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- PDB-5j0n: Lambda excision HJ intermediate -

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Basic information

Entry
Database: PDB / ID: 5j0n
TitleLambda excision HJ intermediate
Components
  • (Integration host factor subunit ...) x 2
  • Excisionase
  • Integrase
  • attB(-19 to +21)
  • attB(-21) to attP(+117)
  • attP(-117 to +79)
  • attP(-79) to attB(+19)
KeywordsTRANSFERASE / HYDROLASE/DNA / bacteriophage lambda / excision / site-specific recombination / Holliday junction / HYDROLASE-DNA complex
Function / homology
Function and homology information


IHF-DNA complex / provirus excision / integrase activity / DNA-binding transcription activator activity / protein-DNA complex / DNA integration / viral genome integration into host DNA / establishment of integrated proviral latency / structural constituent of chromatin / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases ...IHF-DNA complex / provirus excision / integrase activity / DNA-binding transcription activator activity / protein-DNA complex / DNA integration / viral genome integration into host DNA / establishment of integrated proviral latency / structural constituent of chromatin / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / regulation of translation / chromosome / transferase activity / DNA recombination / Hydrolases; Acting on ester bonds / hydrolase activity / transcription cis-regulatory region binding / 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 / Core-binding (CB) domain ...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 / Core-binding (CB) domain / Tyrosine recombinase domain profile. / Core-binding (CB) domain profile. / Phage integrase family / Histone-like DNA-binding protein, conserved site / Bacterial histone-like DNA-binding proteins signature. / Histone-like DNA-binding protein / Integrase, catalytic domain / Bacterial DNA-binding protein / bacterial (prokaryotic) histone like domain / Integrase/recombinase, N-terminal / Integrase-like, catalytic domain superfamily / Integration host factor (IHF)-like DNA-binding domain superfamily / DNA breaking-rejoining enzyme, catalytic core / Putative DNA-binding domain superfamily / DNA-binding domain superfamily
Similarity search - Domain/homology
DNA / DNA (> 10) / DNA (> 100) / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 11 Å
AuthorsVan Duyne, G. / Grigorieff, N. / Landy, A.
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, 2016Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 8, 2017Provider: repository / Type: Initial release
Revision 1.1Jul 18, 2018Group: Data collection / Experimental preparation / Category: em_sample_support / em_software / Item: _em_sample_support.grid_type / _em_software.name
Revision 1.2Mar 6, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
A: attP(-117 to +79)
B: attB(-21) to attP(+117)
C: attB(-19 to +21)
D: attP(-79) to attB(+19)
E: Integrase
F: Integrase
G: Integrase
H: Integrase
I: Integration host factor subunit alpha
J: Integration host factor subunit beta
K: Integration host factor subunit alpha
L: Integration host factor subunit beta
M: Excisionase
N: Excisionase
O: Excisionase


Theoretical massNumber of molelcules
Total (without water)372,04815
Polymers372,04815
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area76660 Å2
ΔGint-381 kcal/mol
Surface area166440 Å2

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Components

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DNA chain , 4 types, 4 molecules ABCD

#1: DNA chain attP(-117 to +79)


Mass: 60790.953 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: 1 / Source: (synth.) Enterobacteria phage lambda (virus)
#2: DNA chain attB(-21) to attP(+117)


Mass: 42883.652 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: 1 / Source: (synth.) Enterobacteria phage lambda (virus)
#3: DNA chain attB(-19 to +21)


Mass: 12607.103 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: 1 / Source: (synth.) Escherichia coli (E. coli)
#4: DNA chain attP(-79) to attB(+19)


Mass: 30552.629 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: 1 / Source: (synth.) Enterobacteria phage lambda (virus)

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Protein , 2 types, 7 molecules EFGHMNO

#5: Protein
Integrase


Mass: 40373.168 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Details: 1 / Source: (gene. exp.) Enterobacteria phage lambda (virus) / Gene: int / Production host: Escherichia coli (E. coli)
References: UniProt: P03700, Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases, Hydrolases; Acting on ester bonds
#8: Protein Excisionase


Mass: 6793.799 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Details: 1 / Source: (gene. exp.) Enterobacteria phage lambda (virus) / Gene: xis / Production host: Escherichia coli (E. coli) / References: UniProt: P03699

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Integration host factor subunit ... , 2 types, 4 molecules IKJL

#6: Protein Integration host factor subunit alpha / IHF-alpha


Mass: 10998.554 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: 1 / Source: (gene. exp.) Escherichia coli (E. coli) / Gene: ihfA, himA, ECS88_1763 / Production host: Escherichia coli (E. coli) / References: UniProt: B7MAS3, UniProt: P0A6X7*PLUS
#7: Protein Integration host factor subunit beta / IHF-beta


Mass: 10671.178 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: 1 / Source: (gene. exp.) Escherichia coli (E. coli) / Gene: ihfB, himD, ECS88_0940 / Production host: Escherichia coli (E. coli) / References: UniProt: B7MHM1, UniProt: P0A6Y1*PLUS

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Details

Sequence detailsChains F, G, and H include 20-residue linkers with essentially arbitrary conformations that were ...Chains F, G, and H include 20-residue linkers with essentially arbitrary conformations that were not determined experimentally. These linkers are retained in the model to aid in visualization of domain connectivity.

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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: Lambda excision HJ intermediate / Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
110.0 mMTrisC4H11NO31
250.0 mMsodium chlorideNaCl1
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: The sample was lightly crosslinked.
Specimen supportDetails: Glow discharge with EMITECH K100X / Grid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat
VitrificationInstrument: FEI VITROBOT MARK II / Cryogen name: ETHANE / Humidity: 85 % / Chamber temperature: 298 K
Details: 10 second blot followed by plunging into liquid ethane (FEI VITROBOT MARK II).

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F30 / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI F30
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 78000 X / Calibrated magnification: 100000 X / Nominal defocus max: 4150 nm / Nominal defocus min: 2600 nm / Calibrated defocus min: 1627 nm / Calibrated defocus max: 4793 nm / Cs: 2 mm / C2 aperture diameter: 50 µm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN
Specimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Temperature (max): 90 K / Temperature (min): 90 K
Image recordingAverage exposure time: 2 sec. / Electron dose: 35.5 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON II (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1359
Image scansSampling size: 14 µm / Movie frames/image: 25 / Used frames/image: 1-25

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Processing

EM software
IDNameVersionCategory
1e2boxerEMAN2particle selection
2SerialEMimage acquisition
4CTFFIND3CTF correction
9CNS1.3model refinement
10e2initialmodelEMAN2initial Euler assignment
11FREALIGN9.11final Euler assignment
12FREALIGN9.11classification
13FREALIGN9.113D reconstruction
CTF correctionDetails: Built-in correction in Frealign v9 / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 66033
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 11 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 10956 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: RECIPROCAL / Target criteria: MLHL / Details: Highly restrained DEN refinement in CNS

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