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- EMDB-9277: Single particle cryoEM structure of a DARPin-aldolase platform in... -

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Basic information

Entry
Database: EMDB / ID: EMD-9277
TitleSingle particle cryoEM structure of a DARPin-aldolase platform in complex with GFP
Map dataDARPin-aldolase platform in complex with GFP, Relion Post processed map with B factor -25 angstrom squared
Sample
  • Complex: DARPin-aldolase platform in complex with GFP
    • Complex: DARPin, Muscle-type aldolase chimeric fusion
    • Complex: Green fluorescent protein
    • Protein or peptide: DARPin, Muscle-type aldolase chimeric fusion
    • Protein or peptide: Green fluorescent protein
Function / homology
Function and homology information


negative regulation of Arp2/3 complex-mediated actin nucleation / fructose-bisphosphate aldolase / fructose-bisphosphate aldolase activity / I band / M band / bioluminescence / generation of precursor metabolites and energy / glycolytic process / protein homotetramerization / positive regulation of cell migration
Similarity search - Function
Fructose-bisphosphate aldolase class-I active site. / Fructose-bisphosphate aldolase class-I active site / Fructose-bisphosphate aldolase, class-I / Fructose-bisphosphate aldolase class-I / Green fluorescent protein, GFP / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein / Aldolase-type TIM barrel
Similarity search - Domain/homology
Fructose-bisphosphate aldolase A / Green fluorescent protein
Similarity search - Component
Biological speciessynthetic construct (unknown) / Aequorea victoria (jellyfish) / Oryctolagus cuniculus (rabbit)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsWeaver SJ / Yao Q
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P50 082545 United States
Citation
Journal: Structure / Year: 2019
Title: Fusion of DARPin to Aldolase Enables Visualization of Small Protein by Cryo-EM.
Authors: Qing Yao / Sara J Weaver / Jee-Young Mock / Grant J Jensen /
Abstract: Solving protein structures by single-particle cryoelectron microscopy (cryo-EM) has become a crucial tool in structural biology. While exciting progress is being made toward the visualization of ...Solving protein structures by single-particle cryoelectron microscopy (cryo-EM) has become a crucial tool in structural biology. While exciting progress is being made toward the visualization of small macromolecules, the median protein size in both eukaryotes and bacteria is still beyond the reach of cryo-EM. To overcome this problem, we implemented a platform strategy in which a small protein target was rigidly attached to a large, symmetric base via a selectable adapter. Of our seven designs, the best construct used a designed ankyrin repeat protein (DARPin) rigidly fused to tetrameric rabbit muscle aldolase through a helical linker. The DARPin retained its ability to bind its target: GFP. We solved the structure of this complex to 3.0 Å resolution overall, with 5-8 Å resolution in the GFP region. As flexibility in the DARPin position limited the overall resolution of the target, we describe strategies to rigidify this element.
#1: Journal: Biorxiv / Year: 2018
Title: Fusion of DARPin to aldolase enables visualization of small protein by cryoEM
Authors: Yao Q / Weaver SJ / Mock JY / Jensen GJ
History
DepositionOct 30, 2018-
Header (metadata) releaseNov 21, 2018-
Map releaseNov 21, 2018-
UpdateJan 8, 2020-
Current statusJan 8, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.004
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.004
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-6mwq
  • Surface level: 0.004
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_9277.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationDARPin-aldolase platform in complex with GFP, Relion Post processed map with B factor -25 angstrom squared
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 360 pix.
= 299.34 Å
0.83 Å/pix.
x 360 pix.
= 299.34 Å
0.83 Å/pix.
x 360 pix.
= 299.34 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.8315 Å
Density
Contour LevelBy AUTHOR: 0.004 / Movie #1: 0.004
Minimum - Maximum-0.025016628 - 0.056058165
Average (Standard dev.)0.0000611498 (±0.001124288)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 299.34 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.83150.83150.8315
M x/y/z360360360
origin x/y/z0.0000.0000.000
length x/y/z299.340299.340299.340
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ240240240
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS360360360
D min/max/mean-0.0250.0560.000

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Supplemental data

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Half map: Half map 1 of a DARPin-aldolase platform in complex with GFP

Fileemd_9277_half_map_1.map
AnnotationHalf map 1 of a DARPin-aldolase platform in complex with GFP
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: Half map 2 of a DARPin-aldolase platform in complex with GFP

Fileemd_9277_half_map_2.map
AnnotationHalf map 2 of a DARPin-aldolase platform in complex with GFP
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire : DARPin-aldolase platform in complex with GFP

EntireName: DARPin-aldolase platform in complex with GFP
Components
  • Complex: DARPin-aldolase platform in complex with GFP
    • Complex: DARPin, Muscle-type aldolase chimeric fusion
    • Complex: Green fluorescent protein
    • Protein or peptide: DARPin, Muscle-type aldolase chimeric fusion
    • Protein or peptide: Green fluorescent protein

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Supramolecule #1: DARPin-aldolase platform in complex with GFP

SupramoleculeName: DARPin-aldolase platform in complex with GFP / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: synthetic construct (unknown)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21 (DE3)
Molecular weightTheoretical: 314 KDa

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Supramolecule #2: DARPin, Muscle-type aldolase chimeric fusion

SupramoleculeName: DARPin, Muscle-type aldolase chimeric fusion / type: complex / ID: 2 / Parent: 1
Source (natural)Organism: Aequorea victoria (jellyfish)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21 (DE3)

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Supramolecule #3: Green fluorescent protein

SupramoleculeName: Green fluorescent protein / type: complex / ID: 3 / Parent: 1

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Macromolecule #1: DARPin, Muscle-type aldolase chimeric fusion

MacromoleculeName: DARPin, Muscle-type aldolase chimeric fusion / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO / EC number: fructose-bisphosphate aldolase
Source (natural)Organism: Oryctolagus cuniculus (rabbit)
Molecular weightTheoretical: 53.113434 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (unknown)
SequenceString: SGSDLGKKLL EAARAGQDDE VRILMANGAD VNALDRFGLT PLHLAAQRGH LEIVEVLLKC GADVNAADLW GQTPLHLAAT AGHLEIVEV LLKYGADVNA LDLIGKTPLH LTAIDGHLEI VEVLLKHGAD VNAQDKFGKT AFDISIDNGN EDLAEILQKL N LSDIAHRI ...String:
SGSDLGKKLL EAARAGQDDE VRILMANGAD VNALDRFGLT PLHLAAQRGH LEIVEVLLKC GADVNAADLW GQTPLHLAAT AGHLEIVEV LLKYGADVNA LDLIGKTPLH LTAIDGHLEI VEVLLKHGAD VNAQDKFGKT AFDISIDNGN EDLAEILQKL N LSDIAHRI VAPGKGILAA DESTGSIAKR LQSIGTENTE ENRRFYRQLL LTADDRVNPC IGGVILFHET LYQKADDGRP FP QVIKSKG GVVGIKVDKG VVPLAGTNGE TTTQGLDGLS ERCAQYKKDG ADFAKWRCVL KIGEHTPSAL AIMENANVLA RYA SICQQN GIVPIVEPEI LPDGDHDLKR CQYVTEKVLA AVYKALSDHH IYLEGTLLKP NMVTPGHACT QKYSHEEIAM ATVT ALRRT VPPAVTGVTF LSGGQSEEEA SINLNAINKC PLLKPWALTF SYGRALQASA LKAWGGKKEN LKAAQEEYVK RALAN SLAC QGKYTPSGQ

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Macromolecule #2: Green fluorescent protein

MacromoleculeName: Green fluorescent protein / type: protein_or_peptide / ID: 2 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Aequorea victoria (jellyfish)
Molecular weightTheoretical: 25.473697 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (unknown)
SequenceString: SKGEELFTGV VPILVELDGD VNGHKFSVSG EGEGDATYGK LTLKFICTTG KLPVPWPTLV TTLVQCFSRY PDHMKQHDFF KSAMPEGYV QERTIFFKDD GNYKTRAEVK FEGDTLVNRI ELKGIDFKED GNILGHKLEY NYNSHNVYIM ADKQKNGIKV N FKIRHNIE ...String:
SKGEELFTGV VPILVELDGD VNGHKFSVSG EGEGDATYGK LTLKFICTTG KLPVPWPTLV TTLVQCFSRY PDHMKQHDFF KSAMPEGYV QERTIFFKDD GNYKTRAEVK FEGDTLVNRI ELKGIDFKED GNILGHKLEY NYNSHNVYIM ADKQKNGIKV N FKIRHNIE DGSVQLADHY QQNTPIGDGP VLLPDNHYLS TQSALSKDPN EKRDHMVLLE FVTAAGIT

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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

Concentration2.5 mg/mL
BufferpH: 8
Component:
ConcentrationNameFormula
25.0 mMTris HCl
150.0 mMsodium chlorideNaClSodium chloride
GridModel: UltrAuFoil / Material: GOLD / Mesh: 300 / Pretreatment - Type: PLASMA CLEANING
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277.15 K / Instrument: HOMEMADE PLUNGER
Details: Grids were frozen on a manual plunger at the Scripps Research Institute Core Microscopy Facility in a 4 degrees C cold room humidified to >95%..
DetailsThe protein complex was then purified with Ni-NTA affinity chromatography (Qiagen), and Superdex 200 chromatography (GE healthcare). The purified GFP-DARPin-aldolase complex was concentrated to 2.5mg/ml in a buffer containing 25 mM Tris-HCl pH 8.0 and 150 mM NaCl.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 30.0 µm / Nominal defocus min: 10.0 µm / Nominal magnification: 165000
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recording#0 - Image recording ID: 1 / #0 - Film or detector model: GATAN K2 SUMMIT (4k x 4k) / #0 - Detector mode: SUPER-RESOLUTION / #0 - Number grids imaged: 1 / #0 - Number real images: 1133 / #0 - Average exposure time: 4.0 sec. / #0 - Average electron dose: 2.3 e/Å2
#0 - Details: All three microscope sessions used grids frozen in the same session. In the first microscopy session the stage was untilted (0 degrees).
#1 - Image recording ID: 2 / #1 - Film or detector model: GATAN K2 SUMMIT (4k x 4k) / #1 - Detector mode: SUPER-RESOLUTION / #1 - Number grids imaged: 1 / #1 - Number real images: 548 / #1 - Average exposure time: 4.0 sec. / #1 - Average electron dose: 2.3 e/Å2
#1 - Details: All three microscope sessions used grids frozen in the same session. In the second microscopy session the stage was untilted (0 degrees).
#2 - Image recording ID: 3 / #2 - Film or detector model: GATAN K2 SUMMIT (4k x 4k) / #2 - Detector mode: SUPER-RESOLUTION / #2 - Digitization - Frames/image: 1-40 / #2 - Number grids imaged: 1 / #2 - Number real images: 1180 / #2 - Average exposure time: 4.0 sec. / #2 - Average electron dose: 1.1 e/Å2
#2 - Details: All three microscope sessions used grids frozen in the same session. In the third microscopy session the stage was tilted to 26 degrees.
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 851776
Details: Manually picked particles were used to generate references for autopicking. Particles from all three microscopy sessions were processed together. Microscope session 1 - 358,381 particles ...Details: Manually picked particles were used to generate references for autopicking. Particles from all three microscopy sessions were processed together. Microscope session 1 - 358,381 particles Microscope session 2 - 64,368 particles Microscope session 3 - 402,427 particles
CTF correctionSoftware:
Namedetails
CTFFIND (ver. 4.1.10)whole micrographs
RELION (ver. 3.0 beta 2)Per particle CtfRefine

Details: First whole micrograph correction with CtfFind4. Then per particle CTF Refinement in Relion.
Startup modelType of model: OTHER / Details: cryoSPARC ab initio initial model generation
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0 beta 2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0 beta 2)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0 beta 2) / Number images used: 236339
DetailsParticles from all three microscopy sessions were processed together.
Image recording ID1
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

chain_id: A

chain_id: A

chain_id: B
DetailsPDB models 5vy5 and 5ma6 were used as starting points. These models were mutated to match the sequence of the DARPin-aldolase platform in complex with GFP that were used. The PDB models were docked into the cryoEM density using Chimera fit into map function. No model refinement was used.
RefinementProtocol: RIGID BODY FIT
Output model

PDB-6mwq:
Single particle cryoEM structure of a DARPin-aldolase platform in complex with GFP

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