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- PDB-6s6v: Resting state of the E. coli Mre11-Rad50 (SbcCD) head complex bou... -

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Basic information

Entry
Database: PDB / ID: 6s6v
TitleResting state of the E. coli Mre11-Rad50 (SbcCD) head complex bound to ATPgS
Components
  • Nuclease SbcCD subunit C
  • Nuclease SbcCD subunit D
KeywordsDNA BINDING PROTEIN / Nuclease / DNA repair / ABC-type ATPase / DNA double-strand breaks
Function / homology
Function and homology information


double-stranded DNA endonuclease activity / DNA replication termination / DNA exonuclease activity / single-stranded DNA endodeoxyribonuclease activity / 3'-5'-DNA exonuclease activity / DNA repair complex / exonuclease activity / 3'-5' exonuclease activity / double-strand break repair / endonuclease activity ...double-stranded DNA endonuclease activity / DNA replication termination / DNA exonuclease activity / single-stranded DNA endodeoxyribonuclease activity / 3'-5'-DNA exonuclease activity / DNA repair complex / exonuclease activity / 3'-5' exonuclease activity / double-strand break repair / endonuclease activity / DNA recombination / DNA replication / DNA repair / ATP hydrolysis activity / DNA binding / ATP binding
Similarity search - Function
Nuclease SbcC, gammaproteobacteria type / Nuclease SbcCD subunit D, C-terminal domain / Type 5 capsule protein repressor C-terminal domain / Nuclease SbcCD subunit D / : / SbcC/RAD50-like, Walker B motif / Mre11 nuclease, N-terminal metallophosphatase domain / Rad50/SbcC-type AAA domain / AAA domain / Calcineurin-like phosphoesterase domain, ApaH type ...Nuclease SbcC, gammaproteobacteria type / Nuclease SbcCD subunit D, C-terminal domain / Type 5 capsule protein repressor C-terminal domain / Nuclease SbcCD subunit D / : / SbcC/RAD50-like, Walker B motif / Mre11 nuclease, N-terminal metallophosphatase domain / Rad50/SbcC-type AAA domain / AAA domain / Calcineurin-like phosphoesterase domain, ApaH type / Calcineurin-like phosphoesterase / Metallo-dependent phosphatase-like / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / : / Nuclease SbcCD subunit C / Nuclease SbcCD subunit D / Nuclease SbcCD subunit D / Nuclease SbcCD subunit C
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsKaeshammer, L. / Saathoff, J.H. / Gut, F. / Bartho, J. / Alt, A. / Kessler, B. / Lammens, K. / Hopfner, K.P.
Funding support Germany, 2items
OrganizationGrant numberCountry
European Research Council
German Research Foundation Germany
CitationJournal: Mol Cell / Year: 2019
Title: Mechanism of DNA End Sensing and Processing by the Mre11-Rad50 Complex.
Authors: Lisa Käshammer / Jan-Hinnerk Saathoff / Katja Lammens / Fabian Gut / Joseph Bartho / Aaron Alt / Brigitte Kessler / Karl-Peter Hopfner /
Abstract: DNA double-strand breaks (DSBs) threaten genome stability throughout life and are linked to tumorigenesis in humans. To initiate DSB repair by end joining or homologous recombination, the Mre11- ...DNA double-strand breaks (DSBs) threaten genome stability throughout life and are linked to tumorigenesis in humans. To initiate DSB repair by end joining or homologous recombination, the Mre11-nuclease Rad50-ATPase complex detects and processes diverse and obstructed DNA ends, but a structural mechanism is still lacking. Here we report cryo-EM structures of the E. coli Mre11-Rad50 homolog SbcCD in resting and DNA-bound cutting states. In the resting state, Mre11's nuclease is blocked by ATP-Rad50, and the Rad50 coiled coils appear flexible. Upon DNA binding, the two coiled coils zip up into a rod and, together with the Rad50 nucleotide-binding domains, form a clamp around dsDNA. Mre11 moves to the side of Rad50, binds the DNA end, and assembles a DNA cutting channel for the nuclease reactions. The structures reveal how Mre11-Rad50 can detect and process diverse DNA ends and uncover a clamping and gating function for the coiled coils.
History
DepositionJul 3, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 4, 2019Provider: repository / Type: Initial release
Revision 1.1Sep 18, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed ..._citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name
Revision 1.2Nov 20, 2019Group: Database references / Derived calculations / Category: citation / pdbx_struct_conn_angle / struct_conn / Item: _citation.journal_volume / _citation.page_first

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Structure visualization

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Assembly

Deposited unit
A: Nuclease SbcCD subunit D
B: Nuclease SbcCD subunit D
C: Nuclease SbcCD subunit C
D: Nuclease SbcCD subunit C
hetero molecules


Theoretical massNumber of molelcules
Total (without water)330,31812
Polymers329,0044
Non-polymers1,3158
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area16260 Å2
ΔGint-115 kcal/mol
Surface area59740 Å2
MethodPISA

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Components

#1: Protein Nuclease SbcCD subunit D


Mass: 45650.297 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli)
Gene: sbcD, A9R57_08515, ACU90_20930, AM270_02320, AM464_18970, AMK83_08850, AML07_05375, APZ14_19360, ARC77_21640, AU473_24540, AUQ13_18520, AUS26_08295, AW106_10255, AWF80_027335, B1K96_09895, ...Gene: sbcD, A9R57_08515, ACU90_20930, AM270_02320, AM464_18970, AMK83_08850, AML07_05375, APZ14_19360, ARC77_21640, AU473_24540, AUQ13_18520, AUS26_08295, AW106_10255, AWF80_027335, B1K96_09895, BANRA_00933, BANRA_02010, BANRA_03599, BB545_15570, BHS81_02440, BHS87_02165, BJJ90_20270, BK292_14605, BMT53_08385, BMT91_05280, BN17_02021, BTQ04_02895, BTQ06_17755, BUE81_15560, BVL39_00845, BW690_06610, BWP17_02185, C2U48_08075, C4J69_09475, C5N07_12775, C5P01_16270, C5P43_02310, C6986_02395, C7235_18845, C7B08_06215, C9E25_04545, CA593_01150, CG691_03340, CG705_03245, CG706_02645, COD30_16020, COD46_04825, CR538_19460, CRE06_07850, CVH05_20670, CWS33_22910, D0X26_07455, D2184_11480, D3821_12865, D3822_22430, D3O91_15270, D3Y67_14385, D4M06_05505, D7K63_09095, D7K66_05465, D9H94_07055, D9I87_03345, D9I97_02300, D9J44_05860, D9L89_06325, D9X97_05375, DIV22_26320, DL455_04405, DL545_19095, DL800_03920, DMI04_05300, DMZ31_02500, DNB37_05400, DNQ41_05900, DOY56_05885, DQF57_07710, DQO13_05535, DS732_06955, DTL43_07380, E2855_00535, E2863_00431, EAI44_12705, EAI52_02980, EB509_06820, EB510_01660, EB515_09310, EC1094V2_3455, EC3234A_4c00670, EC3426_01235, EC95NR1_04617, ECs0448, ED225_07565, ED287_10145, ED600_06285, ED607_18305, ED611_06615, ED903_13690, ED944_09545, EEA45_02820, EEP23_01080, EF173_10600, EFV01_15655, EFV11_06690, EFV17_08300, EIA13_09680, EL75_3353, EL79_3448, EL80_3402, ERS085365_01835, ERS085379_01210, ERS085416_01963, ERS139211_01257, ERS150873_01853, ERS150876_00572, FORC28_4694, HW43_05600, NCTC10090_01877, NCTC10429_03756, NCTC11022_05144, NCTC13125_01885, NCTC13127_05149, NCTC13462_01931, NCTC8009_07006, NCTC8179_01650, NCTC8960_01309, NCTC9036_03833, NCTC9037_03951, NCTC9045_04425, NCTC9055_00697, NCTC9058_03126, NCTC9062_04472, NCTC9111_03972, NCTC9703_03216, NCTC9706_01107, PU06_02925, RG28_02875, RK56_026670, RX35_02069, SAMEA3446340_01461, SAMEA3472043_02687, SAMEA3472044_00460, SAMEA3472047_02108, SAMEA3472055_02158, SAMEA3472070_02276, SAMEA3472114_01252, SAMEA3484427_03538, SAMEA3484429_01894, SAMEA3752553_00281, SAMEA3752559_02892, SAMEA3753064_01241, SAMEA3753097_00507, SAMEA3753290_01730, SAMEA3753300_00463, SK85_00423, WR15_04330
Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: C3TMC7, UniProt: P0AG76*PLUS
#2: Protein Nuclease SbcCD subunit C


Mass: 118851.508 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli)
Gene: sbcC, A9R57_08510, ACU57_12010, AM464_18965, AUQ13_18525, BANRA_02011, BANRA_03598, BHS87_02160, BJJ90_20275, BUE81_15555, BW690_06605, C4J69_09480, C5N07_12770, C9E25_04550, CA593_01155, D0X26_ ...Gene: sbcC, A9R57_08510, ACU57_12010, AM464_18965, AUQ13_18525, BANRA_02011, BANRA_03598, BHS87_02160, BJJ90_20275, BUE81_15555, BW690_06605, C4J69_09480, C5N07_12770, C9E25_04550, CA593_01155, D0X26_07450, D3821_12870, D3Y67_14380, D9D20_11475, D9F57_05650, DNQ41_05895, EAI52_02975, EC3426_01234, EL75_3354, EL79_3449, EL80_3403, ERS085365_01836, ERS085416_01964, ERS139211_01256, ERS150873_01854, NCTC13462_01932, NCTC9037_03952, RK56_026675, SAMEA3446340_01460, SAMEA3472047_02107, SAMEA3484427_03539, SAMEA3484429_01893, SAMEA3752559_02893, SAMEA3753300_00462, SK85_00422
Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A037YDI0, UniProt: P13458*PLUS
#3: Chemical
ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mn
#4: Chemical ChemComp-AGS / PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-GAMMA-S / ADENOSINE 5'-(3-THIOTRIPHOSPHATE) / ADENOSINE 5'-(GAMMA-THIOTRIPHOSPHATE) / ADENOSINE-5'-DIPHOSPHATE MONOTHIOPHOSPHATE


Mass: 523.247 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H16N5O12P3S / Comment: ATP-gamma-S, energy-carrying molecule analogue*YM
#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
Has ligand of interestN

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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: Heterotetrameric complex of full-length Mre11-Rad50 in complex with ATPgS
Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil R2/1
VitrificationInstrument: LEICA PLUNGER / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 288 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
Image recordingElectron dose: 68 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 8186
Image scansMovie frames/image: 50

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 142229 / Symmetry type: POINT
Atomic model building
IDPDB-IDPdb chain-ID 3D fitting-ID
14M0V

4m0v

A1
24M0V

4m0v

B1
33QF7

3qf7

C1
43QF7

3qf7

D1

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