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- PDB-7zj4: Ligand bound state of a brocolli-pepper aptamer FRET tile -

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Basic information

Entry
Database: PDB / ID: 7zj4
TitleLigand bound state of a brocolli-pepper aptamer FRET tile
Componentsbrocolli-pepper aptamer
KeywordsRNA / RNA origami aptamer fret
Function / homologyChem-1TU / Chem-J93 / : / RNA / RNA (> 10) / RNA (> 100)
Function and homology information
Biological speciessynthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.43 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: Rea-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: Tristan Ian Croll /
Abstract: This paper introduces ISOLDE, a new software package designed to provide an intuitive environment for high-fidelity interactive remodelling/refinement of macromolecular models into electron-density ...This paper introduces ISOLDE, a new software package designed to provide an intuitive environment for high-fidelity interactive remodelling/refinement of macromolecular models into electron-density maps. ISOLDE combines interactive molecular-dynamics flexible fitting with modern molecular-graphics visualization and established structural biology libraries to provide an immersive interface wherein the model constantly acts to maintain physically realistic conformations as the user interacts with it by directly tugging atoms with a mouse or haptic interface or applying/removing restraints. In addition, common validation tasks are accelerated and visualized in real time. Using the recently described 3.8 Å resolution cryo-EM structure of the eukaryotic minichromosome maintenance (MCM) helicase complex as a case study, it is demonstrated how ISOLDE can be used alongside other modern refinement tools to avoid common pitfalls of low-resolution modelling and improve the quality of the final model. A detailed analysis of changes between the initial and final model provides a somewhat sobering insight into the dangers of relying on a small number of validation metrics to judge the quality of a low-resolution model.
#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

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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)121,3604
Polymers120,7011
Non-polymers6593
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: RNA chain brocolli-pepper aptamer


Mass: 120700.953 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#2: Chemical ChemComp-J93 / 4-[(~{Z})-1-cyano-2-[5-[2-hydroxyethyl(methyl)amino]thieno[3,2-b]thiophen-2-yl]ethenyl]benzenecarbonitrile


Mass: 365.472 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C19H15N3OS2 / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-1TU / 4-(3,5-difluoro-4-hydroxybenzyl)-1,2-dimethyl-1H-imidazol-5-ol


Mass: 254.233 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C12H12F2N2O2 / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-K / POTASSIUM ION


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

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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: Ligand bound 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.
Specimen supportDetails: 15mA of current. / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
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 FIELDBright-field microscopy / 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: 5354
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
9cryoSPARC3initial Euler assignment
10cryoSPARC3final Euler assignment
11cryoSPARC3classification
12cryoSPARC33D reconstruction
13PHENIXmodel refinement
14ISOLDEmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 729630
Details: Picked from templates generated from an ab initio model
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.43 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 150204
Details: Local refinement was performed using a mask covering the entire 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_d0.00588966
ELECTRON MICROSCOPYf_angle_d0.689913969
ELECTRON MICROSCOPYf_chiral_restr0.03721868
ELECTRON MICROSCOPYf_plane_restr0.0043379
ELECTRON MICROSCOPYf_dihedral_angle_d16.17264495

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