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- EMDB-20447: Cryo-EM structure of AdnAB-AMPPNP-DNA complex -

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Basic information

Entry
Database: EMDB / ID: EMD-20447
TitleCryo-EM structure of AdnAB-AMPPNP-DNA complex
Map dataAdnAB-AMPPNP-DNA complex
Sample
  • Complex: AdnAB-AMPPNP-DNA complex
    • Complex: UvrD/REP helicase
    • Complex: ATP-dependent DNA helicase (UvrD/REP)
    • Complex: DNA
    • Protein or peptide: UvrD/REP helicase
    • Protein or peptide: ATP-dependent DNA helicase (UvrD/REP)
    • DNA: DNA (29-MER)
  • Ligand: MAGNESIUM ION
  • Ligand: IRON/SULFUR CLUSTERIron–sulfur cluster
KeywordsDNA / DNA BINDING PROTEIN / DNA BINDING PROTEIN-DNA complex
Function / homology
Function and homology information


DNA 3'-5' helicase / DNA helicase activity / DNA helicase / hydrolase activity / DNA repair / DNA binding / ATP binding
Similarity search - Function
: / PD-(D/E)XK endonuclease-like domain, AddAB-type / PD-(D/E)XK nuclease superfamily / DExx box DNA helicase domain superfamily / UvrD-like DNA helicase C-terminal domain profile. / UvrD-like DNA helicase, C-terminal / UvrD-like helicase C-terminal domain / UvrD/REP helicase N-terminal domain / UvrD-like helicase, ATP-binding domain / UvrD-like DNA helicase ATP-binding domain profile. ...: / PD-(D/E)XK endonuclease-like domain, AddAB-type / PD-(D/E)XK nuclease superfamily / DExx box DNA helicase domain superfamily / UvrD-like DNA helicase C-terminal domain profile. / UvrD-like DNA helicase, C-terminal / UvrD-like helicase C-terminal domain / UvrD/REP helicase N-terminal domain / UvrD-like helicase, ATP-binding domain / UvrD-like DNA helicase ATP-binding domain profile. / DNA helicase, UvrD/REP type / PD-(D/E)XK endonuclease-like domain superfamily / Restriction endonuclease type II-like / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
DNA helicase / DNA 3'-5' helicase / DNA 3'-5' helicase
Similarity search - Component
Biological speciesMycolicibacterium smegmatis (bacteria) / Bacillus subtilis subsp. subtilis str. 168 (bacteria) / Mycobacterium smegmatis (strain ATCC 700084 / mc(2)155) (bacteria) / Mycobacterium smegmatis (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsJia N / Unciuleac M / Shuman S / Patel DJ
CitationJournal: Proc Natl Acad Sci U S A / Year: 2019
Title: Structures and single-molecule analysis of bacterial motor nuclease AdnAB illuminate the mechanism of DNA double-strand break resection.
Authors: Ning Jia / Mihaela C Unciuleac / Chaoyou Xue / Eric C Greene / Dinshaw J Patel / Stewart Shuman /
Abstract: Mycobacterial AdnAB is a heterodimeric helicase-nuclease that initiates homologous recombination by resecting DNA double-strand breaks (DSBs). The AdnA and AdnB subunits are each composed of an N- ...Mycobacterial AdnAB is a heterodimeric helicase-nuclease that initiates homologous recombination by resecting DNA double-strand breaks (DSBs). The AdnA and AdnB subunits are each composed of an N-terminal motor domain and a C-terminal nuclease domain. Here we report cryoelectron microscopy (cryo-EM) structures of AdnAB in three functional states: in the absence of DNA and in complex with forked duplex DNAs before and after cleavage of the 5' single-strand DNA (ssDNA) tail by the AdnA nuclease. The structures reveal the path of the 5' ssDNA through the AdnA nuclease domain and the mechanism of 5' strand cleavage; the path of the 3' tracking strand through the AdnB motor and the DNA contacts that couple ATP hydrolysis to mechanical work; the position of the AdnA iron-sulfur cluster subdomain at the Y junction and its likely role in maintaining the split trajectories of the unwound 5' and 3' strands. Single-molecule DNA curtain analysis of DSB resection reveals that AdnAB is highly processive but prone to spontaneous pausing at random sites on duplex DNA. A striking property of AdnAB is that the velocity of DSB resection slows after the enzyme experiences a spontaneous pause. Our results highlight shared as well as distinctive properties of AdnAB vis-à-vis the RecBCD and AddAB clades of bacterial DSB-resecting motor nucleases.
History
DepositionJul 8, 2019-
Header (metadata) releaseJul 24, 2019-
Map releaseNov 20, 2019-
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: 0.02
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6ppu
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20447.png

Downloads & links

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Map

FileDownload / File: emd_20447.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationAdnAB-AMPPNP-DNA complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.86 Å/pix.
x 280 pix.
= 241.164 Å		0.86 Å/pix.
x 280 pix.
= 241.164 Å		0.86 Å/pix.
x 280 pix.
= 241.164 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.8613 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.02 / Movie #1: 0.02
Minimum - Maximum-0.08700995 - 0.2261253
Average (Standard dev.)0.0001757072 (±0.0032835205)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 241.164 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.86130.86130.8613
M x/y/z280280280
origin x/y/z0.0000.0000.000
length x/y/z241.164241.164241.164
α/β/γ90.00090.00090.000
start NX/NY/NZ929262
NX/NY/NZ290290360
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS280280280
D min/max/mean-0.0870.2260.000

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Supplemental data

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Sample components

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Entire : AdnAB-AMPPNP-DNA complex

EntireName: AdnAB-AMPPNP-DNA complex
Components
  • Complex: AdnAB-AMPPNP-DNA complex
    • Complex: UvrD/REP helicase
    • Complex: ATP-dependent DNA helicase (UvrD/REP)
    • Complex: DNA
    • Protein or peptide: UvrD/REP helicase
    • Protein or peptide: ATP-dependent DNA helicase (UvrD/REP)
    • DNA: DNA (29-MER)
  • Ligand: MAGNESIUM ION
  • Ligand: IRON/SULFUR CLUSTERIron–sulfur cluster

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Supramolecule #1: AdnAB-AMPPNP-DNA complex

SupramoleculeName: AdnAB-AMPPNP-DNA complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Molecular weightTheoretical: 200 KDa

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Supramolecule #2: UvrD/REP helicase

SupramoleculeName: UvrD/REP helicase / type: complex / ID: 2 / Parent: 1
Source (natural)Organism: Mycolicibacterium smegmatis (bacteria)

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Supramolecule #3: ATP-dependent DNA helicase (UvrD/REP)

SupramoleculeName: ATP-dependent DNA helicase (UvrD/REP) / type: complex / ID: 3 / Parent: 1
Source (natural)Organism: Mycolicibacterium smegmatis (bacteria)

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Supramolecule #4: DNA

SupramoleculeName: DNA / type: complex / ID: 4 / Parent: 1
Source (natural)Organism: Bacillus subtilis subsp. subtilis str. 168 (bacteria)

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Macromolecule #1: UvrD/REP helicase

MacromoleculeName: UvrD/REP helicase / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mycobacterium smegmatis (strain ATCC 700084 / mc(2)155) (bacteria)
Strain: ATCC 700084 / mc(2)155
Molecular weightTheoretical: 118.128547 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MTQVASPVVQ ARYSPVELSA ALGLFPPTDE QAAVIAAPPG PLVVIAGAGA GKTETMAARV VWLVANGFAT PSQVLGLTFT RKAAGQLLR RVRTRLARLA GAGLAPGSGA SDESATVSTY HAFAGTLLRE HGLLLPVEPD TRLLSETELW QLAYDVVCAH P GHLDTEKT ...String:
MTQVASPVVQ ARYSPVELSA ALGLFPPTDE QAAVIAAPPG PLVVIAGAGA GKTETMAARV VWLVANGFAT PSQVLGLTFT RKAAGQLLR RVRTRLARLA GAGLAPGSGA SDESATVSTY HAFAGTLLRE HGLLLPVEPD TRLLSETELW QLAYDVVCAH P GHLDTEKT PAAVTAMVLR LSGALAEHLV DTDQLRDTHV ELERLVHTLP AGPYQRDRGP SQWLLRMLAT QTERTELVPL ID ALHQRMR AEKVMDFGMQ MAAAARLAAR FPQVGEQLRQ RFRVVLLDEY QDTGHAQRIA LSSLFGGGAD DGLALTAVGD PIQ SIYGWR GASATNLPRF TTDFPYSDGT PAPTLELRTS WRNPPSTLHV ANAVSEEARR RSVAVRALRP RPDAEPGTIR CALL NNVAA ERDWVADHLA RAYHGAIGRG EAAPTAAVLV RRNADAAPMA EALTARGVPV EVVGVAGLLA VPEVADLVAM LRLIA DPTA GSAVMRILTG PRWRFGARDI AALWRRAVEL DDRPKGELGT ADIVAQAAPD ADTACVADAI CDPGDAERYS PAGYER IVA LGRELTMLRA HLGHPLPELV AEVRRVLGLD AEARAARPVA AGWAGTENLD RFSDLVSDFA GHAGASVSAL LAYLDAA VE VENGLAPAEL TVSHDRVQIL TVHAAKGLEW QVVAVPHLSA RVFPSTTQAR TWLTDASDLP PLLRGDRATE SEIGVPVL D TSDIYDRKIL SDKISDHKKS LDQRRVDEER RLLYVAITRA EDTLLLSGHH WGATESKPRG PSEFLCELKT ILEEATAAG TPCGEIEHWA PDPAPGETNP LRDQVVEALW PPVASADDHV HRGAQLVAAA MAGEVSAEAD QEGWAADVDA LLAERERPPQ QEDTELPGQ LSVSTLVELS RDPKAALTRL RRRLPQRPDP HALLGTTFHE WVQRYFHAER LFDLDDLPGA VDSDSGRAVE E SLAELQDA FVKSPWAART PVEVEVPFDM VLGETVVRGR IDAVFAEPDG TTMVLDWKTG DPPETPEAKE HAAVQLAVYR LA WAAMRGC PPESVRAAFH YVRSGQTVIP ETLPGAEELV KLLAAAPTET AEEADRIT

UniProtKB: DNA 3'-5' helicase

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Macromolecule #2: ATP-dependent DNA helicase (UvrD/REP)

MacromoleculeName: ATP-dependent DNA helicase (UvrD/REP) / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA helicase
Source (natural)Organism: Mycobacterium smegmatis (bacteria)
Molecular weightTheoretical: 76.049312 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK) ...String:
(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) GTVTVRLAAS THAEGTMIAD ALRRAHLVDG IPWSQMAVIV RSVPRVGTAL ARALTAAGV PVQDNGTDVP VGRQPAAAAL LTVLDVTATG HLDADSAVAL LTGPIGRVDP VTLRQLRRAL RRADGSQPPR D FGDLLVDA IEREPKGLSA EHARTLRRLR AVLTAARRSD ASGADPRYTL WQAWHASGLQ RRWLAASERG GSVGAQADRD LD AVTTLFD VADQYVNRTA GASLRGLVDH VTRLGAAVAR TEPETAAEAV AVLSVHGALA GEWDFVVIAG VQEGLWPNMI PRG GVLGTQ HLVDVLDGVA DMTDRTVSTR APLVAEERRL LMAAMGRART RVMITAVDS(UNK) (UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)PPLVAPR VLAPSALVGR LRAVVCAPDG AVDDDARACA AAQ LARLAA AGVPGADPSQ WHAMTSLTTE EPLWSEPGHV VTLSPSTLQM LTDCPLRWLL ERHGGDDGRD VRSTVGSLVH ALVS EPGKT ESQLVNELEK VWDDLPYDAK WYSDNELARH RAMLETFTRW REDTRRQLTE VATEIPVEGI VVEPGENTPG VRVRG RLDR LERDEAGRLV VVDLKTGKSP VTKDDAQNHA QLAMYQLAVA AGLLDDGDEP GGGKLVYLGK AGAAGATERE QDPLTP DKR AEWLETVGEA AAATAGPRFV ARVNNGCANC PVRSSCPAQA NGDRP

UniProtKB: DNA helicase, DNA helicase

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Macromolecule #3: DNA (29-MER)

MacromoleculeName: DNA (29-MER) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Bacillus subtilis subsp. subtilis str. 168 (bacteria)
Molecular weightTheoretical: 21.477703 KDa
SequenceString: (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DC)(DT)(DA) (DA)(DT)(DG)(DC)(DG)(DA)(DG)(DC)(DA)(DC) (DT)(DG)(DC)(DT)(DA)(DT)(DT)(DC)(DC) (DC)(DT)(DA)(DG)(DC)(DA)(DG)(DT)(DG)(DC) (DT) (DC)(DG)(DC)(DA)(DT)(DT) ...String:
(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DC)(DT)(DA) (DA)(DT)(DG)(DC)(DG)(DA)(DG)(DC)(DA)(DC) (DT)(DG)(DC)(DT)(DA)(DT)(DT)(DC)(DC) (DC)(DT)(DA)(DG)(DC)(DA)(DG)(DT)(DG)(DC) (DT) (DC)(DG)(DC)(DA)(DT)(DT)(DA)(DG) (DA)(DT)(DT)(DT)(DT)(DG)(DT)(DT)(DT)(DT) (DT)(DT) (DT)(DA)(DG)(DC)(DG)(DG)(DT) (DT)(DT)(DT)

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #5: IRON/SULFUR CLUSTER

MacromoleculeName: IRON/SULFUR CLUSTER / type: ligand / ID: 5 / Number of copies: 1 / Formula: SF4
Molecular weightTheoretical: 351.64 Da
Chemical component information

ChemComp-FS1:
IRON/SULFUR CLUSTER / Iron–sulfur cluster

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1.5 mg/mL
BufferpH: 7.5 / Component - Formula: Tris / Details: 20 mM Tris-HCl, pH 7.5, 150 mM NaCl
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 2.16 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1) / Number images used: 60108
FSC plot (resolution estimation)

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