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- PDB-6nhv: Single particle reconstruction of DARPin and its bound GFP on a s... -

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Basic information

Entry
Database: PDB / ID: 6nhv
TitleSingle particle reconstruction of DARPin and its bound GFP on a symmetric scaffold
Components
  • DARP14 - Subunit B
  • Subunit A-DARPin
  • superfolder GFP
KeywordsBIOSYNTHETIC PROTEIN / protein engineering / symmetric scaffold / small protein cryo-EM / display platform
Function / homology
Function and homology information


5-carboxymethyl-2-hydroxymuconate delta-isomerase activity / catabolic process
Similarity search - Function
5-carboxymethyl-2-hydroxymuconate isomerase / 5-carboxymethyl-2-hydroxymuconate isomerase / Hypothetical Protein Ta1238; Chain: A; / Cobalamin adenosyltransferase-like / Macrophage Migration Inhibitory Factor / Macrophage Migration Inhibitory Factor / Tautomerase/MIF superfamily / Up-down Bundle / 2-Layer Sandwich / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
5-carboxymethyl-2-hydroxymuconate isomerase
Similarity search - Component
Biological speciesAequorea victoria (jellyfish)
Pseudomonas aeruginosa (bacteria)
Pyrococcus horikoshii (archaea)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsLiu, Y. / Huynh, D. / Yeates, T.O.
Funding support United States, 6items
OrganizationGrant numberCountry
Department of Energy (DOE, United States)DE-FC02-02ER63421 United States
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)S10RR23057 United States
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)S10OD018111 United States
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)U24GM11679 United States
National Science Foundation (NSF, United States)DBI-1338135 United States
National Science Foundation (NSF, United States)DMR-1548924 United 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.
History
DepositionDec 24, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 8, 2019Provider: repository / Type: Initial release
Revision 1.1Nov 27, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization

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Structure visualization

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  • Deposited structure unit
  • Imaged by Jmol
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  • Superimposition on EM map
  • EMDB-9374
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
A: superfolder GFP
N: DARP14 - Subunit B
O: DARP14 - Subunit B
R: Subunit A-DARPin
S: Subunit A-DARPin
T: Subunit A-DARPin
X: DARP14 - Subunit B


Theoretical massNumber of molelcules
Total (without water)173,8167
Polymers173,8167
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, native gel electrophoresis, microscopy, Negative stain EM
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area14900 Å2
ΔGint-61 kcal/mol
Surface area49140 Å2

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Components

#1: Protein superfolder GFP


Mass: 26623.918 Da / Num. of mol.: 1 / Mutation: V206A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Aequorea victoria (jellyfish) / Production host: Escherichia coli (E. coli)
#2: Protein DARP14 - Subunit B


Mass: 14346.274 Da / Num. of mol.: 3
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
#3: Protein Subunit A-DARPin


Mass: 34717.820 Da / Num. of mol.: 3
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)

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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
IDNameTypeEntity IDParent-IDSource
1Subunit A with DARPin + Subunit B + superfolder GFPCOMPLEXall0MULTIPLE SOURCES
2superfolder GFPCOMPLEX#11RECOMBINANT
3Subunit A with DARPinCOMPLEX#31RECOMBINANT
4Subunit BCOMPLEX#21RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Aequorea victoria (jellyfish)6100
23Pyrococcus horikoshii OT3 (archaea)70601
34Pseudomonas aeruginosa PAO1 (bacteria)208964
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli (E. coli)562
23Escherichia coli (E. coli)562
34Escherichia coli (E. coli)562
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMTrisC4H11NO31
2500 mMsodium chlorideNaCl1
31 mMDTTC4H10O2S21
40.5 %glycerolC3H8O31
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: unspecified
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 FIELD / Nominal magnification: 130000 X / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN / Specimen holder 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
11RELION3final Euler assignment
12RELION3classification
13RELION33D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
Particle selectionNum. of particles selected: 963036
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 91211 / 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 for the symmetric core and DARPin, rigid body refinement for GFP
Atomic model building
IDPDB-ID 3D fitting-ID
16C9K

6c9k

1
25MA8

5ma8

1
34W6B

4w6b

1

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