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- PDB-7rhz: Heterodimer of Cre recombinase mutants D33A/A36V/R192A and R72E/L... -

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Basic information

Entry
Database: PDB / ID: 7rhz
TitleHeterodimer of Cre recombinase mutants D33A/A36V/R192A and R72E/L115D/R119D in complex with loxP DNA.
Components
  • (DNA (44-MER)) x 2
  • (Recombinase cre) x 2
KeywordsRECOMBINATION/DNA / Cre / recombinase / dimer / loxP / RECOMBINATION-DNA complex
Function / homology
Function and homology information


DNA integration / DNA recombination / DNA binding
Similarity search - Function
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
Similarity search - Domain/homology
DNA / DNA (> 10) / Recombinase cre
Similarity search - Component
Biological speciesEscherichia phage P1 (virus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.48 Å
AuthorsStachowski, K. / Foster, M.P.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01-GM122432-04 United States
CitationJournal: Nucleic Acids Res / Year: 2022
Title: Mechanisms of Cre recombinase synaptic complex assembly and activation illuminated by Cryo-EM.
Authors: Kye Stachowski / Andrew S Norris / Devante Potter / Vicki H Wysocki / Mark P Foster /
Abstract: Cre recombinase selectively recognizes DNA and prevents non-specific DNA cleavage through an orchestrated series of assembly intermediates. Cre recombines two loxP DNA sequences featuring a pair of ...Cre recombinase selectively recognizes DNA and prevents non-specific DNA cleavage through an orchestrated series of assembly intermediates. Cre recombines two loxP DNA sequences featuring a pair of palindromic recombinase binding elements and an asymmetric spacer region, by assembly of a tetrameric synaptic complex, cleavage of an opposing pair of strands, and formation of a Holliday junction intermediate. We used Cre and loxP variants to isolate the monomeric Cre-loxP (54 kDa), dimeric Cre2-loxP (110 kDa), and tetrameric Cre4-loxP2 assembly intermediates, and determined their structures using cryo-EM to resolutions of 3.9, 4.5 and 3.2 Å, respectively. Progressive and asymmetric bending of the spacer region along the assembly pathway enables formation of increasingly intimate interfaces between Cre protomers and illuminates the structural bases of biased loxP strand cleavage order and half-the-sites activity. Application of 3D variability analysis to the tetramer data reveals constrained conformational sampling along the pathway between protomer activation and Holliday junction isomerization. These findings underscore the importance of protein and DNA flexibility in Cre-mediated site selection, controlled activation of alternating protomers, the basis for biased strand cleavage order, and recombination efficiency. Such considerations may advance development of site-specific recombinases for use in gene editing applications.
History
DepositionJul 19, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 19, 2022Provider: repository / Type: Initial release
Revision 1.1Aug 3, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name

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Structure visualization

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Assembly

Deposited unit
A: Recombinase cre
B: Recombinase cre
C: DNA (44-MER)
D: DNA (44-MER)


Theoretical massNumber of molelcules
Total (without water)104,1144
Polymers104,1144
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: mass spectrometry, assay for oligomerization, gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area13630 Å2
ΔGint-71 kcal/mol
Surface area47760 Å2

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Components

#1: Protein Recombinase cre


Mass: 38493.094 Da / Num. of mol.: 1 / Mutation: D33A, A36V, R192A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia phage P1 (virus) / Gene: cre / Production host: Escherichia coli (E. coli) / References: UniProt: P06956
#2: Protein Recombinase cre


Mass: 38526.906 Da / Num. of mol.: 1 / Mutation: R72E, L115D, R119D
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia phage P1 (virus) / Gene: cre / Production host: Escherichia coli (E. coli) / References: UniProt: P06956
#3: DNA chain DNA (44-MER)


Mass: 13491.689 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia phage P1 (virus)
#4: DNA chain DNA (44-MER)


Mass: 13602.748 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia phage P1 (virus)

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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: Heterodimer of Cre recombinase mutants D33A/A36V/R192A and R72E/L115D/R119D in complex with loxP DNA
Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES
Molecular weightValue: 0.1005 MDa / Experimental value: YES
Source (natural)Organism: Escherichia phage P1 (virus)
Buffer solutionpH: 7
Details: Buffer was made fresh from solid reagents and filtered with a 0.22 um filter.
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESC8H18N2O4S1
2100 mMSodium ChlorideNaClSodium chloride1
35 mMMagnesium ChlorideMgCl21
416.8 mMn-OctylglucosideC14H28O61
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 20 mA Pelco easiGLOW / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 81000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm / C2 aperture diameter: 100 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 86 K / Temperature (min): 86 K
Image recordingAverage exposure time: 2 sec. / Electron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4306 / Details: 45 total frames
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 15 eV
Spherical aberration corrector: Microscope was modified with a Cs corrector with two hexapoles.
Image scansWidth: 5760 / Height: 4096

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Processing

EM software
IDNameVersionCategory
1Topazparticle selection
2cryoSPARC3.0.1particle selection
3EPU2.7image acquisition
5cryoSPARC3.0.1CTF correction
8ISOLDE1.2model fitting
10cryoSPARC3.0.1initial Euler assignment
11cryoSPARC3.0.1final Euler assignment
13cryoSPARC3.0.13D reconstruction
14PHENIX1.19model refinement
15Coot0.9.4.1model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1277272
3D reconstructionResolution: 4.48 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 256734 / Symmetry type: POINT
Atomic model buildingB value: 150.3 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: Correlation Coefficient
Atomic model buildingPDB-ID: 2HOI

2hoi


Pdb chain-ID: A / Pdb chain residue range: 20-343

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