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- PDB-7zj5: Unbound state of a brocolli-pepper aptamer FRET tile. -

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Basic information

Entry
Database: PDB / ID: 7zj5
TitleUnbound state of a brocolli-pepper aptamer FRET tile.
Componentsbrocolli-pepper aptamer
KeywordsRNA / origami / aptamer / fret
Function / homology: / RNA / RNA (> 10) / RNA (> 100)
Function and homology information
Biological speciessynthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.55 Å
AuthorsMcRae, E.K.S. / Vallina, N.S. / Hansen, B.K. / Boussebayle, A. / Andersen, E.S.
Funding support Denmark, 1items
OrganizationGrant numberCountry
Danish Council for Independent Research Denmark
Citation
Journal: To Be Published
Title: Structure determination of Pepper-Broccoli FRET pair by RNA origami scaffolding
Authors: McRae, E.K.S. / Vallina, N.S. / Hansen, B.K. / Boussebayle, A. / Andersen, E.S.
#1: Journal: Acta Crystallogr D Struct Biol / Year: 2018
Title: Real-space refinement in PHENIX for cryo-EM and crystallography.
Authors: Afonine, P.V. / Poon, B.K. / Read, R.J. / Sobolev, O.V. / Terwilliger, T.C. / Urzhumtsev, A. / Adams, P.D.
#2: Journal: Acta Crystallogr D Struct Biol / Year: 2018
Title: ISOLDE: a physically realistic environment for model building into low-resolution electron-density maps.
Authors: Croll, T.I.
#3: Journal: Nat Methods / Year: 2017
Title: cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination.
Authors: Punjani, A. / Rubinstein, J.L. / Fleet, D.J. / Brubaker, M.A.
History
DepositionApr 8, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 19, 2023Provider: repository / Type: Initial release
Revision 1.1May 24, 2023Group: Structure summary / Category: struct / Item: _struct.title
Revision 1.2Jul 26, 2023Group: Other / Category: pdbx_database_status / Item: _pdbx_database_status.pdb_format_compatible

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
E: brocolli-pepper aptamer
hetero molecules


Theoretical massNumber of molelcules
Total (without water)120,7402
Polymers120,7011
Non-polymers391
Water00
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area0 Å2
ΔGint0 kcal/mol
Surface area62130 Å2
MethodPISA

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Components

#1: RNA chain brocolli-pepper aptamer


Mass: 120700.953 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: synthetic construct (others)
#2: Chemical ChemComp-K / POTASSIUM ION


Mass: 39.098 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: K
Has ligand of interestN

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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

ComponentName: Unbound state of a brocolli-pepper aptamer FRET tile / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.12 MDa / Experimental value: NO
Source (natural)Organism: synthetic construct (others)
Source (recombinant)Organism: synthetic construct (others)
Buffer solutionpH: 7.5
Details: 40mM HEPES pH 7.5, 5mM MgCl2, 50mM KCl. Filtered through 0.22 um filter.
SpecimenConc.: 2.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Sample was purified by size exclusion chromatography.
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 288 K
Details: 3 uL sample, blotted onto double layer of whatman filter paper for 6 seconds.

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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 defocus max: 2000 nm / Nominal defocus min: 700 nm / Cs: 2.7 mm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1605
Details: Collected with a calibrated pixel size of 0.647 Angstrom
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.20.1_4487refinement
PHENIX1.20.1_4487refinement
EM software
IDNameVersionCategory
4cryoSPARC3CTF correction
7UCSF ChimeraXmodel fitting
9PHENIXmodel refinement
10ISOLDEmodel refinement
11cryoSPARC3initial Euler assignment
12cryoSPARC3final Euler assignment
13cryoSPARC3classification
14cryoSPARC33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 478981
Details: Picked from templates generated from an ab initio model
3D reconstructionResolution: 4.55 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 51278 / Algorithm: BACK PROJECTION
Details: Local refinement was performed in cryoSPARC using a mask that covers the entire particle volume
Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model building
IDPDB-ID 3D fitting-ID
17PTQ

7ptq

1
27EOP

7eop

1
RefinementCross valid method: NONE
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00248922
ELECTRON MICROSCOPYf_angle_d0.650713912
ELECTRON MICROSCOPYf_chiral_restr0.04051870
ELECTRON MICROSCOPYf_plane_restr0.0034374
ELECTRON MICROSCOPYf_dihedral_angle_d14.67954485

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