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- EMDB-9374: Single particle reconstruction of DARPin and its bound GFP on a s... -

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Basic information

Entry
Database: EMDB / ID: EMD-9374
TitleSingle particle reconstruction of DARPin and its bound GFP on a symmetric scaffold
Map dataRefinement of DARPin, GFP, and two adjacent trimers
Sample
  • Complex: Subunit A with DARPin + Subunit B + superfolder GFP
    • Complex: superfolder GFP
      • Protein or peptide: superfolder GFP
    • Complex: Subunit A with DARPin
      • Protein or peptide: Subunit A-DARPin
    • Complex: Subunit B
      • Protein or peptide: DARP14 - Subunit B
Function / homology5-carboxymethyl-2-hydroxymuconate isomerase / 5-carboxymethyl-2-hydroxymuconate isomerase / 5-carboxymethyl-2-hydroxymuconate delta-isomerase activity / Tautomerase/MIF superfamily / catabolic process / 5-carboxymethyl-2-hydroxymuconate isomerase
Function and homology information
Biological speciesAequorea victoria (jellyfish) / Pyrococcus horikoshii OT3 (archaea) / Pseudomonas aeruginosa PAO1 (bacteria) / Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1) (bacteria) / Pyrococcus horikoshii (strain ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3) (archaea)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsLiu Y / Huynh D / Yeates TO
Funding support United States, 6 items
OrganizationGrant numberCountry
Department of Energy (DOE, United States)DE-FC02-02ER63421 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)S10RR23057 United States
National Science Foundation (NSF, United States)DBI-1338135 United States
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)U24GM11679 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, 2018-
Header (metadata) releaseJan 23, 2019-
Map releaseMay 8, 2019-
UpdateNov 27, 2019-
Current statusNov 27, 2019Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 7.26
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 7.26
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-6nhv
  • Surface level: 7.26
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_9374.png

Downloads & links

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Map

FileDownload / File: emd_9374.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRefinement of DARPin, GFP, and two adjacent trimers
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
1.06 Å/pix.
x 288 pix.
= 305.28 Å		1.06 Å/pix.
x 288 pix.
= 305.28 Å		1.06 Å/pix.
x 288 pix.
= 305.28 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.06 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy EMDB: 7.26 / Movie #1: 7.26
Minimum - Maximum-28.381075 - 42.377045
Average (Standard dev.)-0.0012176203 (±0.93447405)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions288288288
Spacing288288288
CellA=B=C: 305.27997 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.061.061.06
M x/y/z288288288
origin x/y/z0.0000.0000.000
length x/y/z305.280305.280305.280
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ288288288
MAP C/R/S321
start NC/NR/NS000
NC/NR/NS288288288
D min/max/mean-28.38142.377-0.001

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Supplemental data

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Mask #1

Fileemd_9374_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half body 1

Fileemd_9374_half_map_1.map
AnnotationHalf body 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half body 2

Fileemd_9374_half_map_2.map
AnnotationHalf body 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Subunit A with DARPin + Subunit B + superfolder GFP

EntireName: Subunit A with DARPin + Subunit B + superfolder GFP
Components
  • Complex: Subunit A with DARPin + Subunit B + superfolder GFP
    • Complex: superfolder GFP
      • Protein or peptide: superfolder GFP
    • Complex: Subunit A with DARPin
      • Protein or peptide: Subunit A-DARPin
    • Complex: Subunit B
      • Protein or peptide: DARP14 - Subunit B

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Supramolecule #1: Subunit A with DARPin + Subunit B + superfolder GFP

SupramoleculeName: Subunit A with DARPin + Subunit B + superfolder GFP / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all

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Supramolecule #2: superfolder GFP

SupramoleculeName: superfolder GFP / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Aequorea victoria (jellyfish)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Supramolecule #3: Subunit A with DARPin

SupramoleculeName: Subunit A with DARPin / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: Pyrococcus horikoshii OT3 (archaea)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Supramolecule #4: Subunit B

SupramoleculeName: Subunit B / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Pseudomonas aeruginosa PAO1 (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #1: superfolder GFP

MacromoleculeName: superfolder GFP / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Aequorea victoria (jellyfish)
Molecular weightTheoretical: 26.623918 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MSKGEELFTG VVPILVELDG DVNGHKFSVR GEGEGDATNG KLTLKFICTT GKLPVPWPTL VTTL(CRO)VQCFS RYPDHM KRH DFFKSAMPEG YVQERTISFK DDGTYKTRAE VKFEGDTLVN RIELKGIDFK EDGNILGHKL EYNFNSHNVY ITADKQK NG IKANFKIRHN ...String:
MSKGEELFTG VVPILVELDG DVNGHKFSVR GEGEGDATNG KLTLKFICTT GKLPVPWPTL VTTL(CRO)VQCFS RYPDHM KRH DFFKSAMPEG YVQERTISFK DDGTYKTRAE VKFEGDTLVN RIELKGIDFK EDGNILGHKL EYNFNSHNVY ITADKQK NG IKANFKIRHN VEDGSVQLAD HYQQNTPIGD GPVLLPDNHY LSTQSALSKD PNEKRDHMVL LEFVTAAGIT HHHHHH

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Macromolecule #2: DARP14 - Subunit B

MacromoleculeName: DARP14 - Subunit B / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: 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
Molecular weightTheoretical: 14.346274 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MPHLVIEATA NLRLETSPGE LLEQANKALF ASGQFGEADI KSRFVTLEAY RQGTAAVERA YLHACLSILD GRDIATRTLL GASLCAVLA EAVAGGGEEG VQVSVEVREM ERLSYAKRVV ARQRLEHHHH HH

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Macromolecule #3: Subunit A-DARPin

MacromoleculeName: Subunit A-DARPin / type: protein_or_peptide / ID: 3 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: 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
Molecular weightTheoretical: 34.71782 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MRITTKVGDK GSTRLFGGEE VWKDSPIIEA NGTLDELTSF IGEAKHYVDE EMKGILEEIQ NDIYKIMGEI GSKGKIEGIS EERIAWLLK LILRYMEMVN LKSFVLPGGT LESAKLDVCR TIARRALRKV LTVTREFGIG AEAAAYLLAL SDLLFLLARV I EIEQGKKL ...String:
MRITTKVGDK GSTRLFGGEE VWKDSPIIEA NGTLDELTSF IGEAKHYVDE EMKGILEEIQ NDIYKIMGEI GSKGKIEGIS EERIAWLLK LILRYMEMVN LKSFVLPGGT LESAKLDVCR TIARRALRKV LTVTREFGIG AEAAAYLLAL SDLLFLLARV I EIEQGKKL LEAARAGQDD EVRILMANGA DVNAADDVGV TPLHLAAQRG HLEIVEVLLK CGADVNAADL WGQTPLHLAA TA GHLEIVE VLLKNGADVN ARDNIGHTPL HLAAWAGHLE IVEVLLKYGA DVNAQDKFGK TPFDLAIDNG NEDIAEVLQK AA

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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

Concentration1 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
10.0 mMC4H11NO3Tris
500.0 mMNaClsodium chloride
1.0 mMC4H10O2S2DTT
0.5 %C3H8O3glycerol
GridDetails: unspecified
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
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

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 3-20 / Number grids imaged: 1 / Number real images: 1929 / Average exposure time: 8.0 sec. / Average electron dose: 56.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 963036
CTF correctionSoftware - Name: CTFFIND (ver. 4.1.5)
Startup modelType of model: EMDB MAP
EMDB ID:

9373

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 91211
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final 3D classificationNumber classes: 5 / Software - Name: RELION (ver. 3.0)
Details: 3D classification on symmetry-expanded, signal subtracted particles without alignment
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model(PDB ID:

6c9k

,

5ma8

,

4w6b

)
DetailsInitial local fitting by Chimera and individual residues refined using phenix.real_space_refine for the symmetric core and DARPin, rigid body refinement for GFP
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

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

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