOURCES
2Subunit B21RECOMBINANT
3Subunit A with DARPin11RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Pseudomonas aeruginosa PAO1 (bacteria)208964
23Pyrococcus horikoshii OT3 (archaea)70601
Source (recombinant)

Ncbi tax-ID: 562 / Organism: Escherichia coli (E. coli)

IDEntity assembly-ID
12
23
Buffer solutionpH: 7.5
Buffer component

Buffer-ID: 1

IDConc.NameFormula
110 mMTrisC4H11NO3
2500 mMsodium chlorideNaCl
31 mMDTTC4H10O2S2
40.5 %glycerolC3H8O3
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K
Details: 2.5 microliter of sample, 0 sec wait, 0 sec drain, 3 sec blot, -15 blot force, grids pre-treated with 0.1% poly-lysine for 6 hours

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 130000 X / Cs: 2.7 mm / C2 aperture diameter: 50 µns
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 8 sec. / Electron dose: 56 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1929
Image scansMovie frames/image: 40 / Used frames/image: 3-20

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Processing

SoftwareName: PHENIX / Version: 1.14_3260: / Classification: refinement
EM software
IDNameVersionCategory
1RELION2particle selection
2Leginonimage acquisition
4CTFFIND4.1.5CTF correction
7PHENIXmodel fitting
9PHENIXmodel refinement
10RELION2initial Euler assignment
11cryoSPARCv.2.4.0final Euler assignment
12cryoSPARCv.2.4.0classification
13cryoSPARCv.2.4.03D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
Particle selectionNum. of particles selected: 81319
SymmetryPoint symmetry: T (tetrahedral)
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 80253 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Details: Initial local fitting by Chimera and individual residues refined using phenix.real_space_refine
Atomic model buildingPDB-ID: 6C9K

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