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- PDB-8w0p: BsaXI -- Type IIB R-M system -

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Basic information

Entry
Database: PDB / ID: 8w0p
TitleBsaXI -- Type IIB R-M system
Components
  • RM.BsaXI
  • S.BsaXI
KeywordsANTIMICROBIAL PROTEIN / R-M system / restriction nucleases / Type IIB / protein-DNA complex / BsaXI.
Function / homology: / Type I restriction modification DNA specificity domain / Type I restriction modification DNA specificity domain superfamily / Type I restriction modification DNA specificity domain / DNA restriction-modification system / DNA binding / S-ADENOSYLMETHIONINE / Uncharacterized protein / :
Function and homology information
Biological speciesGeobacillus stearothermophilus (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.26 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: To Be Published
Title: Atomic insights into the mechanism of DNA recognition and methylation by type IIB restriction-modification system: CryoEM structures of DNA free and cognate DNA bound BsaXI.
Authors: Shen, B.W. / Stoddard, B.L.
History
DepositionFeb 13, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 30, 2024Provider: repository / Type: Initial release
Revision 1.0Oct 30, 2024Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Oct 30, 2024Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Oct 30, 2024Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Oct 30, 2024Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Oct 30, 2024Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Oct 30, 2024Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1May 21, 2025Group: Data collection / Category: em_admin / em_software / Item: _em_admin.last_update / _em_software.name
Revision 1.1May 21, 2025Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Data processing / Experimental summary / Data content type: EM metadata / EM metadata / Category: em_admin / em_software / Data content type: EM metadata / EM metadata / Item: _em_admin.last_update / _em_software.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: RM.BsaXI
B: RM.BsaXI
C: S.BsaXI
hetero molecules


Theoretical massNumber of molelcules
Total (without water)270,2265
Polymers269,4293
Non-polymers7972
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein RM.BsaXI / site-specific DNA-methyltransferase (adenine-specific)


Mass: 107195.234 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: RM fusion protein
Source: (gene. exp.) Geobacillus stearothermophilus (bacteria)
Strain: Cpw230 / Gene: E5Z46_18465 / Production host: Escherichia coli (E. coli) / Strain (production host): C2566
References: UniProt: A0A4D7QEP1, 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
#3: Chemical ChemComp-SAM / S-ADENOSYLMETHIONINE


Mass: 398.437 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C15H22N6O5S / Feature type: SUBJECT OF INVESTIGATION
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: heterotrimers of RM fusion and S subunit / Type: COMPLEX
Details: reconstituted RM and S subunits of type IIB R-M systems.
Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.59 MDa / 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, ph 8.0, 2 mM CaCl2, 150 mM NaCl
SpecimenConc.: 0.35 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: 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: 298 K

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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 FIELD / Nominal defocus max: 5000 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Image recordingAverage exposure time: 2 sec. / Electron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 6227
Image scansMovie frames/image: 50 / Used frames/image: 2-50

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Processing

EM software
IDNameVersionCategory
1cryoSPARC3.32particle selection
4cryoSPARC3.32CTF correction
8PHENIXmodel refinement
10cryoSPARC3.32initial Euler assignment
11cryoSPARC3.32final Euler assignment
12cryoSPARC3.32classification
13cryoSPARC3.323D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2487316
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.26 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 194038 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00319506
ELECTRON MICROSCOPYf_angle_d0.5626302
ELECTRON MICROSCOPYf_dihedral_angle_d5.6132555
ELECTRON MICROSCOPYf_chiral_restr0.0422844
ELECTRON MICROSCOPYf_plane_restr0.0043397

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