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- EMDB-44513: Activated wild-type SgrAI endonuclease DNA-bound dimer with Mg2+ ... -

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Basic information

Entry
Database: EMDB / ID: EMD-44513
TitleActivated wild-type SgrAI endonuclease DNA-bound dimer with Mg2+ and cleaved primary site DNA
Map dataCryo-EM full map of wtSgrAI/40-1/Mg
Sample
  • Complex: Activated wild-type filamentous SgrAI endonuclease with Mg2+ and cleaved primary site DNA
    • Protein or peptide: SgraIR restriction enzyme
    • DNA: 40-1 DNA
    • DNA: 40-1 DNA
  • Ligand: MAGNESIUM ION
  • Ligand: water
Keywordsrestriction endonuclease / DNAse / allostery / bacterial innate immunity / filament / hyper-activation / substrate specificity / HYDROLASE-DNA complex / DNA BINDING PROTEIN
Function / homologyRestriction endonuclease, type II, Cfr10I/Bse634I / Cfr10I/Bse634I restriction endonuclease / Restriction endonuclease type II-like / metal ion binding / identical protein binding / SgraIR restriction enzyme
Function and homology information
Biological speciesStreptomyces griseus (bacteria)
Methodhelical reconstruction / cryo EM / Resolution: 3.05 Å
AuthorsShan Z / Lyumkis D / Horton NC
Funding support United States, 2 items
OrganizationGrant numberCountry
National Science Foundation (NSF, United States)MCB-1934291 United States
National Science Foundation (NSF, United States)DBI-2018942 United States
CitationJournal: J Biol Chem / Year: 2024
Title: Two-metal ion mechanism of DNA cleavage by activated, filamentous SgrAI.
Authors: Zelin Shan / Andres Rivero-Gamez / Dmitry Lyumkis / Nancy C Horton /
Abstract: Enzymes that form filamentous assemblies with modulated enzymatic activities have gained increasing attention in recent years. SgrAI is a sequence specific type II restriction endonuclease that forms ...Enzymes that form filamentous assemblies with modulated enzymatic activities have gained increasing attention in recent years. SgrAI is a sequence specific type II restriction endonuclease that forms polymeric filaments with accelerated DNA cleavage activity and expanded DNA sequence specificity. Prior studies have suggested a mechanistic model linking the structural changes accompanying SgrAI filamentation to its accelerated DNA cleavage activity. In this model, the conformational changes that are specific to filamentous SgrAI maximize contacts between different copies of the enzyme within the filament and create a second divalent cation binding site in each subunit, which in turn facilitates the DNA cleavage reaction. However, our understanding of the atomic mechanism of catalysis is incomplete. Herein, we present two new structures of filamentous SgrAI solved using cryo-EM. The first structure, resolved to 3.3 Å, is of filamentous SgrAI containing an active site mutation that is designed to stall the DNA cleavage reaction, which reveals the enzymatic configuration prior to DNA cleavage. The second structure, resolved to 3.1 Å, is of WT filamentous SgrAI containing cleaved substrate DNA, which reveals the enzymatic configuration at the end of the enzymatic cleavage reaction. Both structures contain the phosphate moiety at the cleavage site and the biologically relevant divalent cation cofactor Mg and define how the Mg cation reconfigures during enzymatic catalysis. The data support a model for the activation mechanism that involves binding of a second Mg in the SgrAI active site as a direct result of filamentation induced conformational changes.
History
DepositionApr 18, 2024-
Header (metadata) releaseMar 5, 2025-
Map releaseMar 5, 2025-
UpdateMar 5, 2025-
Current statusMar 5, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_44513.png

Downloads & links

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Map

FileDownload / File: emd_44513.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM full map of wtSgrAI/40-1/Mg
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 320 pix.
= 265.6 Å		0.83 Å/pix.
x 320 pix.
= 265.6 Å		0.83 Å/pix.
x 320 pix.
= 265.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.83 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.2
Minimum - Maximum-0.88134986 - 1.4913872
Average (Standard dev.)0.011708059 (±0.06707558)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 265.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_44513_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Cryo-EM half map B of wtSgrAI/40-1/Mg

Fileemd_44513_half_map_1.map
AnnotationCryo-EM half map B of wtSgrAI/40-1/Mg
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Cryo-EM half map A of wtSgrAI/40-1/Mg

Fileemd_44513_half_map_2.map
AnnotationCryo-EM half map A of wtSgrAI/40-1/Mg
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Activated wild-type filamentous SgrAI endonuclease with Mg2+ and ...

EntireName: Activated wild-type filamentous SgrAI endonuclease with Mg2+ and cleaved primary site DNA
Components
  • Complex: Activated wild-type filamentous SgrAI endonuclease with Mg2+ and cleaved primary site DNA
    • Protein or peptide: SgraIR restriction enzyme
    • DNA: 40-1 DNA
    • DNA: 40-1 DNA
  • Ligand: MAGNESIUM ION
  • Ligand: water

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Supramolecule #1: Activated wild-type filamentous SgrAI endonuclease with Mg2+ and ...

SupramoleculeName: Activated wild-type filamentous SgrAI endonuclease with Mg2+ and cleaved primary site DNA
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Molecular weightTheoretical: 33.6 kDa/nm

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Macromolecule #1: SgraIR restriction enzyme

MacromoleculeName: SgraIR restriction enzyme / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Streptomyces griseus (bacteria)
Molecular weightTheoretical: 39.783895 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: MPFTYSIEAT RNLATTERCI QDIRNAPVRN RSTQFQLAQQ NMLAYTFGEV IPGFASAGIN GMDYRDVIGR PVENAVTEGT HFFRDDFRV DSNAKAKVAG DIFEIVSSAV MWNCAARWNS LMVGEGWRSQ PRYSRPTLSP SPRRQVAVLN LPRSFDWVSL L VPESQEVI ...String:
MPFTYSIEAT RNLATTERCI QDIRNAPVRN RSTQFQLAQQ NMLAYTFGEV IPGFASAGIN GMDYRDVIGR PVENAVTEGT HFFRDDFRV DSNAKAKVAG DIFEIVSSAV MWNCAARWNS LMVGEGWRSQ PRYSRPTLSP SPRRQVAVLN LPRSFDWVSL L VPESQEVI EEFRAGLRKD GLGLPTSTPD LAVVVLPEEF QNDEMWREEI AGLTRPNQIL LSGAYQRLQG RVQPGEISLA VA FKRSLRS DRLYQPLYEA NVMQLLLEGK LGAPKVEFEV HTLAPEGTNA FVTYEAASLY GLAEGRSAVH RAIRELYVPP TAA DLARRF FAFLNERMEL VNGENLYFQS HHHHHH

UniProtKB: SgraIR restriction enzyme

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Macromolecule #2: 40-1 DNA

MacromoleculeName: 40-1 DNA / type: dna / ID: 2 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Streptomyces griseus (bacteria)
Molecular weightTheoretical: 5.547588 KDa
SequenceString:
(DG)(DA)(DT)(DG)(DC)(DG)(DT)(DG)(DG)(DG) (DT)(DC)(DT)(DT)(DC)(DA)(DC)(DA)

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Macromolecule #3: 40-1 DNA

MacromoleculeName: 40-1 DNA / type: dna / ID: 3 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Streptomyces griseus (bacteria)
Molecular weightTheoretical: 6.722343 KDa
SequenceString:
(DC)(DC)(DG)(DG)(DT)(DG)(DT)(DG)(DA)(DA) (DG)(DA)(DC)(DC)(DC)(DA)(DC)(DG)(DC)(DA) (DT)(DC)

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 4 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #5: water

MacromoleculeName: water / type: ligand / ID: 5 / Number of copies: 177 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

BufferpH: 8
Component:
ConcentrationNameFormula
25.0 mMTris-HCl
150.0 mMsodium chlorideNaCl
1.0 mMTCEP
8.7 mMMagnesium chlorideMgCl2
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Instrument: HOMEMADE PLUNGER
Details: Cryo-EM grids were prepared by freezing using a manual plunger in cold room at 4C.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 1-50 / Number real images: 2704 / Average electron dose: 29.6 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated magnification: 60240 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 165000
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

Final reconstructionApplied symmetry - Helical parameters - Δz: 21.3 Å
Applied symmetry - Helical parameters - Δ&Phi: -86.2 °
Applied symmetry - Helical parameters - Axial symmetry: C2 (2 fold cyclic)
Resolution.type: BY AUTHOR / Resolution: 3.05 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.2) / Number images used: 202664
Startup modelType of model: EMDB MAP
EMDB ID:

20015

Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 4.2)

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Atomic model buiding 1

Initial modelPDB ID:

6obj


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 87.9 / Target criteria: Correlation coefficient
Output model

PDB-9bgi:
Activated wild-type SgrAI endonuclease DNA-bound dimer with Mg2+ and cleaved primary site DNA

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