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- PDB-7ss5: Activated SgrAI endonuclease DNA-bound dimer with Ca2+ and intact... -

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Basic information

Entry
Database: PDB / ID: 7ss5
TitleActivated SgrAI endonuclease DNA-bound dimer with Ca2+ and intact primary site DNA
Components
  • DNA (5'-D(P*GP*GP*TP*CP*TP*TP*CP*AP*CP*AP*CP*CP*GP*GP*TP*GP*TP*GP*AP*AP*GP*AP*CP*C)-3')
  • SgraIR restriction enzyme
KeywordsHYDROLASE/DNA / restriction endonuclease / DNAse / allostery / bacterial innate immunity / filament / hyper-activation / substrate specificity / HYDROLASE-DNA complex
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: 2.7 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)MCB-1410355 United States
CitationJournal: J Biol Chem / Year: 2022
Title: Pretransition state and apo structures of the filament-forming enzyme SgrAI elucidate mechanisms of activation and substrate specificity.
Authors: Zelin Shan / Niloofar Ghadirian / Dmitry Lyumkis / Nancy C Horton /
Abstract: Enzyme filamentation is a widespread phenomenon that mediates enzyme regulation and function. For the filament-forming sequence-specific DNA endonuclease SgrAI, the process of filamentation both ...Enzyme filamentation is a widespread phenomenon that mediates enzyme regulation and function. For the filament-forming sequence-specific DNA endonuclease SgrAI, the process of filamentation both accelerates its DNA cleavage activity and expands its DNA sequence specificity, thus allowing for many additional DNA sequences to be rapidly cleaved. Both outcomes-the acceleration of DNA cleavage and the expansion of sequence specificity-are proposed to regulate critical processes in bacterial innate immunity. However, the mechanistic bases underlying these events remain unclear. Herein, we describe two new structures of the SgrAI enzyme that shed light on its catalytic function. First, we present the cryo-EM structure of filamentous SgrAI bound to intact primary site DNA and Ca resolved to ∼2.5 Å within the catalytic center, which represents the trapped enzyme-DNA complex prior to the DNA cleavage reaction. This structure reveals important conformational changes that contribute to the catalytic mechanism and the binding of a second divalent cation in the enzyme active site, which is expected to contribute to increased DNA cleavage activity of SgrAI in the filamentous state. Second, we present an X-ray crystal structure of DNA-free (apo) SgrAI resolved to 2.0 Å resolution, which reveals a disordered loop involved in DNA recognition. Collectively, these multiple new observations clarify the mechanism of expansion of DNA sequence specificity of SgrAI, including the indirect readout of sequence-dependent DNA structure, changes in protein-DNA interactions, and the disorder-to-order transition of a crucial DNA recognition element.
History
DepositionNov 9, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 1, 2021Provider: repository / Type: Initial release
Revision 1.1Jun 15, 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

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
A: SgraIR restriction enzyme
C: DNA (5'-D(P*GP*GP*TP*CP*TP*TP*CP*AP*CP*AP*CP*CP*GP*GP*TP*GP*TP*GP*AP*AP*GP*AP*CP*C)-3')
B: SgraIR restriction enzyme
D: DNA (5'-D(P*GP*GP*TP*CP*TP*TP*CP*AP*CP*AP*CP*CP*GP*GP*TP*GP*TP*GP*AP*AP*GP*AP*CP*C)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)104,43810
Polymers104,1984
Non-polymers2406
Water10,683593
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein SgraIR restriction enzyme


Mass: 39783.895 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptomyces griseus (bacteria) / Gene: sgraIR / Production host: Escherichia coli (E. coli) / References: UniProt: Q9F6L0
#2: DNA chain DNA (5'-D(P*GP*GP*TP*CP*TP*TP*CP*AP*CP*AP*CP*CP*GP*GP*TP*GP*TP*GP*AP*AP*GP*AP*CP*C)-3')


Mass: 12314.889 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Streptomyces griseus (bacteria)
#3: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 593 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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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: Activated filamentous SgrAI endonuclease with Ca2+ and intact primary site DNA
Type: COMPLEX / Entity ID: #1-#2 / Source: MULTIPLE SOURCES
Molecular weightValue: 74 kDa/nm / Experimental value: NO
Source (natural)Organism: Streptomyces griseus (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMTris-HClTris1
2150 mMsodium chlorideNaClSodium chloride1
31 mMTCEP1
410 mMCalcium chlorideCaCl21
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / 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 FIELDBright-field microscopy / Nominal magnification: 165000 X / Calibrated magnification: 60240 X / Nominal defocus max: 2000 nm / Nominal defocus min: 800 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: 55 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 216
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 30 eV
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 60 / Used frames/image: 1-60

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategory
2Leginonimage acquisition
4WarpCTF correction
7Cootmodel fitting
9cryoSPARCinitial Euler assignment
10RELION3.1final Euler assignment
12RELION3.13D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: -85.8 ° / Axial rise/subunit: 21.2 Å / Axial symmetry: C1
3D reconstructionResolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 220067 / Algorithm: FOURIER SPACE / Symmetry type: HELICAL
Atomic model buildingB value: 107.7 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: Correlation coefficient
Atomic model buildingPDB-ID: 6OBJ

6obj

RefinementHighest resolution: 2.7 Å
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0036656
ELECTRON MICROSCOPYf_angle_d0.549252
ELECTRON MICROSCOPYf_dihedral_angle_d21.9741270
ELECTRON MICROSCOPYf_chiral_restr0.041018
ELECTRON MICROSCOPYf_plane_restr0.0041036

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