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- EMDB-3400: Lambda excision HJ intermediate -

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Basic information

Entry
Database: EMDB / ID: EMD-3400
TitleLambda excision HJ intermediate
Map dataMap calculated from 10956 particles and fully CTF corrected
Sample
  • Sample: Lambda excision HJ intermediate
  • DNA: attB(-21) to attP(+117)
  • DNA: attB(-19 to +21)
  • DNA: attP(-79) to attB(+19)
  • DNA: attP (-117 to +79)
  • Protein or peptide: Int
  • Protein or peptide: Int
  • Protein or peptide: IHFa
  • Protein or peptide: IHFb
  • Protein or peptide: Xis
Keywordsbacteriophage lambda / excision / site-specfific recombination / Holliday junction
Function / homology
Function and homology information


IHF-DNA complex / provirus excision / integrase activity / DNA-binding transcription activator activity / protein-DNA complex / DNA integration / structural constituent of chromatin / viral genome integration into host DNA / establishment of integrated proviral latency / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases ...IHF-DNA complex / provirus excision / integrase activity / DNA-binding transcription activator activity / protein-DNA complex / DNA integration / structural constituent of chromatin / viral genome integration into host DNA / establishment of integrated proviral latency / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / regulation of translation / chromosome / transferase activity / DNA recombination / Hydrolases; Acting on ester bonds / transcription cis-regulatory region binding / hydrolase activity / symbiont entry into host cell / DNA-templated transcription / regulation of DNA-templated transcription / DNA binding / identical protein binding / cytosol
Similarity search - Function
Excisionase-like / Integrase, lambda-type, N-terminal DNA-binding / Excisionase-like superfamily / Excisionase-like protein / Bacteriophage lambda integrase, Arm DNA-binding domain / Integration host factor, alpha subunit / Integration host factor, beta subunit / Phage integrase, N-terminal SAM-like domain / Integrase, SAM-like, N-terminal / Histone-like DNA-binding protein, conserved site ...Excisionase-like / Integrase, lambda-type, N-terminal DNA-binding / Excisionase-like superfamily / Excisionase-like protein / Bacteriophage lambda integrase, Arm DNA-binding domain / Integration host factor, alpha subunit / Integration host factor, beta subunit / Phage integrase, N-terminal SAM-like domain / Integrase, SAM-like, N-terminal / Histone-like DNA-binding protein, conserved site / Bacterial histone-like DNA-binding proteins signature. / Histone-like DNA-binding protein / Bacterial DNA-binding protein / bacterial (prokaryotic) histone like domain / Integration host factor (IHF)-like DNA-binding domain superfamily / Core-binding (CB) domain / Tyrosine recombinase domain profile. / Core-binding (CB) domain profile. / Phage integrase family / Integrase, catalytic domain / Integrase/recombinase, N-terminal / Integrase-like, catalytic domain superfamily / DNA breaking-rejoining enzyme, catalytic core / Putative DNA-binding domain superfamily / DNA-binding domain superfamily
Similarity search - Domain/homology
Integration host factor subunit alpha / Integration host factor subunit beta / Excisionase / Integrase / Integration host factor subunit alpha / Integration host factor subunit beta
Similarity search - Component
Biological speciesEnterobacteria phage lambda (virus) / Escherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 11.0 Å
AuthorsLaxmikanthan G / Xu C / Brilot AF / Warren D / Steele L / Seah N / Tong W / Grigorieff N / Landy A / Van Duyne G
CitationJournal: Elife / Year: 2016
Title: Structure of a Holliday junction complex reveals mechanisms governing a highly regulated DNA transaction.
Authors: Gurunathan Laxmikanthan / Chen Xu / Axel F Brilot / David Warren / Lindsay Steele / Nicole Seah / Wenjun Tong / Nikolaus Grigorieff / Arthur Landy / Gregory D Van Duyne /
Abstract: The molecular machinery responsible for DNA expression, recombination, and compaction has been difficult to visualize as functionally complete entities due to their combinatorial and structural ...The molecular machinery responsible for DNA expression, recombination, and compaction has been difficult to visualize as functionally complete entities due to their combinatorial and structural complexity. We report here the structure of the intact functional assembly responsible for regulating and executing a site-specific DNA recombination reaction. The assembly is a 240-bp Holliday junction (HJ) bound specifically by 11 protein subunits. This higher-order complex is a key intermediate in the tightly regulated pathway for the excision of bacteriophage λ viral DNA out of the E. coli host chromosome, an extensively studied paradigmatic model system for the regulated rearrangement of DNA. Our results provide a structural basis for pre-existing data describing the excisive and integrative recombination pathways, and they help explain their regulation.
History
DepositionMar 28, 2016-
Header (metadata) releaseApr 6, 2016-
Map releaseJun 15, 2016-
UpdateJun 15, 2016-
Current statusJun 15, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2.32
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 2.32
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-5j0n
  • Surface level: 2.32
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-3400.jpg

Downloads & links

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Map

FileDownload / File: emd_3400.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap calculated from 10956 particles and fully CTF corrected
Voxel sizeX=Y=Z: 2.8 Å
Density
Contour LevelBy AUTHOR: 2.32 / Movie #1: 2.32
Minimum - Maximum-1.88158226 - 8.851735120000001
Average (Standard dev.)-0.02879323 (±0.45667744)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 358.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.82.82.8
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z358.400358.400358.400
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-1.8828.852-0.029

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Supplemental data

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Sample components

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Entire : Lambda excision HJ intermediate

EntireName: Lambda excision HJ intermediate
Components
  • Sample: Lambda excision HJ intermediate
  • DNA: attB(-21) to attP(+117)
  • DNA: attB(-19 to +21)
  • DNA: attP(-79) to attB(+19)
  • DNA: attP (-117 to +79)
  • Protein or peptide: Int
  • Protein or peptide: Int
  • Protein or peptide: IHFa
  • Protein or peptide: IHFb
  • Protein or peptide: Xis

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Supramolecule #1000: Lambda excision HJ intermediate

SupramoleculeName: Lambda excision HJ intermediate / type: sample / ID: 1000
Oligomeric state: Four DNA molecules, four Int proteins, two IHF dimers, three Xis proteins
Number unique components: 9
Molecular weightTheoretical: 500 KDa

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Macromolecule #1: attB(-21) to attP(+117)

MacromoleculeName: attB(-21) to attP(+117) / type: dna / ID: 1 / Classification: DNA / Structure: SINGLE STRANDED / Synthetic?: Yes
Source (natural)Organism: Enterobacteria phage lambda (virus) / synonym: Lambda Phage
SequenceString:
CCGTTTCGCT CAAGTTAGTA TATTAAAGCT GAACGAGAAA CGTAAAATGA TATAAATATC AATATATTAA ATTAGATTTT GCATAAAAAA CAGACTACAT AATACTGTAA AACACAACAT ATGCAGTCAC TATGCCGAC

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Macromolecule #2: attB(-19 to +21)

MacromoleculeName: attB(-19 to +21) / type: dna / ID: 2 / Classification: DNA / Structure: SINGLE STRANDED / Synthetic?: Yes
Source (natural)Organism: Enterobacteria phage lambda (virus) / synonym: Lambda Phage
SequenceString:
CCGTTGAAGC CTGCTTTTTT ATACTAACTT GAGCGAAACG G

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Macromolecule #3: attP(-79) to attB(+19)

MacromoleculeName: attP(-79) to attB(+19) / type: dna / ID: 3 / Classification: DNA / Structure: SINGLE STRANDED / Synthetic?: Yes
Source (natural)Organism: Enterobacteria phage lambda (virus) / synonym: Lambda Phage
SequenceString:
ATCAATATTT TGACTGATAG TGACCTGTTC GTTGCAACAA ATTGATAAGC AATGCTTTTT TAGAATTCCA ACTTATTGTA AAAAAGCAGG CTTCAACGG

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Macromolecule #4: attP (-117 to +79)

MacromoleculeName: attP (-117 to +79) / type: dna / ID: 4 / Classification: DNA / Structure: SINGLE STRANDED / Synthetic?: Yes
Source (natural)Organism: Enterobacteria phage lambda (virus) / synonym: Lambda Phage
SequenceString: GTCGGCATAG TGACTGCATA TGTTGTGTTT TACAGTATTA TGTAGTCTGT TTTTTATGCA AAATCTAATT TAATATATTG ATATTTATAT CATTTTACGT TTCTCGTTCA GCTTTAATAC AATAAGTTGG AATTCTAAAA AAGCATTGCT TATCAATTTG TTGCAACGAA ...String:
GTCGGCATAG TGACTGCATA TGTTGTGTTT TACAGTATTA TGTAGTCTGT TTTTTATGCA AAATCTAATT TAATATATTG ATATTTATAT CATTTTACGT TTCTCGTTCA GCTTTAATAC AATAAGTTGG AATTCTAAAA AAGCATTGCT TATCAATTTG TTGCAACGAA CAGGTCACTA TCAGTCAAAA TATTGAT

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Macromolecule #5: Int

MacromoleculeName: Int / type: protein_or_peptide / ID: 5 / Number of copies: 4 / Oligomeric state: Tetramer / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #6: Int

MacromoleculeName: Int / type: protein_or_peptide / ID: 6 / Number of copies: 4 / Oligomeric state: Tetramer / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #7: IHFa

MacromoleculeName: IHFa / type: protein_or_peptide / ID: 7 / Number of copies: 2 / Oligomeric state: Heterodimer / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #8: IHFb

MacromoleculeName: IHFb / type: protein_or_peptide / ID: 8 / Number of copies: 2 / Oligomeric state: Heterodimer / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #9: Xis

MacromoleculeName: Xis / type: protein_or_peptide / ID: 9 / Number of copies: 3 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration2 mg/mL
BufferpH: 8 / Details: 10mM Tris, 50mM NaCl
GridDetails: 1.2/1.3 400-mesh copper C-flat
VitrificationCryogen name: ETHANE / Chamber humidity: 85 % / Instrument: FEI VITROBOT MARK II / Method: 10 seconds blotting (two-sided)

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Electron microscopy

MicroscopeFEI TECNAI F30
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 100000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal defocus max: 4.793 µm / Nominal defocus min: 1.627 µm / Nominal magnification: 78000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
TemperatureMin: 90 K / Max: 90 K
DetailsImages were collected as 25-frame movies with a 2-second exposure
DateJun 11, 2014
Image recordingCategory: CCD / Film or detector model: FEI FALCON II (4k x 4k) / Digitization - Sampling interval: 14 µm / Number real images: 1359 / Average electron dose: 35.5 e/Å2
Details: Every image is the average of all 25 aligned movie frames
Experimental equipment
Model: Tecnai F30 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each particle
Final two d classificationNumber classes: 6
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 11.0 Å / Resolution method: OTHER / Software - Name: Frealign, v9
Details: Final map was the best class, out of size 3D classes
Number images used: 10956
DetailsManual particle selection with e2boxer (EMAN2), 2D classification with ISAC, initial map generation with e2initialmodel (EMAN2), 3D refinement and classification with Frealign v9.11

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Atomic model buiding 1

Initial modelPDB ID:

1z1g


Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B / Chain - #2 - Chain ID: C / Chain - #3 - Chain ID: D / Chain - #4 - Chain ID: I / Chain - #5 - Chain ID: J / Chain - #6 - Chain ID: K / Chain - #7 - Chain ID: L
SoftwareName: CNS 1.3
DetailsManual docking followed by highly restrained DEN-refinement in CNS
RefinementSpace: RECIPROCAL / Protocol: FLEXIBLE FIT / Overall B value: 500 / Target criteria: HLML
Output model

PDB-5j0n:
Lambda excision HJ intermediate

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Atomic model buiding 2

Initial modelPDB ID:

2wcc


Chain - #0 - Chain ID: 1 / Chain - #1 - Chain ID: 2 / Chain - #2 - Chain ID: 3
SoftwareName: CNS 1.3
DetailsManual docking followed by highly restrained DEN-refinement in CNS
RefinementSpace: RECIPROCAL / Protocol: FLEXIBLE FIT / Overall B value: 500 / Target criteria: HLML
Output model

PDB-5j0n:
Lambda excision HJ intermediate

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Atomic model buiding 3

Initial modelPDB ID:

2ief


Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B / Chain - #2 - Chain ID: C / Chain - #3 - Chain ID: D / Chain - #4 - Chain ID: E / Chain - #5 - Chain ID: F
SoftwareName: CNS 1.3
DetailsManual docking followed by highly restrained DEN-refinement in CNS
RefinementSpace: RECIPROCAL / Protocol: FLEXIBLE FIT / Overall B value: 500 / Target criteria: HLML
Output model

PDB-5j0n:
Lambda excision HJ intermediate

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