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- PDB-6obj: Structure of a DNA-bound dimer extracted from filamentous SgrAI e... -

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Basic information

Entry
Database: PDB / ID: 6obj
TitleStructure of a DNA-bound dimer extracted from filamentous SgrAI endonuclease in its activated form
Components
  • DNA (26-MER)
  • SgraIR restriction enzyme
KeywordsHYDROLASE/DNA / restriction endonuclease / DNAse / allostery / bacterial innate immunity / filament / hyper-activation / substrate specificity / HYDROLASE-DNA complex
Function / homology
Function and homology information


identical protein binding / metal ion binding
Similarity search - Function
Restriction Endonuclease - #10 / Restriction endonuclease, type II, Cfr10I/Bse634I / Cfr10I/Bse634I restriction endonuclease / Restriction Endonuclease / Restriction endonuclease type II-like / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
DNA / DNA (> 10) / SgraIR restriction enzyme
Similarity search - Component
Biological speciesStreptomyces griseus (bacteria)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsPolley, S. / Lyumkis, D. / Horton, N.C.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)DP5 OD021396 United States
National Science Foundation (NSF, United States)MCB-1410355 United States
CitationJournal: Structure / Year: 2019
Title: Mechanism of Filamentation-Induced Allosteric Activation of the SgrAI Endonuclease.
Authors: Smarajit Polley / Dmitry Lyumkis / Nancy C Horton /
Abstract: Filament formation by enzymes is increasingly recognized as an important phenomenon with potentially unique regulatory properties and biological roles. SgrAI is an allosterically regulated type II ...Filament formation by enzymes is increasingly recognized as an important phenomenon with potentially unique regulatory properties and biological roles. SgrAI is an allosterically regulated type II restriction endonuclease that forms filaments with enhanced DNA cleavage activity and altered sequence specificity. Here, we present the cryoelectron microscopy (cryo-EM) structure of the filament of SgrAI in its activated configuration. The structural data illuminate the mechanistic origin of hyperaccelerated DNA cleavage activity and suggests how indirect DNA sequence readout within filamentous SgrAI may enable recognition of substantially more nucleotide sequences than its low-activity form, thereby altering and partially relaxing its DNA sequence specificity. Together, substrate DNA binding, indirect readout, and filamentation simultaneously enhance SgrAI's catalytic activity and modulate substrate preference. This unusual enzyme mechanism may have evolved to perform the specialized functions of bacterial innate immunity in rapid defense against invading phage DNA without causing damage to the host DNA.
History
DepositionMar 20, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 26, 2020Provider: repository / Type: Initial release
Revision 1.1Sep 9, 2020Group: Database references / Derived calculations / Category: citation / citation_author / struct_conn
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 / _citation_author.identifier_ORCID / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn.ptnr2_label_seq_id
Revision 1.2Mar 20, 2024Group: Data collection / Database references / Derived calculations
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_struct_oper_list
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_oper_list.name / _pdbx_struct_oper_list.symmetry_operation / _pdbx_struct_oper_list.type

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Structure visualization

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  • Biological unit as representative helical assembly
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  • Deposited structure unit
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Assembly

Deposited unit
A: SgraIR restriction enzyme
C: DNA (26-MER)
B: SgraIR restriction enzyme
D: DNA (26-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)100,8256
Polymers100,7764
Non-polymers492
Water0
1
A: SgraIR restriction enzyme
C: DNA (26-MER)
B: SgraIR restriction enzyme
D: DNA (26-MER)
hetero molecules
x 10


Theoretical massNumber of molelcules
Total (without water)1,008,24660
Polymers1,007,76040
Non-polymers48620
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
helical symmetry operation9
2


  • Idetical with deposited unit
  • helical asymmetric unit
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
SymmetryHelical symmetry: (Circular symmetry: 1 / Dyad axis: no / N subunits divisor: 1 / Num. of operations: 10 / Rise per n subunits: 21.6 Å / Rotation per n subunits: -86.2 °)

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Components

#1: Protein SgraIR restriction enzyme / SgrAI


Mass: 38073.102 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, Hydrolases; Acting on ester bonds; Endodeoxyribonucleases producing 5'-phosphomonoesters
#2: DNA chain DNA (26-MER)


Mass: 12314.889 Da / Num. of mol.: 2 / Source method: obtained synthetically / Details: assembled from pre-cleaved DNAs / 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

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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 assembly of SgrAI protein bound to pre-cleaved DNA
Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
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
110 mMTris-HClTris1
2150 mMsodium chlorideNaClSodium chloride1
35 mMmagnesium acetate1
40.5 mMDithiothreitol1
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: UltrAuFoil
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 80 %

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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: 22500 X / Calibrated magnification: 38167 X / Nominal defocus max: 2600 nm / Nominal defocus min: 600 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
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 60 / Used frames/image: 1-60

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Processing

EM software
IDNameVersionCategory
1FindEMparticle selection
2Leginonimage acquisition
4CTFFINDCTF correction
7Cootmodel fitting
9PHENIXmodel refinement
10RELIONinitial Euler assignment
11RELION3final Euler assignment
12RELION3classification
13RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: -86.2 ° / Axial rise/subunit: 21.6 Å / Axial symmetry: C1
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 7 / Algorithm: FOURIER SPACE / Symmetry type: HELICAL
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL / Target criteria: Correlation coefficient

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