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- EMDB-24471: Cre recombinase mutant (D33A/A36V/R192A) in complex with loxA DNA... -

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Basic information

Entry
Database: EMDB / ID: EMD-24471
TitleCre recombinase mutant (D33A/A36V/R192A) in complex with loxA DNA hairpin
Map dataStructure of Cre recombinase mutant (D33A/A36V/R192A) in complex with a loxA DNA hairpin.
Sample
  • Complex: Complex of Cre recombinase mutant D33A/A36V/R192A and loxA DNA hairpin
    • Protein or peptide: Recombinase cre
    • DNA: DNA (49-MER)
KeywordsCre / recombinase / monomer / hairpin / RECOMBINATION-DNA complex
Function / homology
Function and homology information


DNA integration / DNA recombination / DNA binding
Similarity search - Function
: / Phage integrase family / Core-binding (CB) domain / Core-binding (CB) domain profile. / Integrase, catalytic domain / Tyrosine recombinase domain profile. / 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: 3.91 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-
UpdateJun 5, 2024-
Current statusJun 5, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.55
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.55
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7rhy
  • Surface level: 0.55
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_24471.png

Downloads & links

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Map

FileDownload / File: emd_24471.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationStructure of Cre recombinase mutant (D33A/A36V/R192A) in complex with a loxA DNA hairpin.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.96 Å/pix.
x 256 pix.
= 245.7 Å		0.96 Å/pix.
x 256 pix.
= 245.7 Å		0.96 Å/pix.
x 256 pix.
= 245.7 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.95977 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.55 / Movie #1: 0.55
Minimum - Maximum-1.8044534 - 3.1718903
Average (Standard dev.)0.00057812256 (±0.055428695)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 245.70001 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.9597656250.9597656250.959765625
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z245.700245.700245.700
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-1.8043.1720.001

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Supplemental data

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Mask #1

Fileemd_24471_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_24471_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_24471_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 : Complex of Cre recombinase mutant D33A/A36V/R192A and loxA DNA hairpin

EntireName: Complex of Cre recombinase mutant D33A/A36V/R192A and loxA DNA hairpin
Components
  • Complex: Complex of Cre recombinase mutant D33A/A36V/R192A and loxA DNA hairpin
    • Protein or peptide: Recombinase cre
    • DNA: DNA (49-MER)

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Supramolecule #1: Complex of Cre recombinase mutant D33A/A36V/R192A and loxA DNA hairpin

SupramoleculeName: Complex of Cre recombinase mutant D33A/A36V/R192A and loxA DNA hairpin
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: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia phage P1 (virus)
Molecular weightTheoretical: 38.493094 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MSNLLTVHQN LPALPVDATS DEVRKNLMDM FRARQVFSEH TWKMLLSVCR SWAAWCKLNN RKWFPAEPED VRDYLLYLQA RGLAVKTIQ QHLGQLNMLH RRSGLPRPSD SNAVSLVMRR IRKENVDAGE RAKQALAFER TDFDQVRSLM ENSDRCQDIR N LAFLGIAY ...String:
MSNLLTVHQN LPALPVDATS DEVRKNLMDM FRARQVFSEH TWKMLLSVCR SWAAWCKLNN RKWFPAEPED VRDYLLYLQA RGLAVKTIQ QHLGQLNMLH RRSGLPRPSD SNAVSLVMRR IRKENVDAGE RAKQALAFER TDFDQVRSLM ENSDRCQDIR N LAFLGIAY NTLLRIAEIA RIRVKDISRT DGGAMLIHIG RTKTLVSTAG VEKALSLGVT KLVERWISVS GVADDPNNYL FC RVRKNGV AAPSATSQLS TRALEGIFEA THRLIYGAKD DSGQRYLAWS GHSARVGAAR DMARAGVSIP EIMQAGGWTN VNI VMNYIR NLDSETGAMV RLLEDGD

UniProtKB: Recombinase cre

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Macromolecule #2: DNA (49-MER)

MacromoleculeName: DNA (49-MER) / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Escherichia phage P1 (virus)
Molecular weightTheoretical: 15.119743 KDa
SequenceString:
(DG)(DC)(DA)(DT)(DA)(DA)(DC)(DT)(DT)(DC) (DG)(DT)(DA)(DT)(DA)(DG)(DC)(DA)(DT)(DA) (DT)(DG)(DC)(DG)(DA)(DA)(DG)(DC)(DA) (DT)(DA)(DT)(DG)(DC)(DT)(DA)(DT)(DA)(DC) (DG) (DA)(DA)(DG)(DT)(DT)(DA)(DT)(DG) (DC)

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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

Concentration7.5 mg/mL
BufferpH: 7
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHEPES
100.0 mMNaClSodium Chloride
5.0 mMMgCl2Magnesium Chloride
4.0 mMC32H58N2O8SCHAPSO

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 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR / Details: Pelco easiGLOW at 20 mA
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
TemperatureMin: 86.0 K / Max: 86.0 K
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
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: 6235 / Average exposure time: 2.0 sec. / Average electron dose: 60.0 e/Å2 / Details: 45 total frames
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.2 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 898039
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.91 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.0.1) / Number images used: 146715
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.0.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.0.1)

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Atomic model buiding 1

Initial modelPDB ID:

2hoi


Chain - Chain ID: A / Chain - Residue range: 20-343 / Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 248.4 / Target criteria: correlation coefficient
Output model

PDB-7rhy:
Cre recombinase mutant (D33A/A36V/R192A) in complex with loxA DNA hairpin

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