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- PDB-9bgj: SgrAI mutant K242A endonuclease DNA-bound dimer with Mg2+ and int... -

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Basic information

Entry
Database: PDB / ID: 9bgj
TitleSgrAI mutant K242A endonuclease DNA-bound dimer with Mg2+ and intact primary site DNA
Components
  • 40-1 DNA
  • SgraIR restriction enzyme
KeywordsHYDROLASE/DNA / restriction 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 / identical protein binding / metal ion binding / DNA / DNA (> 10) / SgraIR restriction enzyme
Function and homology information
Biological speciesStreptomyces griseus (bacteria)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.34 Å
AuthorsShan, Z. / Lyumkis, D. / Horton, N.C.
Funding support United States, 2items
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, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 5, 2025Provider: repository / Type: Initial release
Revision 1.0Mar 5, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Mar 5, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Mar 5, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Mar 5, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Mar 5, 2025Data content type: Mask / Part number: 1 / Data content type: Mask / Provider: repository / Type: Initial release
Revision 1.0Mar 5, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: SgraIR restriction enzyme
C: 40-1 DNA
B: SgraIR restriction enzyme
D: 40-1 DNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)128,8826
Polymers128,8334
Non-polymers492
Water1629
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein SgraIR restriction enzyme


Mass: 39741.816 Da / Num. of mol.: 2 / Mutation: K242A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptomyces griseus (bacteria) / Gene: sgraIR
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: Q9F6L0
#2: DNA chain 40-1 DNA


Mass: 24674.738 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Streptomyces griseus (bacteria)
#3: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 9 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: helical reconstruction

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Sample preparation

ComponentName: Filamentous SgrAI mutant K242A endonuclease with Mg2+ and intact primary site DNA
Type: COMPLEX / Entity ID: #1-#2 / Source: MULTIPLE SOURCES
Molecular weightValue: 33.6 kDa/nm / Experimental value: NO
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMTris-HCl1
2150 mMsodium chlorideNaCl1
31 mMTCEP1
48.7 mMMagnesium chlorideMgCl21
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE
Details: Cryo-EM grids were prepared by freezing using a manual plunger in cold room at 4C

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 42.8 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 3488
Image scansWidth: 5760 / Height: 4092 / Movie frames/image: 50 / Used frames/image: 1-50

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Processing

EM software
IDNameVersionCategory
2Leginonimage acquisition
4cryoSPARCCTF correction
7Cootmodel fitting
9cryoSPARCinitial Euler assignment
10RELION4.2final Euler assignment
12cryoSPARC4.23D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: -86.2 ° / Axial rise/subunit: 20.9 Å / Axial symmetry: C2
3D reconstructionResolution: 3.34 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 357489 / Symmetry type: HELICAL
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: Correlation coefficient
Atomic model buildingPDB-ID: 6OBJ

6obj


Accession code: 6OBJ / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0036590
ELECTRON MICROSCOPYf_angle_d0.5159160
ELECTRON MICROSCOPYf_dihedral_angle_d23.4241305
ELECTRON MICROSCOPYf_chiral_restr0.0391010
ELECTRON MICROSCOPYf_plane_restr0.0041028

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