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- EMDB-2441: Cryo-EM structure of activated and oligomeric restriction endonuc... -

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Basic information

Entry
Database: EMDB / ID: EMD-2441
TitleCryo-EM structure of activated and oligomeric restriction endonuclease SgrAI
Map datacryo-EM reconstruction of activated and oligomeric restriction endonuclease SgrAI
Sample
  • Sample: cryo-EM reconstruction of activated and oligomeric restriction endonuclease SgrAI
  • Protein or peptide: SgrAI
  • DNA: SgrAI recognition sequence
Keywordsrestriction endonuclease / helix / oligomer / allostery / DNA cleavage / parasite-host interaction
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)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 8.6 Å
AuthorsLyumkis D / Talley H / Stewart A / Shah S / Park CK / Tama F / Potter CS / Carragher B / Horton NC
CitationJournal: Structure / Year: 2013
Title: Allosteric regulation of DNA cleavage and sequence-specificity through run-on oligomerization.
Authors: Dmitry Lyumkis / Heather Talley / Andrew Stewart / Santosh Shah / Chad K Park / Florence Tama / Clinton S Potter / Bridget Carragher / Nancy C Horton /
Abstract: SgrAI is a sequence specific DNA endonuclease that functions through an unusual enzymatic mechanism that is allosterically activated 200- to 500-fold by effector DNA, with a concomitant expansion of ...SgrAI is a sequence specific DNA endonuclease that functions through an unusual enzymatic mechanism that is allosterically activated 200- to 500-fold by effector DNA, with a concomitant expansion of its DNA sequence specificity. Using single-particle transmission electron microscopy to reconstruct distinct populations of SgrAI oligomers, we show that in the presence of allosteric, activating DNA, the enzyme forms regular, repeating helical structures characterized by the addition of DNA-binding dimeric SgrAI subunits in a run-on manner. We also present the structure of oligomeric SgrAI at 8.6 Å resolution, demonstrating the conformational state of SgrAI in its activated form. Activated and oligomeric SgrAI displays key protein-protein interactions near the helix axis between its N termini, as well as allosteric protein-DNA interactions that are required for enzymatic activation. The hybrid approach reveals an unusual mechanism of enzyme activation that explains SgrAI's oligomerization and allosteric behavior.
History
DepositionAug 16, 2013-
Header (metadata) releaseSep 4, 2013-
Map releaseSep 4, 2013-
UpdateApr 20, 2016-
Current statusApr 20, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3.25
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 3.25
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-4c3g
  • Surface level: 3.25
  • Imaged by UCSF Chimera
  • Download
  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-4c3g
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2441-EMD...

Downloads & links

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Map

FileDownload / File: emd_2441.map.gz / Format: CCP4 / Size: 26.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationcryo-EM reconstruction of activated and oligomeric restriction endonuclease SgrAI
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.42 Å/pix.
x 192 pix.
= 272.64 Å		1.42 Å/pix.
x 192 pix.
= 272.64 Å		1.42 Å/pix.
x 192 pix.
= 272.64 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.42 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 3.25 / Movie #1: 3.25
Minimum - Maximum-5.64923096 - 9.381752970000001
Average (Standard dev.)-0.18320161 (±1.22895467)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions192192192
Spacing192192192
CellA=B=C: 272.63998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.421.421.42
M x/y/z192192192
origin x/y/z0.0000.0000.000
length x/y/z272.640272.640272.640
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ454586
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS192192192
D min/max/mean-5.6499.382-0.183

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Supplemental data

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Sample components

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Entire : cryo-EM reconstruction of activated and oligomeric restriction en...

EntireName: cryo-EM reconstruction of activated and oligomeric restriction endonuclease SgrAI
Components
  • Sample: cryo-EM reconstruction of activated and oligomeric restriction endonuclease SgrAI
  • Protein or peptide: SgrAI
  • DNA: SgrAI recognition sequence

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Supramolecule #1000: cryo-EM reconstruction of activated and oligomeric restriction en...

SupramoleculeName: cryo-EM reconstruction of activated and oligomeric restriction endonuclease SgrAI
type: sample / ID: 1000 / Details: 1 megadalton (100 kDa) per DNA-bound dimer
Oligomeric state: oligomeric SgrAI forms a helical array, whereby each asymmetric unit is composed of a DNA-bound SgrAI dimer
Number unique components: 2
Molecular weightTheoretical: 1 MDa

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Macromolecule #1: SgrAI

MacromoleculeName: SgrAI / type: protein_or_peptide / ID: 1 / Name.synonym: SgraIR restriction enzyme
Details: 2 copies of pre-cleaved DNA containing sticky ends and the SgrAI recognition sequence is bound to an SgrAI dimer. The oligomeric helix is formed from multiple copies of such DNA-bound dimers
Oligomeric state: DNA-bound dimer / Recombinant expression: Yes
Source (natural)Organism: Streptomyces griseus (bacteria)
Molecular weightTheoretical: 38 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: ER2566 / Recombinant plasmid: pET21a_SgrA1R
SequenceUniProtKB: SgraIR restriction enzyme

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Macromolecule #2: SgrAI recognition sequence

MacromoleculeName: SgrAI recognition sequence / type: dna / ID: 2
Details: 2 copies of pre-cleaved DNA containing sticky ends and the SgrAI recognition sequence is bound to an SgrAI dimer. The oligomeric helix is formed from multiple copies of such DNA-bound dimers
Classification: DNA / Structure: DOUBLE HELIX / Synthetic?: No
Source (natural)Organism: Streptomyces griseus (bacteria)
SequenceString:
GATGCGTGGG TCTTCACACC GGTGTGAAGA CCCACGCATC

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsingle particle reconstruction
Aggregation statehelical array

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Sample preparation

Concentration0.2 mg/mL
BufferpH: 8
Details: 10 mM Tris-HCl pH 8.0, 150 mM NaCl, 5 mM Mg(OAc)2, 0.5 mM DTT
StainingType: NEGATIVE
Details: Specimens were prepared for cryo-EM by applying 3 microliters of sample in binding buffer to a holey carbon C-flat grid (CF-2/2-400) (Protochips, inc.) that had been plasma cleaned (Gatan, ...Details: Specimens were prepared for cryo-EM by applying 3 microliters of sample in binding buffer to a holey carbon C-flat grid (CF-2/2-400) (Protochips, inc.) that had been plasma cleaned (Gatan, Solarus) for 5 sec. The sample was allowed to adsorb to the grid for 30 sec., then plunge-frozen into liquid ethane using a manual cryo-plunger at 4 C.
GridDetails: CF-2/2-400
VitrificationCryogen name: ETHANE / Instrument: HOMEMADE PLUNGER
Method: Specimens were prepared for cryo-EM by applying 3 microliters of sample in binding buffer to a holey carbon C-flat grid (CF-2/2-400 (Protochips, inc.) that had been plasma cleaned (Gatan, ...Method: Specimens were prepared for cryo-EM by applying 3 microliters of sample in binding buffer to a holey carbon C-flat grid (CF-2/2-400 (Protochips, inc.) that had been plasma cleaned (Gatan, Solarus) for 5 sec. The sample was allowed to adsorb to the grid for 30 sec., then plunge-frozen into liquid ethane using a manual cryo-plunger at 4 C.

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Electron microscopy

MicroscopeFEI TECNAI F20
Alignment procedureLegacy - Astigmatism: objective lens astigmatism was corrected at 120,000 times magnification
DateSep 19, 2012
Image recordingCategory: FILM / Film or detector model: DIRECT ELECTRON DE-12 (4k x 3k) / Digitization - Scanner: OTHER / Digitization - Sampling interval: 6.0 µm / Number real images: 655 / Average electron dose: 16 e/Å2
Tilt angle min0
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 42134 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 62000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

DetailsThe final reconstruction was obtained from 1,918 filament segments, averaged and inserted into the final reconstruction 8 times using Frealign
CTF correctionDetails: Frealign
Final reconstructionApplied symmetry - Helical parameters - Δz: 21.6 Å
Applied symmetry - Helical parameters - Δ&Phi: 86.2 °
Applied symmetry - Helical parameters - Axial symmetry: D1 (2x1 fold dihedral)
Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 8.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: Xmipp, and, Frealign / Number images used: 1918

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Atomic model buiding 1

Initial modelPDB ID:

3dvo


Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B
SoftwareName: Direx
DetailsA model of SgrAI bound to PC DNA generated using the x-ray crystal structure of SgrAI bound to an 18 bp DNA containing a primary site (PDB ID: 3DVO) and additional modeled flanking DNA in B form was flexibly fitted using Direx, a geometry-based conformational sampling approach under low-resolution restraints. The refinement procedure was run for 500 steps followed by the minimization procedure of 300 steps
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-4c3g:
cryo-EM structure of activated and oligomeric restriction endonuclease SgrAI

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