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- PDB-8w2p: BsaXI-DNA complex I -

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Basic information

Entry
Database: PDB / ID: 8w2p
TitleBsaXI-DNA complex I
Components
  • (DNA (41-MER) ...) x 2
  • S.BsaXI
  • site-specific DNA-methyltransferase (adenine-specific)
KeywordsDNA BINDING PROTEIN/DNA / restriction nuclease / restriction-modification systems / Type IIB / R-M complex / DNA BINDING PROTEIN / DNA BINDING PROTEIN-DNA complex
Function / homology
Function and homology information


N-methyltransferase activity / DNA restriction-modification system / methylation / DNA binding
Similarity search - Function
Type I restriction enzyme R protein, N-terminal domain / : / Type I restriction enzyme R protein N terminus (HSDR_N) / : / Type I restriction modification DNA specificity domain / Type I restriction modification DNA specificity domain superfamily / Type I restriction modification DNA specificity domain / N-6 DNA Methylase / DNA methylase, adenine-specific / N-6 Adenine-specific DNA methylases signature. ...Type I restriction enzyme R protein, N-terminal domain / : / Type I restriction enzyme R protein N terminus (HSDR_N) / : / Type I restriction modification DNA specificity domain / Type I restriction modification DNA specificity domain superfamily / Type I restriction modification DNA 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-ADENOSYL-L-HOMOCYSTEINE / S-ADENOSYLMETHIONINE / DNA / DNA (> 10) / Uncharacterized protein / Site-specific DNA-methyltransferase (adenine-specific)
Similarity search - Component
Biological speciesGeobacillus stearothermophilus (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.14 Å
AuthorsShen, B.W. / Stoddard, B.L. / Xu, S.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)Ro1-GM105691 United States
CitationJournal: To Be Published
Title: Structural and biochemical analysis of subunit assembly, DNA recognition and cleavage by a Type IIB restriction-modification enzyme: BsaXI
Authors: Shen, B.W. / Heiter, D. / Xu, S. / Stoddard, B.L.
History
DepositionFeb 20, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 30, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: site-specific DNA-methyltransferase (adenine-specific)
B: site-specific DNA-methyltransferase (adenine-specific)
C: S.BsaXI
D: DNA (41-MER) Top strand of BsaXI cognate DNA substrate
E: DNA (41-MER) Complementary strand of BsaXI DNA substrate
hetero molecules


Theoretical massNumber of molelcules
Total (without water)302,2998
Polymers301,4765
Non-polymers8233
Water905
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, co-elution of RM2S and DNA duplex on SEC chromatography.
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Protein , 2 types, 3 molecules ABC

#1: Protein site-specific DNA-methyltransferase (adenine-specific)


Mass: 107195.234 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Geobacillus stearothermophilus (bacteria)
Strain: Cpw230 / Gene: B9L19_03590 / Production host: Escherichia coli (E. coli) / Strain (production host): C2566
References: UniProt: A0AA91YUR9, site-specific DNA-methyltransferase (adenine-specific)
#2: Protein S.BsaXI / Type I restriction modification DNA specificity domain-containing protein


Mass: 55038.824 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Geobacillus stearothermophilus (bacteria)
Strain: Cpw230 / Gene: GT3921_04730 / Production host: Escherichia coli (E. coli) / Strain (production host): C2566 / References: UniProt: A0A226QBA7

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DNA (41-MER) ... , 2 types, 2 molecules DE

#3: DNA chain DNA (41-MER) Top strand of BsaXI cognate DNA substrate


Mass: 16083.387 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Geobacillus stearothermophilus (bacteria)
#4: DNA chain DNA (41-MER) Complementary strand of BsaXI DNA substrate


Mass: 15963.281 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Geobacillus stearothermophilus (bacteria)

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Non-polymers , 4 types, 8 molecules

#5: Chemical ChemComp-SAM / S-ADENOSYLMETHIONINE


Mass: 398.437 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C15H22N6O5S / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION
#7: Chemical ChemComp-SAH / S-ADENOSYL-L-HOMOCYSTEINE


Mass: 384.411 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C14H20N6O5S / Feature type: SUBJECT OF INVESTIGATION
#8: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 5 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY
Has protein modificationN

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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: Quaternary complex of RM, S and DNA complex / Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weightValue: 2.69 kDa/nm / Experimental value: NO
Source (natural)Organism: Geobacillus stearothermophilus (bacteria) / Strain: Cpw230
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: C2566
Buffer solutionpH: 8 / Details: 20 mM TrisHCl, 250 mM Nacl, 2 mM CaCl2
Buffer componentConc.: 20 mM / Name: TrisHCl
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Sample was monodisperse.
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 278 K

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company /
Model: Titan Krios / Image courtesy: FEI Company
EM imaging

Alignment procedure: COMA FREE / C2 aperture diameter: 100 µm / Cryogen: NITROGEN / Electron source: FIELD EMISSION GUN / Illumination mode: FLOOD BEAM / Mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 5000 nm / Nominal defocus min: 1200 nm / Temperature (min): 170 K / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Specimen-ID: 1

IDAccelerating voltage (kV)Calibrated defocus max (nm)Calibrated defocus min (nm)ModelTemperature (max) (K)
120050001200FEI TALOS ARCTICA
23000FEI TITAN KRIOS170
Image recording

Average exposure time: 2 sec. / Electron dose: 40 e/Å2 / Detector mode: COUNTING

IDImaging-IDFilm or detector modelNum. of grids imagedNum. of real images
11DIRECT ELECTRON DE-10 (5k x 4k)31579
22DIRECT ELECTRON DE-20 (5k x 3k)12238
EM imaging optics
IDImaging-IDPhase plate
11VOLTA PHASE PLATE
22OTHER
Image scans
Movie frames/imageUsed frames/imageIDImage recording-IDEntry-ID
502-50118W2P
502-50228W2P

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Processing

EM software
IDNameVersionCategoryImaging-ID
1cryoSPARC3.2particle selection
2SerialEM3image acquisition1
4cryoSPARC3.2CTF correction
7Coot0.9.8.9.2ELmodel fitting
9SerialEM4image acquisition2
10cryoSPARC3.3initial Euler assignment
11cryoSPARC3.3final Euler assignment
12cryoSPARC3.3classification
13cryoSPARC3.33D reconstruction
14PHENIX1.19.2model refinement
CTF correctionType: NONE
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.14 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 245111 / Algorithm: FOURIER SPACE / Num. of class averages: 2 / Symmetry type: POINT
Atomic model buildingProtocol: BACKBONE TRACE / Space: REAL
Atomic model buildingDetails: manual built model in coot / Source name: Other / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00321359
ELECTRON MICROSCOPYf_angle_d0.66229149
ELECTRON MICROSCOPYf_dihedral_angle_d19.2413346
ELECTRON MICROSCOPYf_chiral_restr0.0423168
ELECTRON MICROSCOPYf_plane_restr0.0053475

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