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- EMDB-25164: Cryo-EM structure of AdnA-AdnB(W325A) in complex with DNA and AMPPNP -

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Basic information

Entry
Database: EMDB / ID: EMD-25164
TitleCryo-EM structure of AdnA-AdnB(W325A) in complex with DNA and AMPPNP
Map data
Sample
  • Complex: Cryo-EM map of AdnA-AdnB(W325A) in complex with DNA and AMPPNP
    • Protein or peptide: DNA helicase
    • Protein or peptide: DNA helicase
    • DNA: DNA (70-MER)
  • Ligand: MAGNESIUM ION
  • Ligand: IRON/SULFUR CLUSTER
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
Keywordshomologous recombination / DNA end resection / cryoelectron microscopy / DNA curtain / DNA BINDING PROTEIN / DNA BINDING PROTEIN-DNA complex
Function / homology
Function and homology information


exonuclease activity / DNA helicase activity / DNA helicase / hydrolase activity / DNA repair / ATP hydrolysis activity / 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 helicase
Similarity search - Component
Biological speciesMycolicibacterium smegmatis (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsWang J / Warren GM
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)P30CA008748 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI64693 United States
CitationJournal: Nucleic Acids Res / Year: 2022
Title: Structure-activity relationships at a nucleobase-stacking tryptophan required for chemomechanical coupling in the DNA resecting motor-nuclease AdnAB.
Authors: Garrett M Warren / Aviv Meir / Juncheng Wang / Dinshaw J Patel / Eric C Greene / Stewart Shuman /
Abstract: Mycobacterial AdnAB is a heterodimeric helicase-nuclease that initiates homologous recombination by resecting DNA double-strand breaks. The AdnB subunit hydrolyzes ATP to drive single-nucleotide ...Mycobacterial AdnAB is a heterodimeric helicase-nuclease that initiates homologous recombination by resecting DNA double-strand breaks. The AdnB subunit hydrolyzes ATP to drive single-nucleotide steps of 3'-to-5' translocation of AdnAB on the tracking DNA strand via a ratchet-like mechanism. Trp325 in AdnB motif III, which intercalates into the tracking strand and makes a π stack on a nucleobase 5' of a flipped-out nucleoside, is the putative ratchet pawl without which ATP hydrolysis is mechanically futile. Here, we report that AdnAB mutants wherein Trp325 was replaced with phenylalanine, tyrosine, histidine, leucine, or alanine retained activity in ssDNA-dependent ATP hydrolysis but displayed a gradient of effects on DSB resection. The resection velocities of Phe325 and Tyr325 mutants were 90% and 85% of the wild-type AdnAB velocity. His325 slowed resection rate to 3% of wild-type and Leu325 and Ala325 abolished DNA resection. A cryo-EM structure of the DNA-bound Ala325 mutant revealed that the AdnB motif III peptide was disordered and the erstwhile flipped out tracking strand nucleobase reverted to a continuous base-stacked arrangement with its neighbors. We conclude that π stacking of Trp325 on a DNA nucleobase triggers and stabilizes the flipped-out conformation of the neighboring nucleoside that underlies formation of a ratchet pawl.
History
DepositionOct 18, 2021-
Header (metadata) releaseDec 22, 2021-
Map releaseDec 22, 2021-
UpdateJun 5, 2024-
Current statusJun 5, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.018
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.018
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7sjr
  • Surface level: 0.018
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_25164.png

Downloads & links

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Map

FileDownload / File: emd_25164.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.064 Å
Density
Contour LevelBy AUTHOR: 0.015 / Movie #1: 0.018
Minimum - Maximum-0.089118406 - 0.24863328
Average (Standard dev.)0.00001032289 (±0.0039714836)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 272.384 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0641.0641.064
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z272.384272.384272.384
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0890.2490.000

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Supplemental data

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Sample components

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Entire : Cryo-EM map of AdnA-AdnB(W325A) in complex with DNA and AMPPNP

EntireName: Cryo-EM map of AdnA-AdnB(W325A) in complex with DNA and AMPPNP
Components
  • Complex: Cryo-EM map of AdnA-AdnB(W325A) in complex with DNA and AMPPNP
    • Protein or peptide: DNA helicase
    • Protein or peptide: DNA helicase
    • DNA: DNA (70-MER)
  • Ligand: MAGNESIUM ION
  • Ligand: IRON/SULFUR CLUSTER
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER

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Supramolecule #1: Cryo-EM map of AdnA-AdnB(W325A) in complex with DNA and AMPPNP

SupramoleculeName: Cryo-EM map of AdnA-AdnB(W325A) in complex with DNA and AMPPNP
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Mycolicibacterium smegmatis (bacteria)

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Macromolecule #1: DNA helicase

MacromoleculeName: DNA helicase / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA helicase
Source (natural)Organism: Mycolicibacterium smegmatis (bacteria)
Molecular weightTheoretical: 118.013406 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
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 SIYGAR 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 helicase

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Macromolecule #2: DNA helicase

MacromoleculeName: DNA helicase / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA helicase
Source (natural)Organism: Mycolicibacterium smegmatis (bacteria)
Molecular weightTheoretical: 111.030656 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: SMTTRPAESA PQTASTLLEP GSNGVVRLLG GPGTGKSSLL VDTAVQHILA GADPESVLLL TGSARLRTAA RAAITARLLG AGTVGVVRE PLVRTVHSYA FAVLRLAAQR NGDPPPRLIT SAEQDGIIRE LLAGDLEDGH RSPVGWPEQL WPALTTAGFA T ELRDLMAR ...String:
SMTTRPAESA PQTASTLLEP GSNGVVRLLG GPGTGKSSLL VDTAVQHILA GADPESVLLL TGSARLRTAA RAAITARLLG AGTVGVVRE PLVRTVHSYA FAVLRLAAQR NGDPPPRLIT SAEQDGIIRE LLAGDLEDGH RSPVGWPEQL WPALTTAGFA T ELRDLMAR CTERGVDPIA LQRLGRTAKR PEWLAAGRFA QAYEQIMLLR SAVGMAAPQA TVPALGAAEL VGAALEALGA DD ELLDTER NRIKLLLVDD AQHLDPQAAR LVRALAAGTG LTVIAGDPDQ SVFGYRGADP VLLRDDTHPA ITLTQSYRCA PEI ASAITG LGQRLPGVSD TRHWTGNPQR EGTVTVRLAA STHAEGTMIA DALRRAHLVD GIPWSQMAVI VRSVPRVGTA LARA LTAAG VPVQDNGTDV PVGRQPAAAA LLTVLDVTAT GHLDADSAVA LLTGPIGRVD PVTLRQLRRA LRRADGSQPP RDFGD LLVD AIEREPKGLS AEHARTLRRL RAVLTAARRS DASGADPRYT LWQAWHASGL QRRWLAASER GGSVGAQADR DLDAVT TLF DVADQYVNRT AGASLRGLVD HVTRLGAAVA RTEPETAAEA VAVLSVHGAL AGEWDFVVIA GVQEGLWPNM IPRGGVL GT QHLVDVLDGV ADMTDRTVST RAPLVAEERR LLMAAMGRAR TRVMITAVDS DTGDESLLPS PFCAEISAWA TEPVAEPP L VAPRVLAPSA LVGRLRAVVC APDGAVDDDA RACAAAQLAR LAAAGVPGAD PSQWHAMTSL TTEEPLWSEP GHVVTLSPS TLQMLTDCPL RWLLERHGGD DGRDVRSTVG SLVHALVSEP GKTESQLVNE LEKVWDDLPY DAKWYSDNEL ARHRAMLETF TRWREDTRR QLTEVATEIP VEGIVVEPGE NTPGVRVRGR LDRLERDEAG RLVVVDLKTG KSPVTKDDAQ NHAQLAMYQL A VAAGLLDD GDEPGGGKLV YLGKAGAAGA TEREQDPLTP DKRAEWLETV GEAAAATAGP RFVARVNNGC ANCPVRSSCP AQ ANGDRP

UniProtKB: DNA helicase

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Macromolecule #3: DNA (70-MER)

MacromoleculeName: DNA (70-MER) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Mycolicibacterium smegmatis (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

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Macromolecule #6: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / type: ligand / ID: 6 / 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

BufferpH: 8
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 %

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 53.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6ppj

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 98408
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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Atomic model buiding 1

Initial modelPDB ID:

6ppj


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-7sjr:
Cryo-EM structure of AdnA-AdnB(W325A) in complex with DNA and AMPPNP

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