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- PDB-6mwq: Single particle cryoEM structure of a DARPin-aldolase platform in... -

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Basic information

Entry
Database: PDB / ID: 6mwq
TitleSingle particle cryoEM structure of a DARPin-aldolase platform in complex with GFP
Components
  • DARPin, Muscle-type aldolase chimeric fusion
  • Green fluorescent protein
KeywordsLYASE/FLUORESCENT PROTEIN / synthetic construct / platform / single particle cryoEM / small protein display / LYASE-FLUORESCENT PROTEIN complex
Function / homology
Function and homology information


negative regulation of Arp2/3 complex-mediated actin nucleation / fructose-bisphosphate aldolase / fructose-bisphosphate aldolase activity / M band / I 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 / Green fluorescent protein / Green fluorescent protein, GFP / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein ...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 / Green fluorescent protein / Green fluorescent protein, GFP / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein / Aldolase-type TIM barrel / Beta Barrel / Mainly Beta
Similarity search - Domain/homology
Fructose-bisphosphate aldolase A / Green fluorescent protein
Similarity search - Component
Biological speciessynthetic construct (others)
Oryctolagus cuniculus (rabbit)
Aequorea victoria (jellyfish)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsWeaver, S.J. / Yao, Q.
Funding support United States, 1items
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, S.J. / Mock, J.Y. / Jensen, G.J.
History
DepositionOct 30, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 21, 2018Provider: repository / Type: Initial release
Revision 1.1May 29, 2019Group: Data collection / Database references / Category: citation / citation_author
Revision 1.2Jul 17, 2019Group: Data collection / Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first
Revision 1.3Jan 8, 2020Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.4Mar 13, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _citation.journal_id_ISSN / _database_2.pdbx_DOI ..._citation.journal_id_ISSN / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

Movie
  • Biological unit as author_defined_assembly
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  • Deposited structure unit
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  • Superimposition on EM map
  • EMDB-9277
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: DARPin, Muscle-type aldolase chimeric fusion
G: Green fluorescent protein
B: DARPin, Muscle-type aldolase chimeric fusion
J: Green fluorescent protein
C: DARPin, Muscle-type aldolase chimeric fusion
I: Green fluorescent protein
D: DARPin, Muscle-type aldolase chimeric fusion
H: Green fluorescent protein


Theoretical massNumber of molelcules
Total (without water)314,3498
Polymers314,3498
Non-polymers00
Water0
1
A: DARPin, Muscle-type aldolase chimeric fusion
G: Green fluorescent protein


Theoretical massNumber of molelcules
Total (without water)78,5872
Polymers78,5872
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_5551
2
B: DARPin, Muscle-type aldolase chimeric fusion
J: Green fluorescent protein


Theoretical massNumber of molelcules
Total (without water)78,5872
Polymers78,5872
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_5551
3
C: DARPin, Muscle-type aldolase chimeric fusion
I: Green fluorescent protein


Theoretical massNumber of molelcules
Total (without water)78,5872
Polymers78,5872
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_5551
4
D: DARPin, Muscle-type aldolase chimeric fusion
H: Green fluorescent protein


Theoretical massNumber of molelcules
Total (without water)78,5872
Polymers78,5872
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
DARPin, Muscle-type aldolase chimeric fusion / / Fructose-bisphosphate aldolase A


Mass: 53113.434 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Details: Rabbit (Oryctolagus cuniculus) muscle aldolase and a synthetic DARPin were fused to create this synthetic construct
Source: (gene. exp.) synthetic construct (others), (gene. exp.) Oryctolagus cuniculus (rabbit)
Plasmid: pET21b / Gene: ALDOA
Details (production host): C-terminal His-tag of DARPin-aldolase chimeric fusion
Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: P00883, fructose-bisphosphate aldolase
#2: Protein
Green fluorescent protein /


Mass: 25473.697 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Aequorea victoria (jellyfish) / Gene: GFP / Plasmid: pACYCDuet / Details (production host): no tag / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: P42212

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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
1DARPin-aldolase platform in complex with GFPCOMPLEXall0RECOMBINANT
2DARPin, Muscle-type aldolase chimeric fusionCOMPLEX1RECOMBINANT
3Green fluorescent proteinCOMPLEX1RECOMBINANT
Molecular weightValue: 0.314 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
11synthetic construct (others)32630
22Aequorea victoria (jellyfish)6100
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrain
11Escherichia coli (E. coli)469008BL21 (DE3)
22Escherichia coli (E. coli)469008BL21 (DE3)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMTris HCl1
2150 mMsodium chlorideNaClSodium chloride1
SpecimenConc.: 2.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: The 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 ...Details: The 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.
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277.15 K
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%.

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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: 165000 X / Nominal defocus max: 30000 nm / Nominal defocus min: 10000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recording

Imaging-ID: 1 / Average exposure time: 4 sec. / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1

IDElectron dose (e/Å2)Num. of real imagesDetails
12.31133All three microscope sessions used grids frozen in the same session. In the first microscopy session the stage was untilted (0 degrees).
22.3548All three microscope sessions used grids frozen in the same session. In the second microscopy session the stage was untilted (0 degrees).
31.11180All three microscope sessions used grids frozen in the same session. In the third microscopy session the stage was tilted to 26 degrees.
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV
Image scansMovie frames/image: 40 / Used frames/image: 1-40

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Processing

EM software
IDNameVersionCategoryDetails
2SerialEMimage acquisition
3EPUimage acquisition
5CTFFIND4.1.10CTF correctionwhole micrographs
6RELION3.0 beta 2CTF correctionPer particle CtfRefine
9UCSF Chimera1.12model fittingFit into map
11RELION3.0 beta 2initial Euler assignment
12RELION3.0 beta 2final Euler assignment
14RELION3.0 beta 23D reconstruction
Image processingDetails: Particles from all three microscopy sessions were processed together.
CTF correctionDetails: First whole micrograph correction with CtfFind4. Then per particle CTF Refinement in Relion.
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles 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
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 236339 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Details: PDB 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 ...Details: PDB 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.
Atomic model building

3D fitting-ID: 1 / Source name: PDB / Type: experimental model

IDPDB-IDPdb chain-IDAccession codeInitial refinement model-ID
15VY5

5vy5

A5VY51
25MA6

5ma6

A5MA62
35MA6

5ma6

B5MA62

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