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- EMDB-34010: Cryo-EM structure of baculovirus LEF-3 in complex with ssDNA -

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Basic information

Entry
Database: EMDB / ID: EMD-34010
TitleCryo-EM structure of baculovirus LEF-3 in complex with ssDNA
Map data
Sample
  • Complex: Baculovirus LEF-3 in complex with ssDNA
    • Complex: LEF-3
      • Protein or peptide: Lef3
    • Complex: ssDNA
      • DNA: DNA (28-MER)
KeywordsssDNA / SSB / Baculovirus / DNA BINDING PROTEIN
Function / homologyNucleopolyhedrovirus late expression factor 3 / Nucleopolyhedrovirus late expression factor 3 (LEF-3) / regulation of DNA-templated transcription / DNA binding / Lef3
Function and homology information
Biological speciesHelicoverpa armigera nucleopolyhedrovirus / synthetic construct (others)
Methodhelical reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsYin J / Fu Y / Rao G / Li Z / Cao S
Funding support China, 1 items
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, 2022-
Header (metadata) releaseJan 25, 2023-
Map releaseJan 25, 2023-
UpdateJul 3, 2024-
Current statusJul 3, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_34010.png

Downloads & links

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Map

FileDownload / File: emd_34010.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.95 Å
Density
Contour LevelBy AUTHOR: 0.41
Minimum - Maximum-2.4675062 - 3.7571864
Average (Standard dev.)0.0020681848 (±0.11082076)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 228.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_34010_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_34010_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Baculovirus LEF-3 in complex with ssDNA

EntireName: Baculovirus LEF-3 in complex with ssDNA
Components
  • Complex: Baculovirus LEF-3 in complex with ssDNA
    • Complex: LEF-3
      • Protein or peptide: Lef3
    • Complex: ssDNA
      • DNA: DNA (28-MER)

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Supramolecule #1: Baculovirus LEF-3 in complex with ssDNA

SupramoleculeName: Baculovirus LEF-3 in complex with ssDNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all

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Supramolecule #2: LEF-3

SupramoleculeName: LEF-3 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Helicoverpa armigera nucleopolyhedrovirus

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Supramolecule #3: ssDNA

SupramoleculeName: ssDNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2

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Macromolecule #1: Lef3

MacromoleculeName: Lef3 / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Helicoverpa armigera nucleopolyhedrovirus
Molecular weightTheoretical: 47.637707 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: MGSSHHHHHH SSGLVPRGSH MASMTGGQQM GRGSMSNMDI SPVKQLIDIE NDDAMNTPEK GMKRPLMRTM SSVEEPQAKM AKLRTLNVK GQLLTKTTMS INNEDYYLFK FLVNNKSIDY YGTQTQFFSL INNKTYELVL QYSRKKLLIK SYEQCEDEDL L MTVCKSVT ...String:
MGSSHHHHHH SSGLVPRGSH MASMTGGQQM GRGSMSNMDI SPVKQLIDIE NDDAMNTPEK GMKRPLMRTM SSVEEPQAKM AKLRTLNVK GQLLTKTTMS INNEDYYLFK FLVNNKSIDY YGTQTQFFSL INNKTYELVL QYSRKKLLIK SYEQCEDEDL L MTVCKSVT FQEFCANEIK SLLAKFLYGF KIYGSSNVYK LVFVILLEDN NGTINGVQVE MMSDFKRLSG AFKNHVIENE ND LFDCMYK SEEKYFNLYR IKCNHNANNY KSLSLSSNSQ LERLETDDSM FEYEFQYDYT VNISRSNKII QKHRVTGNFT SER NIYQNS DRFVISYDTA NEKIKTSIYN RMENAESKTD YDTSITLKDV TLSQLNSLIE SNLVQVDVYL VTDPNNVKNN VIAG ITKIE IDGTYEPL

UniProtKB: Lef3

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Macromolecule #2: DNA (28-MER)

MacromoleculeName: DNA (28-MER) / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 8.724834 KDa
SequenceString:
(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA) (DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA) (DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statehelical array

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Sample preparation

BufferpH: 6.3
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeJEOL CRYO ARM 300
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm

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Image processing

Final reconstructionApplied symmetry - Helical parameters - Δz: 15.105 Å
Applied symmetry - Helical parameters - Δ&Phi: -108.281 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 530580
Startup modelType of model: NONE
Final angle assignmentType: NOT APPLICABLE

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