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- PDB-7ljy: Cryo-EM structure of the B dENE construct complexed with a 28-mer... -

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Database: PDB / ID: 7ljy
TitleCryo-EM structure of the B dENE construct complexed with a 28-mer poly(A)
  • B dENE construct
  • poly(A)Polyadenylation
KeywordsRNA / RNA triple helix / RNA stability / poly(A) / SAXS and cryo-EM
Biological speciesOryza sativa (rice)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 5.6 Å
AuthorsTorabi, S. / Chen, Y. / Zhang, K. / Wang, J. / DeGregorio, S. / Vaidya, A. / Su, Z. / Pabit, S. / Chiu, W. / Pollack, L. / Steitz, J.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P41GM103832 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P01AI120943 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)S10OD021600 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2021
Title: Structural analyses of an RNA stability element interacting with poly(A).
Authors: Seyed-Fakhreddin Torabi / Yen-Lin Chen / Kaiming Zhang / Jimin Wang / Suzanne J DeGregorio / Anand T Vaidya / Zhaoming Su / Suzette A Pabit / Wah Chiu / Lois Pollack / Joan A Steitz /
Abstract: Cis-acting RNA elements are crucial for the regulation of polyadenylated RNA stability. The element for nuclear expression (ENE) contains a U-rich internal loop flanked by short helices. An ENE ...Cis-acting RNA elements are crucial for the regulation of polyadenylated RNA stability. The element for nuclear expression (ENE) contains a U-rich internal loop flanked by short helices. An ENE stabilizes RNA by sequestering the poly(A) tail via formation of a triplex structure that inhibits a rapid deadenylation-dependent decay pathway. Structure-based bioinformatic studies identified numerous ENE-like elements in evolutionarily diverse genomes, including a subclass containing two ENE motifs separated by a short double-helical region (double ENEs [dENEs]). Here, the structure of a dENE derived from a rice transposable element (TWIFB1) before and after poly(A) binding (∼24 kDa and ∼33 kDa, respectively) is investigated. We combine biochemical structure probing, small angle X-ray scattering (SAXS), and cryo-electron microscopy (cryo-EM) to investigate the dENE structure and its local and global structural changes upon poly(A) binding. Our data reveal 1) the directionality of poly(A) binding to the dENE, and 2) that the dENE-poly(A) interaction involves a motif that protects the 3'-most seven adenylates of the poly(A). Furthermore, we demonstrate that the dENE does not undergo a dramatic global conformational change upon poly(A) binding. These findings are consistent with the recently solved crystal structure of a dENE+poly(A) complex [S.-F. Torabi , 371, eabe6523 (2021)]. Identification of additional modes of poly(A)-RNA interaction opens new venues for better understanding of poly(A) tail biology.
Validation Report
SummaryFull reportAbout validation report
DepositionFeb 1, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 14, 2021Provider: repository / Type: Initial release

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Deposited unit
B: B dENE construct
A: poly(A)

Theoretical massNumber of molelcules
Total (without water)33,4152

TypeNameSymmetry operationNumber
identity operation1_5551
Buried area3730 Å2
ΔGint-26 kcal/mol
Surface area16750 Å2


#1: RNA chain B dENE construct

Mass: 24242.186 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Oryza sativa (rice)
#2: RNA chain poly(A) / Polyadenylation

Mass: 9172.806 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

Experimental details


EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

Sample preparation

ComponentName: dENE construct complexed with a 28-mer poly(A) / Type: COMPLEX / Entity ID: #1-#2 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.033 MDa / Experimental value: YES
Source (natural)Organism: Oryza sativa (rice)
Buffer solutionpH: 7
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/1
VitrificationCryogen name: ETHANE / Humidity: 100 %

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
Image recordingElectron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)


SoftwareName: REFMAC / Version: 5.8.0258 / Classification: refinement
EM software
1EMAN22.3particle selection
2EPU2.5image acquisition
4CTFFIND4.1CTF correction
7UCSF Chimeramodel fitting
12cryoSPARC2.15.03D reconstruction
13REFMACmodel refinement
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 5.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 283486 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
RefinementResolution: 6.5→120 Å / Cor.coef. Fo:Fc: 0.945 / Cor.coef. Fo:Fc free: 0.885 / SU ML: 110.55 / Cross valid method: THROUGHOUT / ESU R Free: 2.176
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
RfactorNum. reflection% reflectionSelection details
Rfree0.45134 279 6.4 %RANDOM
Rwork0.44358 ---
Obs0.4441 4078 100 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 79.714 Å2
Baniso -1Baniso -2Baniso -3
1--28.05 Å2-1.78 Å2-14.17 Å2
2--8.86 Å28.58 Å2
3---19.19 Å2
Refinement stepCycle: 1 / Total: 2147
Refine LS restraints
Refinement-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0190.0142456
ELECTRON MICROSCOPYr_bond_other_d1.0040.02978
ELECTRON MICROSCOPYr_angle_refined_deg0.8981.2893739
ELECTRON MICROSCOPYr_angle_other_deg1.68432389
ELECTRON MICROSCOPYr_dihedral_angle_1_deg12.09310.277524
ELECTRON MICROSCOPYr_dihedral_angle_2_deg
ELECTRON MICROSCOPYr_dihedral_angle_3_deg
ELECTRON MICROSCOPYr_dihedral_angle_4_deg
ELECTRON MICROSCOPYr_chiral_restr0.1610.214424
ELECTRON MICROSCOPYr_gen_planes_refined1.0040.021178
ELECTRON MICROSCOPYr_gen_planes_other1.0030.02514
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_long_range_B_refined
ELECTRON MICROSCOPYr_long_range_B_other
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 6.5→6.668 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.816 22 -
Rwork0.736 319 -
Obs--100 %

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