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- EMDB-24295: Cryo-EM structure of DNMT5 quaternary complex with hemimethylated... -

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Basic information

Entry
Database: EMDB / ID: EMD-24295
TitleCryo-EM structure of DNMT5 quaternary complex with hemimethylated DNA, AMP-PNP and SAH
Map dataEM structure of DNMT5 quaternary complex with hemimethylated DNA, AMP-PNP and SAH
Sample
  • Complex: DNMT5 in complex with hemimethylated DNA, AMP-PNP and SAH
    • DNA: DNA (5'-D(P*CP*AP*GP*(5CM)P*GP*CP*AP*T)-3')
    • DNA: DNA (5'-D(*TP*GP*CP*GP*CP*TP*GP*AP*CP*A)-3')
  • Protein or peptide: DNA repair protein Rad8
  • Ligand: S-ADENOSYL-L-HOMOCYSTEINE
  • Ligand: ZINC ION
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION
KeywordsCytosine-5 methyltransferase / SNF2 ATPase / TRANSFERASE / hemimethylated DNA / ATP / SAM / TRANSFERASE-DNA complex
Function / homology
Function and homology information


ATP-dependent DNA (cytosine-5-)-methyltransferase activity / DNA (cytosine-5-)-methyltransferase activity / DNA methylation-dependent constitutive heterochromatin formation / ATP-dependent chromatin remodeler activity / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / chromosome / sequence-specific DNA binding / methylation / chromatin binding / ATP hydrolysis activity ...ATP-dependent DNA (cytosine-5-)-methyltransferase activity / DNA (cytosine-5-)-methyltransferase activity / DNA methylation-dependent constitutive heterochromatin formation / ATP-dependent chromatin remodeler activity / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / chromosome / sequence-specific DNA binding / methylation / chromatin binding / ATP hydrolysis activity / ATP binding / nucleus
Similarity search - Function
: / C-5 cytosine methyltransferase / C-5 cytosine-specific DNA methylase / Chromo domain / Chromo (CHRromatin Organisation MOdifier) domain / Chromo and chromo shadow domain profile. / Chromo/chromo shadow domain / Chromatin organization modifier domain / : / SNF2-like, N-terminal domain superfamily ...: / C-5 cytosine methyltransferase / C-5 cytosine-specific DNA methylase / Chromo domain / Chromo (CHRromatin Organisation MOdifier) domain / Chromo and chromo shadow domain profile. / Chromo/chromo shadow domain / Chromatin organization modifier domain / : / SNF2-like, N-terminal domain superfamily / SNF2, N-terminal / SNF2-related domain / Chromo-like domain superfamily / Helicase conserved C-terminal domain / Superfamilies 1 and 2 helicase C-terminal domain profile. / Superfamilies 1 and 2 helicase ATP-binding type-1 domain profile. / DEAD-like helicases superfamily / Helicase, C-terminal / Helicase superfamily 1/2, ATP-binding domain / S-adenosyl-L-methionine-dependent methyltransferase superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
DNA (cytosine-5-)-methyltransferase DMT5
Similarity search - Component
Biological speciesCryptococcus neoformans (fungus) / Cryptococcus neoformans var. grubii serotype A (strain H99 / ATCC 208821 / CBS 10515 / FGSC 9487) (fungus)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsWang J / Patel DJ
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)P30CA008748 United States
CitationJournal: Mol Cell / Year: 2022
Title: Structural insights into DNMT5-mediated ATP-dependent high-fidelity epigenome maintenance.
Authors: Juncheng Wang / Sandra Catania / Chongyuan Wang / M Jason de la Cruz / Beiduo Rao / Hiten D Madhani / Dinshaw J Patel /
Abstract: Epigenetic evolution occurs over million-year timescales in Cryptococcus neoformans and is mediated by DNMT5, the first maintenance type cytosine methyltransferase identified in the fungal or protist ...Epigenetic evolution occurs over million-year timescales in Cryptococcus neoformans and is mediated by DNMT5, the first maintenance type cytosine methyltransferase identified in the fungal or protist kingdoms, the first dependent on adenosine triphosphate (ATP), and the most hemimethyl-DNA-specific enzyme known. To understand these novel properties, we solved cryo-EM structures of CnDNMT5 in three states. These studies reveal an elaborate allosteric cascade in which hemimethylated DNA binding first activates the SNF2 ATPase domain by a large rigid body rotation while the target cytosine partially flips out of the DNA duplex. ATP binding then triggers striking structural reconfigurations of the methyltransferase catalytic pocket to enable cofactor binding, completion of base flipping, and catalysis. Bound unmethylated DNA does not open the catalytic pocket and is instead ejected upon ATP binding, driving high fidelity. This unprecedented chaperone-like, enzyme-remodeling role of the SNF2 ATPase domain illuminates how energy is used to enable faithful epigenetic memory.
History
DepositionJun 24, 2021-
Header (metadata) releaseFeb 23, 2022-
Map releaseFeb 23, 2022-
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: 0.02
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.02
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  • Surface view with fitted model
  • Atomic models: PDB-7r78
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_24295.png

Downloads & links

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Map

FileDownload / File: emd_24295.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationEM structure of DNMT5 quaternary complex with hemimethylated DNA, AMP-PNP and SAH
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 256 pix.
= 272.384 Å		1.06 Å/pix.
x 256 pix.
= 272.384 Å		1.06 Å/pix.
x 256 pix.
= 272.384 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.064 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.02 / Movie #1: 0.02
Minimum - Maximum-0.07819214 - 0.14570029
Average (Standard dev.)0.000038415175 (±0.005042348)
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-0.0780.1460.000

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Supplemental data

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Sample components

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Entire : DNMT5 in complex with hemimethylated DNA, AMP-PNP and SAH

EntireName: DNMT5 in complex with hemimethylated DNA, AMP-PNP and SAH
Components
  • Complex: DNMT5 in complex with hemimethylated DNA, AMP-PNP and SAH
    • DNA: DNA (5'-D(P*CP*AP*GP*(5CM)P*GP*CP*AP*T)-3')
    • DNA: DNA (5'-D(*TP*GP*CP*GP*CP*TP*GP*AP*CP*A)-3')
  • Protein or peptide: DNA repair protein Rad8
  • Ligand: S-ADENOSYL-L-HOMOCYSTEINE
  • Ligand: ZINC ION
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION

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Supramolecule #1: DNMT5 in complex with hemimethylated DNA, AMP-PNP and SAH

SupramoleculeName: DNMT5 in complex with hemimethylated DNA, AMP-PNP and SAH
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #2-#3
Source (natural)Organism: Cryptococcus neoformans (fungus)
Molecular weightTheoretical: 260 KDa

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Macromolecule #1: DNA repair protein Rad8

MacromoleculeName: DNA repair protein Rad8 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Cryptococcus neoformans var. grubii serotype A (strain H99 / ATCC 208821 / CBS 10515 / FGSC 9487) (fungus)
Strain: H99 / ATCC 208821 / CBS 10515 / FGSC 9487
Molecular weightTheoretical: 263.811656 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MSYYHHHHHH DYDIPTTENL YFQGAMGSGD DWYEIDYIAD SRVIRRKGRQ ILQYLIHWAG YAVHERTWED EDGIGGEDCA LVQEFYRKN PGKPRLSPSS VRKEVKLARM VEVVITTRRI DGKSRAASST DQPSPHRLGI TSPQANNIGG EDPNPSLTRR P VRSTVSEI ...String:
MSYYHHHHHH DYDIPTTENL YFQGAMGSGD DWYEIDYIAD SRVIRRKGRQ ILQYLIHWAG YAVHERTWED EDGIGGEDCA LVQEFYRKN PGKPRLSPSS VRKEVKLARM VEVVITTRRI DGKSRAASST DQPSPHRLGI TSPQANNIGG EDPNPSLTRR P VRSTVSEI AKRPTSKKVH PNKKCKASSD DESDFVFEEG EWDEDEDDDN DVDFRSSEDD EDDEQERSAE EPESDEEIIK PA KKTKSSL PKAKLRPKPA NLGGFVTGVR PLNQGLDIKA AVRNMSDDLP PISDIEAMFD HLVSRIPDIV ELVRQLNGRK LRV ATMCSG TESPLLALNM IAKAIKAQHG LTLAFEHVFS CEIEPFKQAY IERNFTPPIL FRDVTELGKK RAHTAYGSMV DVPG DVDIL IAGTSCVDYS NLNNVQQDID ANGESGRTFR GMLQWVKKHQ PPIVILENVC NAPWDKVVEY FGQIDYDAQY TRLDT KEFY IPHTRTRVYL FATPSSSESD DLPEKWAQTV KDLRRPWSSP FEAFLLHTDD PNIHRARLEL ASARAQTDGT SRKTTD WNR CESRHQRARQ DEALGLLRPL TSWQEAGVCK GLDWTWNDWL LAQTERVVDL LEISTLRMAK DGIDSGFKAC IWNVSQN VD RQTGSSKTAL APCLTPNMIP WVTIRGGPVT GREALALQGI PVRELLLTSE NEDQLADLAG NAMTTTVVGS AMIAALKV A CHKITEGANP EKEAALILEK EAVDDEQVAN RIIGEDYLEH HDLDLAKVTK SNLSEILDLA CRSSRHCQCE GQSGTAPNI LECQECSYRA CKSCGGRPEH VYAPCANQRV EPAEFEKRFK GLLPMRVRIA GLTDQCLNAV RKAAEKSNKG SVNDNDWQLW STALLEGIH DAEFRFRYLK RQSTWTAVYE ARRAMLSLVL RNQIPEWRLT IKAPASEPNN SQLRALLLHP VARLQIDIAG Q DVLCGPWE LCIPSMKTID IEITGKGELL PSWQASLGLQ GPFANTTRWS EVEISLQAED ENTLDRKLSG TYQLLPRCGQ AM SSLHKKR PDLSDDGLPQ LYFFLDPTRC GESREDRYVF STSTERLDYG TERPVIARLD SHWREGNEKQ RKVKLDVSGA WVK CPEAHL TAIGGDDIAV VANDAAANEI HRDRATFAIP SSASAISASL TTEGCSHAMA LLSCRVPLDP THSESMWRRG AWAE IDLSH QGNTTFANLA WITERLPPLD GLKNWAHIAD DVSEHVCERC APRPPKIHWI KREGKANKKG NKTKSTIIAF EDKLE AGQY EHALKHRPSP FVVQLRLDQD IGSFRIGLNI VSLAHRALSR LPPTTSEHKI SLSWRLTPGH VTESPQPRRV FILPSN KQD PENSQPEAFK LPLRKEQLRS LWWMLEQEKA TGKTHTFVEE EISESLLPAV GWRAEGKAER PVMVRGGVIA DQVGYGK TV ISIALVAQTL SLPAPEPATP GLIDLKATLI VVPGHLSKQW PNEIARFTGS MFKVIVIQGM KDLQEKTIAE LGKADIIV M ASEIFESDVY WSRLEYLSAQ PREWLHDTQG GRFFCDRLDA AMESLVSQTK ILKEKGSEAA MRAMEDKKKS LVDNVGSKK EVHTAVNFGK RMKGQAYRDK HDSDSKAKPI TKEELERWEA SEDEDDDENS KTYIPIPKFH SFTGSESIFS ASVKKDYKLL PNPVLHMFR FRRVIADEFT YLQKKSLAAV LRLSSSYRWI LSGTPPVSDF AAIRSIATFM GIHLGVEDDG EGDVQYQKAR A KDQTQAEK FHAFREVHSR AWHNRRDELA QEFLNVFVRQ NIAEIEDIPT VEHIHTFKLP ASEGAVYLEL EHHLQALEMQ AR KETKFKN VSQGDRNARL EEALSDSKTA EEALLKRCCH FTLDLSDKTQ DAKSAQEACD HITSARARQL LACQEDLSRS VNQ AIALHG WIKKKGGFSK NDDERQPFAE WIAFSSNISK HQGDIEAARI LLKVIEKCGV KDGNIPPSPS DKQSPSIASG AKMD DVKWQ LREQTHLLRK LVKELVARVR SLRFFEVVRK IQKGKSDAQI VLESSECGHK PSTNPDIEMA ILSCCGHVAC HKCMR KAAA SQRCVKSGEC QAAVRPTNIV KVSSLGVEGE LSSGRYGAKL EHLVNLIHSI PKNERVLVFL QWEDLAGKVS EALSAG RIP HVTLSGSAKS RANTLDRFQS TNADSARVLL LKMNDASAAG SNLTTANHAV FLGPLFTNSL FNYRAVETQA IGRVRRY GQ QKKVHIHRLL ALDTIDMTIF NARRTELKEK TDWEEIPQEE YKGRGSSISM TNEKRTPTLT VKSNPFKRSS SWALASSF R SKKRSMEARD AEGVSDDDEN SELSDII

UniProtKB: DNA (cytosine-5-)-methyltransferase DMT5

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Macromolecule #2: DNA (5'-D(P*CP*AP*GP*(5CM)P*GP*CP*AP*T)-3')

MacromoleculeName: DNA (5'-D(P*CP*AP*GP*(5CM)P*GP*CP*AP*T)-3') / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Cryptococcus neoformans (fungus)
Molecular weightTheoretical: 11.168184 KDa
SequenceString:
(DT)(DG)(DT)(DA)(DT)(DG)(DG)(DT)(DC)(DT) (DT)(DA)(DG)(DG)(DC)(DA)(DA)(DT)(DT)(DC) (DT)(DA)(DG)(DT)(DG)(DT)(DC)(DA)(DG) (5CM)(DG)(DC)(DA)(DT)(DG)(DG)

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Macromolecule #3: DNA (5'-D(*TP*GP*CP*GP*CP*TP*GP*AP*CP*A)-3')

MacromoleculeName: DNA (5'-D(*TP*GP*CP*GP*CP*TP*GP*AP*CP*A)-3') / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Cryptococcus neoformans (fungus)
Molecular weightTheoretical: 10.999107 KDa
SequenceString:
(DC)(DC)(DA)(DT)(DG)(DC)(DG)(DC)(DT)(DG) (DA)(DC)(DA)(DC)(DT)(DA)(DG)(DA)(DA)(DT) (DT)(DG)(DC)(DC)(DT)(DA)(DA)(DG)(DA) (DC)(DC)(DA)(DT)(DA)(DC)(DA)

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Macromolecule #4: S-ADENOSYL-L-HOMOCYSTEINE

MacromoleculeName: S-ADENOSYL-L-HOMOCYSTEINE / type: ligand / ID: 4 / Number of copies: 1 / Formula: SAH
Molecular weightTheoretical: 384.411 Da
Chemical component information

ChemComp-SAH:
S-ADENOSYL-L-HOMOCYSTEINE

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Macromolecule #5: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 5 / Number of copies: 5 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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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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Macromolecule #7: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 7 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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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

Concentration0.19 mg/mL
BufferpH: 8
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 120 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 278 K / Instrument: FEI VITROBOT MARK IV

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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 opticsCalibrated magnification: 47262 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal magnification: 22500
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 23621
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-7r78:
cryo-EM structure of DNMT5 quaternary complex with hemimethylated DNA, AMP-PNP and SAH

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