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- EMDB-20015: Structure of filamentous SgrAI endonuclease in its activated form -

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Basic information

Entry
Database: EMDB / ID: EMD-20015
TitleStructure of filamentous SgrAI endonuclease in its activated form
Map dataCryo-EM reconstruction of filamentous SgrAI in its activated form
Sample
  • Complex: Filamentous assembly of SgrAI protein bound to pre-cleaved DNA
    • Protein or peptide: SgraIR restriction enzyme
    • DNA: DNA (26-MER)
  • Ligand: MAGNESIUM ION
Keywordsrestriction endonuclease / DNAse / allostery / bacterial innate immunity / filament / hyper-activation / substrate specificity / HYDROLASE-DNA complex
Function / homologyRestriction endonuclease, type II, Cfr10I/Bse634I / Cfr10I/Bse634I restriction endonuclease / Restriction endonuclease type II-like / metal ion binding / identical protein binding / SgraIR restriction enzyme
Function and homology information
Biological speciesStreptomyces griseus (bacteria)
Methodhelical reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsPolley S / Lyumkis D
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)DP5 OD021396 United States
National Science Foundation (NSF, United States)MCB-1410355 United States
CitationJournal: Structure / Year: 2019
Title: Mechanism of Filamentation-Induced Allosteric Activation of the SgrAI Endonuclease.
Authors: Smarajit Polley / Dmitry Lyumkis / Nancy C Horton /
Abstract: Filament formation by enzymes is increasingly recognized as an important phenomenon with potentially unique regulatory properties and biological roles. SgrAI is an allosterically regulated type II ...Filament formation by enzymes is increasingly recognized as an important phenomenon with potentially unique regulatory properties and biological roles. SgrAI is an allosterically regulated type II restriction endonuclease that forms filaments with enhanced DNA cleavage activity and altered sequence specificity. Here, we present the cryoelectron microscopy (cryo-EM) structure of the filament of SgrAI in its activated configuration. The structural data illuminate the mechanistic origin of hyperaccelerated DNA cleavage activity and suggests how indirect DNA sequence readout within filamentous SgrAI may enable recognition of substantially more nucleotide sequences than its low-activity form, thereby altering and partially relaxing its DNA sequence specificity. Together, substrate DNA binding, indirect readout, and filamentation simultaneously enhance SgrAI's catalytic activity and modulate substrate preference. This unusual enzyme mechanism may have evolved to perform the specialized functions of bacterial innate immunity in rapid defense against invading phage DNA without causing damage to the host DNA.
History
DepositionMar 20, 2019-
Header (metadata) releaseApr 3, 2019-
Map releaseFeb 26, 2020-
UpdateMar 20, 2024-
Current statusMar 20, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.045
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.045
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6obj
  • Surface level: 0.045
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6obj
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_20015.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM reconstruction of filamentous SgrAI in its activated form
Voxel sizeX=Y=Z: 1.31 Å
Density
Contour LevelBy AUTHOR: 0.045 / Movie #1: 0.045
Minimum - Maximum-0.10986078 - 0.20589763
Average (Standard dev.)0.0009992273 (±0.011426612)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 335.36 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.311.311.31
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z335.360335.360335.360
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ500500500
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.1100.2060.001

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Supplemental data

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Mask #1

Fileemd_20015_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: half-map 1 for the Cryo-EM reconstruction of filamentous...

Fileemd_20015_half_map_1.map
Annotationhalf-map 1 for the Cryo-EM reconstruction of filamentous SgrAI in its activated form
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: half-map 2 for the Cryo-EM reconstruction of filamentous...

Fileemd_20015_half_map_2.map
Annotationhalf-map 2 for the Cryo-EM reconstruction of filamentous SgrAI in its activated form
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire : Filamentous assembly of SgrAI protein bound to pre-cleaved DNA

EntireName: Filamentous assembly of SgrAI protein bound to pre-cleaved DNA
Components
  • Complex: Filamentous assembly of SgrAI protein bound to pre-cleaved DNA
    • Protein or peptide: SgraIR restriction enzyme
    • DNA: DNA (26-MER)
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Filamentous assembly of SgrAI protein bound to pre-cleaved DNA

SupramoleculeName: Filamentous assembly of SgrAI protein bound to pre-cleaved DNA
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Streptomyces griseus (bacteria)
Molecular weightTheoretical: 74 kDa/nm

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Macromolecule #1: SgraIR restriction enzyme

MacromoleculeName: SgraIR restriction enzyme / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
EC number: Hydrolases; Acting on ester bonds; Endodeoxyribonucleases producing 5'-phosphomonoesters
Source (natural)Organism: Streptomyces griseus (bacteria)
Molecular weightTheoretical: 38.073102 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MPFTYSIEAT RNLATTERCI QDIRNAPVRN RSTQFQLAQQ NMLAYTFGEV IPGFASAGIN GMDYRDVIGR PVENAVTEGT HFFRDDFRV DSNAKAKVAG DIFEIVSSAV MWNCAARWNS LMVGEGWRSQ PRYSRPTLSP SPRRQVAVLN LPRSFDWVSL L VPESQEVI ...String:
MPFTYSIEAT RNLATTERCI QDIRNAPVRN RSTQFQLAQQ NMLAYTFGEV IPGFASAGIN GMDYRDVIGR PVENAVTEGT HFFRDDFRV DSNAKAKVAG DIFEIVSSAV MWNCAARWNS LMVGEGWRSQ PRYSRPTLSP SPRRQVAVLN LPRSFDWVSL L VPESQEVI EEFRAGLRKD GLGLPTSTPD LAVVVLPEEF QNDEMWREEI AGLTRPNQIL LSGAYQRLQG RVQPGEISLA VA FKRSLRS DRLYQPLYEA NVMQLLLEGK LGAPKVEFEV HTLAPEGTNA FVTYEAASLY GLAEGRSAVH RAIRELYVPP TAA DLARRF FAFLNERMEL VNG

UniProtKB: SgraIR restriction enzyme

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Macromolecule #2: DNA (26-MER)

MacromoleculeName: DNA (26-MER) / type: dna / ID: 2 / Details: assembled from pre-cleaved DNAs / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Streptomyces griseus (bacteria)
Molecular weightTheoretical: 12.314889 KDa
SequenceString:
(DG)(DA)(DT)(DG)(DC)(DG)(DT)(DG)(DG)(DG) (DT)(DC)(DT)(DT)(DC)(DA)(DC)(DA)(DC)(DC) (DG)(DG)(DT)(DG)(DT)(DG)(DA)(DA)(DG) (DA)(DC)(DC)(DC)(DA)(DC)(DG)(DC)(DA)(DT) (DC)

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Macromolecule #3: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 3 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration1 mg/mL
BufferpH: 8
Component:
ConcentrationNameFormula
10.0 mMTris-HClTris
150.0 mMsodium chlorideNaClSodium chloride
5.0 mMmagnesium acetate
0.5 mMDithiothreitol
GridModel: UltrAuFoil / Material: GOLD / Mesh: 400 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 6 sec. / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 80 % / Instrument: HOMEMADE PLUNGER

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated magnification: 38167 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.6 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 22500
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 1-60 / Number real images: 216 / Average electron dose: 55.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 3.0)
Final reconstructionApplied symmetry - Helical parameters - Δz: 21.6 Å
Applied symmetry - Helical parameters - Δ&Phi: -86.2 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 7

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL / Target criteria: Correlation coefficient
Output model

PDB-6obj:
Structure of a DNA-bound dimer extracted from filamentous SgrAI endonuclease in its activated form

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