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- EMDB-23543: cryoEM structure DrdV-DNA complex -

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Basic information

Entry
Database: EMDB / ID: EMD-23543
TitlecryoEM structure DrdV-DNA complex
Map dataDrdVDNA complex dimer map from Glacios
Sample
  • Complex: DrdVDNA complex
    • 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 (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.25 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 26, 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: 0.375
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.375
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-7lvv
  • Surface level: 0.375
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23543.png

Downloads & links

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Map

FileDownload / File: emd_23543.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationDrdVDNA complex dimer map from Glacios
Voxel sizeX=Y=Z: 1.16 Å
Density
Contour LevelBy AUTHOR: 0.375 / Movie #1: 0.375
Minimum - Maximum-0.7445625 - 2.2025354
Average (Standard dev.)0.00035449766 (±0.05004532)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 464.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.161.161.16
M x/y/z400400400
origin x/y/z0.0000.0000.000
length x/y/z464.000464.000464.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS400400400
D min/max/mean-0.7452.2030.000

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Supplemental data

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Sample components

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Entire : DrdVDNA complex

EntireName: DrdVDNA complex
Components
  • Complex: DrdVDNA complex
    • 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: DrdVDNA complex

SupramoleculeName: DrdVDNA complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3 / Details: dimer of DrdVDNA complex
Source (natural)Organism: Deinococcus wulumuqiensis (bacteria)
Molecular weightTheoretical: 0.24 kDa/nm

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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: 2 / 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: 2 / 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: 2 / 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

Concentration0.25 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormula
20.0 mMHEPES
150.0 mMNaCLSodium chloride

Details: 20 nM HEPES pH7.5 150 NaCl, 2 mM CaCl2
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Pressure: 0.015 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS GLACIOS
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.3000000000000003 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 37000
Specialist opticsEnergy 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: FEI CETA (4k x 4k) / Number grids imaged: 3 / Number real images: 1200 / Average exposure time: 2.0 sec. / Average electron dose: 40.0 e/Å2
Details: images were collected in movie-mode at 5 frames per second

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Image processing

Particle selectionNumber selected: 224095
Startup modelType of model: OTHER
Details: Phyre2 model of MTase-TRD domain was used as template.
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.1.0) / Software - details: ab-initio 3D reconstruction
Final 3D classificationNumber classes: 4 / Software - Name: cryoSPARC (ver. 3.1.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.25 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.1.0) / Number images used: 34554

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