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- PDB-7lvv: cryoEM structure DrdV-DNA complex -

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Basic information

Entry
Database: PDB / ID: 7lvv
TitlecryoEM structure DrdV-DNA complex
Components
  • DNA (27-MER)
  • DNA (28-MER)
  • Site-specific DNA-methyltransferase (adenine-specific)DNA methyltransferase
KeywordsHYDROLASE/DNA / inhibitor / Complex / endonuclease / methyl transferase / TypeIIL RM system / HYDROLASE / HYDROLASE-DNA complex
Function / homology
Function and homology information


N-methyltransferase activity / site-specific DNA-methyltransferase (adenine-specific) / site-specific DNA-methyltransferase (adenine-specific) activity / endonuclease activity / DNA binding
Similarity search - Function
Type ISP restriction-modification enzyme LLaBIII, C-terminal specificity domain / Type ISP C-terminal specificity domain / N-6 DNA Methylase / DNA methylase, adenine-specific / N-6 Adenine-specific DNA methylases signature. / DNA methylase, N-6 adenine-specific, conserved site / S-adenosyl-L-methionine-dependent methyltransferase superfamily
Similarity search - Domain/homology
S-ADENOSYLMETHIONINE / DNA / DNA (> 10) / site-specific DNA-methyltransferase (adenine-specific)
Similarity search - Component
Biological speciesDeinococcus wulumuqiensis (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.25 Å
AuthorsShen, B.W. / Stoddard, B.L.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM105691 United States
CitationJournal: Structure / Year: 2021
Title: Coordination of phage genome degradation versus host genome protection by a bifunctional restriction-modification enzyme visualized by CryoEM.
Authors: Betty W Shen / Joel D Quispe / Yvette Luyten / Benjamin E McGough / Richard D Morgan / Barry L Stoddard /
Abstract: Restriction enzymes that combine methylation and cleavage into a single assemblage and modify one DNA strand are capable of efficient adaptation toward novel targets. However, they must reliably ...Restriction enzymes that combine methylation and cleavage into a single assemblage and modify one DNA strand are capable of efficient adaptation toward novel targets. However, they must reliably cleave invasive DNA and methylate newly replicated unmodified host sites. One possible solution is to enforce a competition between slow methylation at a single unmodified host target, versus faster cleavage that requires multiple unmodified target sites in foreign DNA to be brought together in a reaction synapse. To examine this model, we have determined the catalytic behavior of a bifunctional type IIL restriction-modification enzyme and determined its structure, via cryoelectron microscopy, at several different stages of assembly and coordination with bound DNA targets. The structures demonstrate a mechanism in which an initial dimer is formed between two DNA-bound enzyme molecules, positioning the endonuclease domain from each enzyme against the other's DNA and requiring further additional DNA-bound enzyme molecules to enable cleavage.
History
DepositionFeb 26, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 17, 2021Provider: repository / Type: Initial release
Revision 1.1Apr 21, 2021Group: 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.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Jun 16, 2021Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Mar 6, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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  • Deposited structure unit
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Site-specific DNA-methyltransferase (adenine-specific)
B: Site-specific DNA-methyltransferase (adenine-specific)
C: Site-specific DNA-methyltransferase (adenine-specific)
D: Site-specific DNA-methyltransferase (adenine-specific)
E: DNA (28-MER)
F: DNA (27-MER)
G: DNA (28-MER)
H: DNA (27-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)509,65314
Polymers508,6968
Non-polymers9576
Water0
1


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

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Components

#1: Protein
Site-specific DNA-methyltransferase (adenine-specific) / DNA methyltransferase


Mass: 118256.859 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Deinococcus wulumuqiensis (bacteria) / Gene: drdVRM, DVJ83_12125 / Production host: Escherichia coli (E. coli)
References: UniProt: A0A345IJ72, site-specific DNA-methyltransferase (adenine-specific)
#2: DNA chain DNA (28-MER)


Mass: 8748.646 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: DNA chain DNA (27-MER)


Mass: 9085.788 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: Chemical ChemComp-SAM / S-ADENOSYLMETHIONINE / S-Adenosyl methionine


Mass: 398.437 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C15H22N6O5S / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: DrdVDNA complex / Type: COMPLEX / Details: dimer of DrdVDNA complex / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightValue: 0.24 kDa/nm / Experimental value: NO
Source (natural)Organism: Deinococcus wulumuqiensis (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5 / Details: 20 nM HEPES pH7.5 150 NaCl, 2 mM CaCl2
Buffer component
IDConc.FormulaBuffer-ID
120 mMHEPES1
2150 mMNaCLSodium chloride1
SpecimenConc.: 0.25 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 298 K

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 37000 X / Nominal defocus max: 2300 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 70 K / Temperature (min): 70 K
Image recordingAverage exposure time: 2 sec. / Electron dose: 40 e/Å2 / Film or detector model: FEI CETA (4k x 4k) / Num. of grids imaged: 3 / Num. of real images: 1200
Details: images were collected in movie-mode at 5 frames per second
EM imaging opticsEnergyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategoryDetails
2Leginon4image acquisition
4cryoSPARC3.1.0CTF correctioncryoSPARC patch CTF was used to estimate CTF parameter.
10cryoSPARC3.1.0initial Euler assignmentab-initio 3D reconstruction
11cryoSPARCfinal Euler assignment
12cryoSPARC3.1.0classification
13cryoSPARC3.1.03D reconstruction
CTF correctionType: NONE
Particle selectionNum. of particles selected: 224095
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.25 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 34554 / Symmetry type: POINT

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