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- EMDB-25404: Activated filamentous SgrAI endonuclease with Ca2+ and intact pri... -

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Basic information

Entry
Database: EMDB / ID: EMD-25404
TitleActivated filamentous SgrAI endonuclease with Ca2+ and intact primary site DNA
Map dataFull reconstruction of filamentous SgrAI bound to Ca2 and intact primary site DNA
Sample
  • Complex: Activated filamentous SgrAI endonuclease with Ca2+ and intact primary site DNA
    • Protein or peptide: SgraIR restriction enzyme
    • DNA: DNA (5'-D(P*GP*GP*TP*CP*TP*TP*CP*AP*CP*AP*CP*CP*GP*GP*TP*GP*TP*GP*AP*AP*GP*AP*CP*C)-3')
  • Ligand: CALCIUM ION
  • Ligand: water
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 / identical protein binding / metal ion binding / SgraIR restriction enzyme
Function and homology information
Biological speciesStreptomyces griseus (bacteria)
Methodhelical reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsShan Z / Lyumkis D
Funding support United States, 2 items
OrganizationGrant numberCountry
National Science Foundation (NSF, United States)MCB-1934291 United States
National Science Foundation (NSF, United States)MCB-1410355 United States
CitationJournal: J Biol Chem / Year: 2022
Title: Pretransition state and apo structures of the filament-forming enzyme SgrAI elucidate mechanisms of activation and substrate specificity.
Authors: Zelin Shan / Niloofar Ghadirian / Dmitry Lyumkis / Nancy C Horton /
Abstract: Enzyme filamentation is a widespread phenomenon that mediates enzyme regulation and function. For the filament-forming sequence-specific DNA endonuclease SgrAI, the process of filamentation both ...Enzyme filamentation is a widespread phenomenon that mediates enzyme regulation and function. For the filament-forming sequence-specific DNA endonuclease SgrAI, the process of filamentation both accelerates its DNA cleavage activity and expands its DNA sequence specificity, thus allowing for many additional DNA sequences to be rapidly cleaved. Both outcomes-the acceleration of DNA cleavage and the expansion of sequence specificity-are proposed to regulate critical processes in bacterial innate immunity. However, the mechanistic bases underlying these events remain unclear. Herein, we describe two new structures of the SgrAI enzyme that shed light on its catalytic function. First, we present the cryo-EM structure of filamentous SgrAI bound to intact primary site DNA and Ca resolved to ∼2.5 Å within the catalytic center, which represents the trapped enzyme-DNA complex prior to the DNA cleavage reaction. This structure reveals important conformational changes that contribute to the catalytic mechanism and the binding of a second divalent cation in the enzyme active site, which is expected to contribute to increased DNA cleavage activity of SgrAI in the filamentous state. Second, we present an X-ray crystal structure of DNA-free (apo) SgrAI resolved to 2.0 Å resolution, which reveals a disordered loop involved in DNA recognition. Collectively, these multiple new observations clarify the mechanism of expansion of DNA sequence specificity of SgrAI, including the indirect readout of sequence-dependent DNA structure, changes in protein-DNA interactions, and the disorder-to-order transition of a crucial DNA recognition element.
History
DepositionNov 9, 2021-
Header (metadata) releaseDec 1, 2021-
Map releaseDec 1, 2021-
UpdateJun 5, 2024-
Current statusJun 5, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.002
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.002
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7ss5
  • Surface level: 0.003
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7ss5
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_25404.png

Downloads & links

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Map

FileDownload / File: emd_25404.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFull reconstruction of filamentous SgrAI bound to Ca2 and intact primary site DNA
Voxel sizeX=Y=Z: 0.83 Å
Density
Contour LevelBy AUTHOR: 0.002 / Movie #1: 0.002
Minimum - Maximum-0.009719854 - 0.02049586
Average (Standard dev.)0.0000057352086 (±0.0011086083)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 265.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.830.830.83
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z265.600265.600265.600
α/β/γ90.00090.00090.000
start NX/NY/NZ-140-140-140
NX/NY/NZ280280280
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.0100.0200.000

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Supplemental data

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Mask #1

Fileemd_25404_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Reconstruction of filamentous SgrAI bound to Ca2 and...

Fileemd_25404_additional_1.map
AnnotationReconstruction of filamentous SgrAI bound to Ca2 and intact primary site DNA, density-modified, 2-fold symmetrized, and averaged along Z-axis
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map #1 of filamentous SgrAI bound to...

Fileemd_25404_half_map_1.map
Annotationhalf map #1 of filamentous SgrAI bound to Ca2 and intact primary site DNA
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map #2 of filamentous SgrAI bound to...

Fileemd_25404_half_map_2.map
Annotationhalf map #2 of filamentous SgrAI bound to Ca2 and intact primary site DNA
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Activated filamentous SgrAI endonuclease with Ca2+ and intact pri...

EntireName: Activated filamentous SgrAI endonuclease with Ca2+ and intact primary site DNA
Components
  • Complex: Activated filamentous SgrAI endonuclease with Ca2+ and intact primary site DNA
    • Protein or peptide: SgraIR restriction enzyme
    • DNA: DNA (5'-D(P*GP*GP*TP*CP*TP*TP*CP*AP*CP*AP*CP*CP*GP*GP*TP*GP*TP*GP*AP*AP*GP*AP*CP*C)-3')
  • Ligand: CALCIUM ION
  • Ligand: water

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Supramolecule #1: Activated filamentous SgrAI endonuclease with Ca2+ and intact pri...

SupramoleculeName: Activated filamentous SgrAI endonuclease with Ca2+ and intact primary site 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
Source (natural)Organism: Streptomyces griseus (bacteria)
Molecular weightTheoretical: 39.783895 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 VNGENLYFQS HHHHHH

UniProtKB: SgraIR restriction enzyme

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Macromolecule #2: DNA (5'-D(P*GP*GP*TP*CP*TP*TP*CP*AP*CP*AP*CP*CP*GP*GP*TP*GP*TP*GP...

MacromoleculeName: DNA (5'-D(P*GP*GP*TP*CP*TP*TP*CP*AP*CP*AP*CP*CP*GP*GP*TP*GP*TP*GP*AP*AP*GP*AP*CP*C)-3')
type: dna / ID: 2 / 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: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 3 / Number of copies: 6 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Macromolecule #4: water

MacromoleculeName: water / type: ligand / ID: 4 / Number of copies: 593 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

BufferpH: 8
Component:
ConcentrationNameFormula
25.0 mMTris-HCl
150.0 mMsodium chlorideNaCl
1.0 mMTCEP
10.0 mMCalcium chlorideCaCl2
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 7 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Instrument: HOMEMADE PLUNGER
Details: Cryo-EM grids were prepared by freezing using a manual plunger in cold room at 4C.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 30 eV
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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated magnification: 60240 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 165000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Helical parameters - Δz: 21.2 Å
Applied symmetry - Helical parameters - Δ&Phi: -85.8 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 220067
Startup modelType of model: EMDB MAP
EMDB ID:

20015

Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 3.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6obj


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 107.7 / Target criteria: Correlation coefficient
Output model

PDB-7ss5:
Activated SgrAI endonuclease DNA-bound dimer with Ca2+ and intact primary site DNA

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