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- PDB-4c3g: cryo-EM structure of activated and oligomeric restriction endonuc... -

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Basic information

Entry
Database: PDB / ID: 4c3g
Titlecryo-EM structure of activated and oligomeric restriction endonuclease SgrAI
Components
  • 5'-D(*CP*CP*GP*GP*TP*GP*TP*GP*AP*AP*GP*AP*CP*CP *CP*AP*CP*GP*CP*AP*TP*CP)-3'
  • 5'-D(*GP*AP*TP*GP*CP*GP*TP*GP*GP*GP*TP*CP*TP*TP *CP*AP*CP*AP)-3'
  • SGRAIR RESTRICTION ENZYME
KeywordsHYDROLASE / RESTRICTION ENDONUCLEASE / 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 / DNA / DNA (> 10) / SgraIR restriction enzyme
Function and homology information
Biological speciesSTREPTOMYCES GRISEUS (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 8.6 Å
Model type detailsCA ATOMS ONLY, CHAIN A, B
AuthorsLyumkis, D. / Talley, H. / Stewart, A. / Shah, S. / Park, C.K. / Tama, F. / Potter, C.S. / Carragher, B. / Horton, N.C.
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 23, 2013Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 11, 2013Provider: repository / Type: Initial release
Revision 1.1Sep 18, 2013Group: Other
Revision 1.2Oct 9, 2013Group: Database references
Revision 1.3Oct 30, 2013Group: Database references
Revision 1.4Aug 23, 2017Group: Data collection / Category: em_software
Item: _em_software.fitting_id / _em_software.image_processing_id / _em_software.name

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Structure visualization

Movie
  • Deposited structure unit
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  • Simplified surface model + fitted atomic model
  • EMDB-2441
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  • EMDB-2441
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Structure viewerMolecule:
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Assembly

Deposited unit
A: SGRAIR RESTRICTION ENZYME
B: SGRAIR RESTRICTION ENZYME
C: 5'-D(*GP*AP*TP*GP*CP*GP*TP*GP*GP*GP*TP*CP*TP*TP *CP*AP*CP*AP)-3'
E: 5'-D(*CP*CP*GP*GP*TP*GP*TP*GP*AP*AP*GP*AP*CP*CP *CP*AP*CP*GP*CP*AP*TP*CP)-3'
D: 5'-D(*GP*AP*TP*GP*CP*GP*TP*GP*GP*GP*TP*CP*TP*TP *CP*AP*CP*AP)-3'
F: 5'-D(*CP*CP*GP*GP*TP*GP*TP*GP*AP*AP*GP*AP*CP*CP *CP*AP*CP*GP*CP*AP*TP*CP)-3'


Theoretical massNumber of molelcules
Total (without water)100,3106
Polymers100,3106
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein SGRAIR RESTRICTION ENZYME / SGRAI / Coordinate model: Cα atoms only


Mass: 37884.855 Da / Num. of mol.: 2 / Fragment: DNA-BINDING DOMAIN, RESIDUES 2-338
Source method: isolated from a genetically manipulated source
Details: AN SGRAI DIMER. THE OLIGOMERIC HELIX IS FORMED FROM MULTIPLE COPIES OF SUCH SGRAI DIMERS BOUND TO DNA.
Source: (gene. exp.) STREPTOMYCES GRISEUS (bacteria) / Plasmid: PET21A_SGRA1R / Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): ER2566 / References: UniProt: Q9F6L0
#2: DNA chain 5'-D(*GP*AP*TP*GP*CP*GP*TP*GP*GP*GP*TP*CP*TP*TP *CP*AP*CP*AP)-3'


Mass: 5547.588 Da / Num. of mol.: 2 / Source method: obtained synthetically
Details: 2 COPIES OF PRE-CLEAVED DNA CONTAINING STICKY ENDS AND THE SGRAI RECOGNITION SEQUENCE
Source: (synth.) STREPTOMYCES GRISEUS (bacteria)
#3: DNA chain 5'-D(*CP*CP*GP*GP*TP*GP*TP*GP*AP*AP*GP*AP*CP*CP *CP*AP*CP*GP*CP*AP*TP*CP)-3'


Mass: 6722.343 Da / Num. of mol.: 2 / Source method: obtained synthetically
Details: 2 COPIES OF PRE-CLEAVED DNA CONTAINING STICKY ENDS AND THE SGRAI RECOGNITION SEQUENCE
Source: (synth.) STREPTOMYCES GRISEUS (bacteria)

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: HELICAL ARRAY / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: HELICAL ASYMMETRIC UNIT OF AN SGRAI DNA-BOUND DIMER / Type: COMPLEX
Buffer solutionName: 10 MM TRIS-HCL PH 8.0, 150 MM NACL, 5 MM CA(OAC) 2, 0.5 MM DTT
pH: 8
Details: 10 MM TRIS-HCL PH 8.0, 150 MM NACL, 5 MM CA(OAC) 2, 0.5 MM DTT
SpecimenConc.: 0.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: HOLEY CARBON
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE
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.

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI F20 / Date: Sep 19, 2012
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 60000 X / Calibrated magnification: 41000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm / Cs: 2 mm
Image recordingElectron dose: 16 e/Å2 / Film or detector model: DIRECT ELECTRON DE-12 (4k x 3k)
Image scansNum. digital images: 656
Radiation wavelengthRelative weight: 1

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Processing

EM software
IDNameCategory
1DireXmodel fitting
2FREALIGN3D reconstruction
3Xmipp3D reconstruction
CTF correctionDetails: INDIVIDUAL PARTICLES
3D reconstructionMethod: PROJECTION-MATCHING / Resolution: 8.6 Å / Num. of particles: 1918 / Nominal pixel size: 1.42 Å / Actual pixel size: 1.42 Å / Magnification calibration: CATALASE CRYSTALS
Details: WE FIRST CONDUCTED 25 ITERATIONS OF THE IHRSR ROUTINE TO REFINE THE FULL CRYODATA SET AND OBTAIN FINAL HELICAL PARAMETERS -- -86.2 HELICAL TWIST AND 21.6 A RISE. SUBSEQUENTLY, THE MODEL FROM ...Details: WE FIRST CONDUCTED 25 ITERATIONS OF THE IHRSR ROUTINE TO REFINE THE FULL CRYODATA SET AND OBTAIN FINAL HELICAL PARAMETERS -- -86.2 HELICAL TWIST AND 21.6 A RISE. SUBSEQUENTLY, THE MODEL FROM IHRSR WAS USED FOR REFINEMENT IN FREALIGN SPECIFYING DIFFERENT DOSE- -FRACTIONATED STACKS FOR THE REFINEMENT (32 E- PER A2) AND RECONSTRUCTION (16 E- PER A2) ROUTINES. THE FINAL RECONSTRUCTION WAS OBTAINED FROM 1,918 FILAMENT SEGMENTS, AVERAGED 8 TIMES TO ACCOUNT FOR THE HELICAL SYMMETRY BASES WERE OMITTED FROM THE FITTED DNA. ONLY THE PHOSPHATE-SUGAR BACKBONE IS SHOWN. SUBMISSION BASED ON EXPERIMENTAL DATA FROM EMDB EMD-2441. (DEPOSITION ID: 11901).
Symmetry type: HELICAL
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Details: METHOD--DIREX REFINEMENT PROTOCOL--FLEXIBLE
Atomic model buildingPDB-ID: 3DVO

3dvo

RefinementHighest resolution: 8.6 Å
Refinement stepCycle: LAST / Highest resolution: 8.6 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms666 868 0 0 1534

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