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- EMDB-23461: cryoEM structure DrdV-DNA complex -

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Basic information

Entry
Database: EMDB / ID: EMD-23461
TitlecryoEM structure DrdV-DNA complex
Map dataTetramer II of DrdV-DNA complex
Sample
  • Complex: DrdV-DNA tetramer II
    • Protein or peptide: Site-specific DNA-methyltransferase (adenine-specific)DNA methyltransferase
    • DNA: DNA (28-MER)
    • DNA: DNA (27-MER)
  • Ligand: S-ADENOSYLMETHIONINES-Adenosyl methionine
  • Ligand: CALCIUM IONCalcium
Keywordsinhibitor / 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
site-specific DNA-methyltransferase (adenine-specific)
Similarity search - Component
Biological speciesDeinococcus wulumuqiensis 479 (bacteria) / Deinococcus wulumuqiensis (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.86 Å
AuthorsShen BW / Stoddard BL
Funding support United States, 1 items
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 9, 2021-
Header (metadata) releaseMar 17, 2021-
Map releaseMar 17, 2021-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.18
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.18
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7lo5
  • Surface level: 0.18
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23461.png

Downloads & links

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Map

FileDownload / File: emd_23461.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationTetramer II of DrdV-DNA complex
Voxel sizeX=Y=Z: 1.0275 Å
Density
Contour LevelBy AUTHOR: 0.18 / Movie #1: 0.18
Minimum - Maximum-0.38112658 - 1.0199002
Average (Standard dev.)0.00020250402 (±0.022025628)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 526.08 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.02751.02751.0275
M x/y/z512512512
origin x/y/z0.0000.0000.000
length x/y/z526.080526.080526.080
α/β/γ90.00090.00090.000
start NX/NY/NZ-200-200-200
NX/NY/NZ400400400
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS512512512
D min/max/mean-0.3811.0200.000

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Supplemental data

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Sample components

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Entire : DrdV-DNA tetramer II

EntireName: DrdV-DNA tetramer II
Components
  • Complex: DrdV-DNA tetramer II
    • Protein or peptide: Site-specific DNA-methyltransferase (adenine-specific)DNA methyltransferase
    • DNA: DNA (28-MER)
    • DNA: DNA (27-MER)
  • Ligand: S-ADENOSYLMETHIONINES-Adenosyl methionine
  • Ligand: CALCIUM IONCalcium

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Supramolecule #1: DrdV-DNA tetramer II

SupramoleculeName: DrdV-DNA tetramer II / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Details: DrdV-DNA complex after SEC Biorad 650 fractionation
Source (natural)Organism: Deinococcus wulumuqiensis 479 (bacteria)
Molecular weightTheoretical: 450 KDa

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Macromolecule #1: Site-specific DNA-methyltransferase (adenine-specific)

MacromoleculeName: Site-specific DNA-methyltransferase (adenine-specific)
type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
EC number: site-specific DNA-methyltransferase (adenine-specific)
Source (natural)Organism: Deinococcus wulumuqiensis (bacteria)
Molecular weightTheoretical: 118.256859 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MSLQLVKKFQ KRLEDIVAYG GTRNESSVRA AFQQLLSDWA EGSGLRLITE VTQKAVAGNN VRPDGTLKDS LQQSRGYWES KDEADTLDD EIQKKLAKGY PRDNIIFEDS RLAVLMQNGE EVQRVDMGDA GALAGLLKLF FEFEPPQVLE FRKAVDHFKD E MPHLLKIL ...String:
MSLQLVKKFQ KRLEDIVAYG GTRNESSVRA AFQQLLSDWA EGSGLRLITE VTQKAVAGNN VRPDGTLKDS LQQSRGYWES KDEADTLDD EIQKKLAKGY PRDNIIFEDS RLAVLMQNGE EVQRVDMGDA GALAGLLKLF FEFEPPQVLE FRKAVDHFKD E MPHLLKIL REAADAAEQK ADYRGERDHF VEIAKEAINP DFSPRDAREM LIQHILTGDL FTSVFDNAQY HEDNNIAQQL QQ LAATFYK GPVKRDIAER TKRYYGAIQA AAAQIADHHE KQRFLKALYE NFYRAYNPAG AERLGIFYTP GEIVRFMIEA TDT LLEKHF QKELADKGVE ILDPATGTGT FITELIDFLP KAKLEQKYRE ELHCNELALL PYYIANLNIE ATYAQKMGRY EEFR NIVLV DTLDNTGFGV HGQQSGLFGS VTAENLERAK RQNARPVRVI IGNPPYRANQ ANENDNNKNR EYKEIDRRIK ATYVA ASTA QKTKLYDMYS RFLRWATDRL KEDGIVAFVS NSSFIDSRTF DGFRKEVVKD FDHIYILDMK GNANTSGERR KREGGN VFN DQIKVGVAVY FLVRSAAGKR KSKDTKIWYH AVPDFWRARE KLEWLKTTKF EDIEFDHIRP DAKHNWLGQV DEENDWN EF LPVADKDTKQ AKGLGQERAI FKLYSLGVVT NRDEWVYSRA EDELADKVRY FIGRYNEIIK LPLGDLMSRN WEGDIKMT R ATIADAQSRK SYSLEKNSIV PSLYRPFDVL KMYFSKNLNE MQYQMPSIFP KGVGENVVIA LSGSPAAKPF QVLATDILP SLDLLEKTQC LPFYRYTMNG ERLNNITDYA LKAFQTHYAD TSISREDIFH YVYAVLHHPA YREKYALNLR QEFPRIPFYP EFGRWAAWG RELMALHIGF ESVAPYPLKR TDEPPKNDTP EALALAKKAR LKVQRDAAKQ PTGAVELDGL TTLAGIPAAA W AYKLGNRS ALEWVLERHK ETTPKDATIR EKFNTYRFAD HKERVIDLLA RVTTVSVETV RIVGEMPAET M

UniProtKB: site-specific DNA-methyltransferase (adenine-specific)

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Macromolecule #2: DNA (28-MER)

MacromoleculeName: DNA (28-MER) / type: dna / ID: 2 / Number of copies: 4 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 8.748646 KDa
SequenceString:
(DC)(DA)(DG)(DC)(DC)(DC)(DA)(DT)(DG)(DG) (DA)(DC)(DC)(DC)(DA)(DG)(DA)(DA)(DC)(DC) (DA)(DC)(DC)(DC)(DA)(DC)(DC)(DC)(DG)

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Macromolecule #3: DNA (27-MER)

MacromoleculeName: DNA (27-MER) / type: dna / ID: 3 / Number of copies: 4 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 9.085788 KDa
SequenceString:
(DG)(DG)(DG)(DT)(DG)(DG)(DG)(DT)(DG)(DG) (DT)(DT)(DC)(DT)(DG)(DG)(DG)(DT)(DC)(DC) (DA)(DT)(DG)(DG)(DG)(DC)(DT)(DG)(DC)

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Macromolecule #4: S-ADENOSYLMETHIONINE

MacromoleculeName: S-ADENOSYLMETHIONINE / type: ligand / ID: 4 / Number of copies: 4 / Formula: SAM
Molecular weightTheoretical: 398.437 Da
Chemical component information

ChemComp-SAM:
S-ADENOSYLMETHIONINE / S-Adenosyl methionine

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Macromolecule #5: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 5 / Number of copies: 4 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.4 mg/mL
BufferpH: 8 / Details: 20 mM TrismaHCl ph 8.0/150 NaCl/2CaCl2
GridModel: Quantifoil / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 15 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated defocus max: 0.5 µm / Calibrated defocus min: 0.8 µm / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.2 µm
Specialist opticsSpherical aberration corrector: Cs corrector with two hexapod elements
Chromatic aberration corrector: CEOS manufactured Cc corrector
Energy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 70.0 K / Max: 70.0 K
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 4300 / Average exposure time: 2.0 sec. / Average electron dose: 30.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cryoSPARC (ver. V3.1.0)
Final 3D classificationNumber classes: 100 / Software - Name: cryoSPARC (ver. v3.1.0)
Final angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cryoSPARC (ver. v3.1.0)
Final reconstructionNumber classes used: 4 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.86 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. V3.1.0) / Number images used: 94920

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