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- EMDB-20440: Cryo-EM structure of AdnA(D934A)-AdnB(D1014A) in complex with AMPPNP -

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Basic information

Entry
Database: EMDB / ID: EMD-20440
TitleCryo-EM structure of AdnA(D934A)-AdnB(D1014A) in complex with AMPPNP
Map dataAdnA(D934A)-AdnB(D1014A) in complex with AMPPNP
Sample
  • Complex: AdnAB-D934A-D1014A mutant in complex with AMPPNP
    • Protein or peptide: UvrD/REP helicase
    • Protein or peptide: ATP-dependent DNA helicase (UvrD/REP)
  • Ligand: IRON/SULFUR CLUSTER
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
KeywordsDNA BINDING PROTEIN
Function / homology
Function and homology information


DNA 3'-5' helicase / exonuclease activity / DNA helicase activity / isomerase 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 / PD-(D/E)XK endonuclease-like domain superfamily / UvrD-like helicase, ATP-binding domain ...: / 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 / PD-(D/E)XK endonuclease-like domain superfamily / UvrD-like helicase, ATP-binding domain / UvrD-like DNA helicase ATP-binding domain profile. / DNA helicase, UvrD/REP type / Restriction endonuclease type II-like / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
DNA helicase / DNA helicase / DNA 3'-5' helicase / DNA 3'-5' helicase
Similarity search - Component
Biological speciesMycolicibacterium smegmatis (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 7, 2019-
Header (metadata) releaseNov 20, 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: 0.015
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.015
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6ppj
  • Surface level: 0.015
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20440.png

Downloads & links

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Map

FileDownload / File: emd_20440.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationAdnA(D934A)-AdnB(D1014A) in complex with AMPPNP
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: 0.01 / Movie #1: 0.015
Minimum - Maximum-0.06802275 - 0.17296569
Average (Standard dev.)0.00019601877 (±0.0030465727)
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-0.0680.1730.000

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Supplemental data

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Sample components

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Entire : AdnAB-D934A-D1014A mutant in complex with AMPPNP

EntireName: AdnAB-D934A-D1014A mutant in complex with AMPPNP
Components
  • Complex: AdnAB-D934A-D1014A mutant in complex with AMPPNP
    • Protein or peptide: UvrD/REP helicase
    • Protein or peptide: ATP-dependent DNA helicase (UvrD/REP)
  • Ligand: IRON/SULFUR CLUSTER
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER

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Supramolecule #1: AdnAB-D934A-D1014A mutant in complex with AMPPNP

SupramoleculeName: AdnAB-D934A-D1014A mutant in complex with AMPPNP / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Mycolicibacterium smegmatis (bacteria)
Molecular weightTheoretical: 200 KDa

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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 / EC number: DNA helicase
Source (natural)Organism: Mycobacterium smegmatis (bacteria)
Molecular weightTheoretical: 114.735938 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 DTDQLR(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)LLRML ATQTERTELV PLIDALHQRM RAEKVMDFGM QMAAAARLAA RFPQVGEQLR QRFRVVLLDE YQDTG HAQR IALSSLFGGG ADDGLALTAV GDPIQSIYGW RGASATNLPR FTTDFPYSDG TPAPTLELRT (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)TIRCALLNN VAAERDWVAD HLARAYHGAI GRGEAAPTAA VLVRRNADAA PMAEALTARG VPVEVV GVA GLLAVPEVAD LVAMLRLIAD PTAGSAVMRI LTGPRWRFGA RDIAALWRRA VELDDRPKGE LGTADIVAQA APDADTA CV ADAICDPGDA ERYSPAGYER IVALGRELTM LRAHLGHPLP ELVAEVRRVL GLDAEARAAR PVAAGWAGTE NLDRFSDL V SDFAGHAGAS VSALLAYLDA AVEVENGLAP AELTVSHDRV QILTVHAAKG LEWQVVAVPH LSARVFPSTT QARTWLTDA SDLPPLLRGD RATESEIGVP VLDTSDIYDR KILSDKISDH KKSLDQRRVD EERRLLYVAI TRAEDTLLLS GHHWGATESK PRGPSEFLC ELKTILEEAT AAGTPCGEIE HWAPDPAPGE TNPLRDQVVE ALWPPVASAD DHVHRGAQLV AAAMAGEVSA E ADQEGWAA DVDALLAERE RP(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)(UNK)(UNK)(UNK)HALLG TTFHEWVQRY FHAERLFDL DDLPGAVDSD SGRAVEESLA ELQDAFVKSP WAARTPVEVE VPFDMVLGET VVRGRIDAVF AEPDGTTMVL A WKTGDPPE TPEAKEHAAV QLAVYRLAWA AMRGCPPESV RAAFHYVRSG (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)LA AAPTETAEEA DRIT

UniProtKB: DNA helicase, DNA helicase, DNA helicase, DNA 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: 110.899562 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MTTRPAESAP QTASTLLEPG SNGVVRLLGG PGTGKSSLLV DTAVQHILAG ADPESVLLLT GSARLRTAAR AAITARLLGA GTVGVVREP LVRTVHSYAF AVLRLAAQRN GDPPPRLITS AEQDGIIREL LAGDLEDGHR SPVGWPEQLW PALTTAGFAT E LRDLMARC ...String:
MTTRPAESAP QTASTLLEPG SNGVVRLLGG PGTGKSSLLV DTAVQHILAG ADPESVLLLT GSARLRTAAR AAITARLLGA GTVGVVREP LVRTVHSYAF AVLRLAAQRN GDPPPRLITS AEQDGIIREL LAGDLEDGHR SPVGWPEQLW PALTTAGFAT E LRDLMARC TERGVDPIAL QRLGRTAKRP EWLAAGRFAQ AYEQIMLLRS AVGMAAPQAT VPALGAAELV GAALEALGAD DE LLDTERN RIKLLLVDDA QHLDPQAARL VRALAAGTGL TVIAGDPDQS VFGYRGADPV LLRDDTHPAI TLTQSYRCAP EIA SAITGL GQRLPGVSDT RHWTGNPQRE GTVTVRLAAS THAEGTMIAD ALRRAHLVDG IPWSQMAVIV RSVPRVGTAL ARAL TAAGV PVQDNGTDVP VGRQPAAAAL LTVLDVTATG HLDADSAVAL LTGPIGRVDP VTLRQLRRAL RRADGSQPPR DFGDL LVDA IEREPKGLSA EHARTLRRLR AVLTAARRSD ASGADPRYTL WQAWHASGLQ RRWLAASERG GSVGAQADRD LDAVTT LFD VADQYVNRTA GASLRGLVDH VTRLGAAVAR TEPETAAEAV AVLSVHGALA GEWDFVVIAG VQEGLWPNMI PRGGVLG TQ HLVDVLDGVA DMTDRTVSTR APLVAEERRL LMAAMGRART RVMITAVDSD TGDESLLPSP FCAEISAWAT EPVAEPPL V APRVLAPSAL VGRLRAVVCA PDGAVDDDAR ACAAAQLARL AAAGVPGADP SQWHAMTSLT TEEPLWSEPG HVVTLSPST LQMLTDCPLR WLLERHGGDD GRDVRSTVGS LVHALVSEPG KTESQLVNEL EKVWDDLPYD AKWYSDNELA RHRAMLETFT RWREDTRRQ LTEVATEIPV EGIVVEPGEN TPGVRVRGRL DRLERDEAGR LVVVALKTGK SPVTKDDAQN HAQLAMYQLA V AAGLLDDG DEPGGGKLVY LGKAGAAGAT EREQDPLTPD KRAEWLETVG EAAAATAGPR FVARVNNGCA NCPVRSSCPA QA NGDRP

UniProtKB: DNA helicase

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Macromolecule #3: IRON/SULFUR CLUSTER

MacromoleculeName: IRON/SULFUR CLUSTER / type: ligand / ID: 3 / Number of copies: 1 / Formula: SF4
Molecular weightTheoretical: 351.64 Da
Chemical component information

ChemComp-FS1:
IRON/SULFUR CLUSTER

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Macromolecule #4: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / type: ligand / ID: 4 / Number of copies: 1 / Formula: ANP
Molecular weightTheoretical: 506.196 Da
Chemical component information

ChemComp-ANP:
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / AMP-PNP, energy-carrying molecule analogue*YM

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

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Image processing

Startup modelType of model: PDB ENTRY
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: 80396
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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