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- EMDB-24470: Cryo-EM structure of precleavage Cre tetrameric complex -

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Basic information

Entry
Database: EMDB / ID: EMD-24470
TitleCryo-EM structure of precleavage Cre tetrameric complex
Map dataPrecleavage synaptic complex of Cre recombinase mutant in complex with duplex loxP DNA
Sample
  • Complex: Precleavage synaptic complex of Cre recombinase mutant K201A and loxP DNA
    • Protein or peptide: Recombinase cre
    • DNA: DNA (42-MER)
    • DNA: DNA (42-MER)
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
Biological speciesEscherichia phage P1 (virus)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.23 Å
AuthorsStachowski K / Foster MP
Funding support United States, 1 items
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, 2021-
Header (metadata) releaseJan 19, 2022-
Map releaseJan 19, 2022-
UpdateAug 3, 2022-
Current statusAug 3, 2022Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.4
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.4
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7rhx
  • Surface level: 0.4
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7rhx
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_24470.png

Downloads & links

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Map

FileDownload / File: emd_24470.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPrecleavage synaptic complex of Cre recombinase mutant in complex with duplex loxP DNA
Voxel sizeX=Y=Z: 0.899 Å
Density
Contour LevelBy AUTHOR: 0.4 / Movie #1: 0.4
Minimum - Maximum-0.9636405 - 2.259666
Average (Standard dev.)0.00012676278 (±0.08057347)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 230.144 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8990.8990.899
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z230.144230.144230.144
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.9642.2600.000

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Supplemental data

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Mask #1

Fileemd_24470_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_24470_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_24470_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 : Precleavage synaptic complex of Cre recombinase mutant K201A and ...

EntireName: Precleavage synaptic complex of Cre recombinase mutant K201A and loxP DNA
Components
  • Complex: Precleavage synaptic complex of Cre recombinase mutant K201A and loxP DNA
    • Protein or peptide: Recombinase cre
    • DNA: DNA (42-MER)
    • DNA: DNA (42-MER)

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Supramolecule #1: Precleavage synaptic complex of Cre recombinase mutant K201A and ...

SupramoleculeName: Precleavage synaptic complex of Cre recombinase mutant K201A and loxP DNA
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Escherichia phage P1 (virus)
Molecular weightTheoretical: 54 KDa

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Macromolecule #1: Recombinase cre

MacromoleculeName: Recombinase cre / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Escherichia phage P1 (virus)
Molecular weightTheoretical: 38.537062 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MSNLLTVHQN LPALPVDATS DEVRKNLMDM FRDRQAFSEH TWKMLLSVCR SWAAWCKLNN RKWFPAEPED VRDYLLYLQA RGLAVKTIQ QHLGQLNMLH RRSGLPRPSD SNAVSLVMRR IRKENVDAGE RAKQALAFER TDFDQVRSLM ENSDRCQDIR N LAFLGIAY ...String:
MSNLLTVHQN LPALPVDATS DEVRKNLMDM FRDRQAFSEH TWKMLLSVCR SWAAWCKLNN RKWFPAEPED VRDYLLYLQA RGLAVKTIQ QHLGQLNMLH RRSGLPRPSD SNAVSLVMRR IRKENVDAGE RAKQALAFER TDFDQVRSLM ENSDRCQDIR N LAFLGIAY NTLLRIAEIA RIRVKDISRT DGGRMLIHIG RTATLVSTAG VEKALSLGVT KLVERWISVS GVADDPNNYL FC RVRKNGV AAPSATSQLS TRALEGIFEA THRLIYGAKD DSGQRYLAWS GHSARVGAAR DMARAGVSIP EIMQAGGWTN VNI VMNYIR NLDSETGAMV RLLEDGD

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Macromolecule #2: DNA (42-MER)

MacromoleculeName: DNA (42-MER) / type: dna / ID: 2 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Escherichia phage P1 (virus)
Molecular weightTheoretical: 12.833278 KDa
SequenceString:
(DC)(DC)(DG)(DC)(DA)(DT)(DA)(DA)(DC)(DT) (DT)(DC)(DG)(DT)(DA)(DT)(DA)(DG)(DC)(DA) (DT)(DA)(DC)(DA)(DT)(DT)(DA)(DT)(DA) (DC)(DG)(DA)(DA)(DG)(DT)(DT)(DA)(DT)(DC) (DG) (DC)(DC)

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Macromolecule #3: DNA (42-MER)

MacromoleculeName: DNA (42-MER) / type: dna / ID: 3 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Escherichia phage P1 (virus)
Molecular weightTheoretical: 13.024384 KDa
SequenceString:
(DG)(DG)(DC)(DG)(DA)(DT)(DA)(DA)(DC)(DT) (DT)(DC)(DG)(DT)(DA)(DT)(DA)(DA)(DT)(DG) (DT)(DA)(DT)(DG)(DC)(DT)(DA)(DT)(DA) (DC)(DG)(DA)(DA)(DG)(DT)(DT)(DA)(DT)(DG) (DC) (DG)(DG)

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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

Concentration4 mg/mL
BufferpH: 7
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHEPES
100.0 mMNaClSodium chlorideSodium Chloride
5.0 mMMgCl2Magnesium Chloride
16.8 mMC14H28O6octyl glucoside

Details: Buffer was made fresh from solid reagents and filtered with a 0.22 um filter.
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 81000
Specialist opticsSpherical aberration corrector: Microscope was modified with a Cs corrector with two hexapoles.
Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 15 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 86.0 K / Max: 86.0 K
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 3662 / Average exposure time: 2.0 sec. / Average electron dose: 60.0 e/Å2 / Details: 45 total frames
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 737980
CTF correctionSoftware - Name: cryoSPARC (ver. 3.0.1)
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.0.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.0.1)
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.23 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.0.1) / Number images used: 315574

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Atomic model buiding 1

Initial modelPDB ID:

2hoi

RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 130 / Target criteria: correlation coefficient
Output model

PDB-7rhx:
Cryo-EM structure of precleavage Cre tetrameric complex

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