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- PDB-7ypo: Cryo-EM structure of baculovirus LEF-3 in complex with ssDNA -

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Basic information

Entry
Database: PDB / ID: 7ypo
TitleCryo-EM structure of baculovirus LEF-3 in complex with ssDNA
Components
  • DNA (28-MER)
  • Lef3
KeywordsDNA BINDING PROTEIN / ssDNA / SSB / Baculovirus
Function / homologyNucleopolyhedrovirus late expression factor 3 / Nucleopolyhedrovirus late expression factor 3 (LEF-3) / regulation of DNA-templated transcription / DNA binding / DNA / DNA (> 10) / Lef3
Function and homology information
Biological speciesHelicoverpa armigera nucleopolyhedrovirus
synthetic construct (others)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsYin, J. / Fu, Y. / Rao, G. / Li, Z. / Cao, S.
Funding support China, 1items
OrganizationGrant numberCountry
Ministry of Science and Technology (MoST, China)2018YFA0507200 China
CitationJournal: Nucleic Acids Res / Year: 2022
Title: Structural transitions during the cooperative assembly of baculovirus single-stranded DNA-binding protein on ssDNA.
Authors: Jiayi Yin / Yan Fu / Guibo Rao / Zhiqiang Li / Kexing Tian / Tingting Chong / Kai Kuang / Manli Wang / Zhihong Hu / Sheng Cao /
Abstract: Single-stranded DNA-binding proteins (SSBs) interact with single-stranded DNA (ssDNA) to form filamentous structures with various degrees of cooperativity, as a result of intermolecular interactions ...Single-stranded DNA-binding proteins (SSBs) interact with single-stranded DNA (ssDNA) to form filamentous structures with various degrees of cooperativity, as a result of intermolecular interactions between neighboring SSB subunits on ssDNA. However, it is still challenging to perform structural studies on SSB-ssDNA filaments at high resolution using the most studied SSB models, largely due to the intrinsic flexibility of these nucleoprotein complexes. In this study, HaLEF-3, an SSB protein from Helicoverpa armigera nucleopolyhedrovirus, was used for in vitro assembly of SSB-ssDNA filaments, which were structurally studied at atomic resolution using cryo-electron microscopy. Combined with the crystal structure of ssDNA-free HaLEF-3 octamers, our results revealed that the three-dimensional rearrangement of HaLEF-3 induced by an internal hinge-bending movement is essential for the formation of helical SSB-ssDNA complexes, while the contacting interface between adjacent HaLEF-3 subunits remains basically intact. We proposed a local cooperative SSB-ssDNA binding model, in which, triggered by exposure to oligonucleotides, HaLEF-3 molecules undergo ring-to-helix transition to initiate continuous SSB-SSB interactions along ssDNA. Unique structural features revealed by the assembly of HaLEF-3 on ssDNA suggest that HaLEF-3 may represent a new class of SSB.
History
DepositionAug 4, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jan 25, 2023Provider: repository / Type: Initial release
Revision 1.1Jul 3, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond / em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Lef3
B: Lef3
C: Lef3
D: Lef3
Q: DNA (28-MER)


Theoretical massNumber of molelcules
Total (without water)199,2765
Polymers199,2765
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1chain "B"
d_2ens_1chain "D"
d_3ens_1chain "C"
d_4ens_1chain "A"

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg label comp-IDEnd label comp-IDLabel asym-IDLabel seq-ID
d_11ens_1LEULEUB1 - 327
d_21ens_1LEULEUD1 - 327
d_31ens_1LEULEUC1 - 327
d_41ens_1LEULEUA1 - 327

NCS oper:
IDCodeMatrixVector
1given(-0.803402657221, -0.595435898114, 0.000511476546116), (0.595435618711, -0.803402799599, -0.000604621743237), (0.000770935179782, -0.000181203361437, 0.999999686412)132.746913488, 62.5804329749, 30.2015169865
2given(-0.31331662351, 0.949648297785, 0.00089663113366), (-0.949648351432, -0.313317194955, 0.000586487882342), (0.000837887170845, -0.000667727874898, 0.999999426042)23.0644005768, 123.07940926, 15.1288122223
3given(0.817681189045, -0.575671270064, -0.000248805713794), (0.575671153762, 0.81768120333, -0.000415266929683), (0.000442500996276, 0.000196325684512, 0.999999882825)40.8618324676, -22.5637313831, 45.287260335

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Components

#1: Protein
Lef3


Mass: 47637.707 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Helicoverpa armigera nucleopolyhedrovirus
Production host: Escherichia coli BL21 (bacteria) / References: UniProt: Q91BW6
#2: DNA chain DNA (28-MER)


Mass: 8724.834 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: HELICAL ARRAY / 3D reconstruction method: helical reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1Baculovirus LEF-3 in complex with ssDNACOMPLEXall0MULTIPLE SOURCES
2LEF-3COMPLEX#11RECOMBINANT
3ssDNACOMPLEX#21SYNTHETIC
Source (natural)Organism: Helicoverpa armigera nucleopolyhedrovirus
Source (recombinant)Organism: Escherichia coli BL21 (bacteria)
Buffer solutionpH: 6.3
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

MicroscopyModel: JEOL CRYO ARM 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 500 nm
Image recordingElectron dose: 40 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: -108.281 ° / Axial rise/subunit: 15.105 Å / Axial symmetry: C1
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 530580 / Symmetry type: HELICAL
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 51.02 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00411579
ELECTRON MICROSCOPYf_angle_d0.55215744
ELECTRON MICROSCOPYf_dihedral_angle_d12.5431713
ELECTRON MICROSCOPYf_chiral_restr0.0441772
ELECTRON MICROSCOPYf_plane_restr0.0021908
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2BELECTRON MICROSCOPYNCS constraints0.0613602157971
ens_1d_3BELECTRON MICROSCOPYNCS constraints0.000625275800487
ens_1d_4BELECTRON MICROSCOPYNCS constraints0.0613740870943

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