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- PDB-8fim: Structure of APOBEC3A (E72A inactive mutant) in complex with TTC-... -

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Basic information

Entry
Database: PDB / ID: 8fim
TitleStructure of APOBEC3A (E72A inactive mutant) in complex with TTC-hairpin DNA substrate
Components
  • DNA (5'-D(*TP*GP*CP*GP*CP*TP*TP*CP*GP*CP*GP*CP*T)-3')
  • DNA dC->dU-editing enzyme APOBEC-3A
KeywordsDNA BINDING PROTEIN / APOBEC / APOBEC3A / A3A / APOBEC3A E72A mutant / cancer / DNA / mutation / cytidine deaminase / hairpin DNA
Function / homology
Function and homology information


mRNA Editing: C to U Conversion / Formation of the Editosome / single-stranded DNA cytosine deaminase / negative regulation of single stranded viral RNA replication via double stranded DNA intermediate / DNA cytosine deamination / cytidine to uridine editing / clearance of foreign intracellular DNA / cytidine deaminase activity / transposable element silencing / positive regulation of gene expression via chromosomal CpG island demethylation ...mRNA Editing: C to U Conversion / Formation of the Editosome / single-stranded DNA cytosine deaminase / negative regulation of single stranded viral RNA replication via double stranded DNA intermediate / DNA cytosine deamination / cytidine to uridine editing / clearance of foreign intracellular DNA / cytidine deaminase activity / transposable element silencing / positive regulation of gene expression via chromosomal CpG island demethylation / negative regulation of viral genome replication / P-body / defense response to virus / innate immune response / RNA binding / zinc ion binding / nucleoplasm / nucleus / cytoplasm
Similarity search - Function
Novel AID APOBEC clade 2 / : / APOBEC/CMP deaminase, zinc-binding / Cytidine and deoxycytidylate deaminases zinc-binding region signature. / Cytidine and deoxycytidylate deaminase domain / Cytidine and deoxycytidylate deaminases domain profile. / Cytidine deaminase-like
Similarity search - Domain/homology
INOSITOL HEXAKISPHOSPHATE / PHOSPHATE ION / DNA / DNA (> 10) / DNA dC->dU-editing enzyme APOBEC-3A
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.22 Å
AuthorsHarjes, S. / Jameson, G.B. / Harjes, E. / Filichev, V.V. / Kurup, H.M.
Funding support New Zealand, 1items
OrganizationGrant numberCountry
Health Research Council (HRC)20/1355 New Zealand
CitationJournal: Nat Commun / Year: 2023
Title: Structure-guided inhibition of the cancer DNA-mutating enzyme APOBEC3A.
Authors: Harjes, S. / Kurup, H.M. / Rieffer, A.E. / Bayarjargal, M. / Filitcheva, J. / Su, Y. / Hale, T.K. / Filichev, V.V. / Harjes, E. / Harris, R.S. / Jameson, G.B.
History
DepositionDec 16, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 6, 2023Provider: repository / Type: Initial release
Revision 1.1Sep 18, 2024Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: DNA dC->dU-editing enzyme APOBEC-3A
B: DNA dC->dU-editing enzyme APOBEC-3A
H: DNA (5'-D(*TP*GP*CP*GP*CP*TP*TP*CP*GP*CP*GP*CP*T)-3')
C: DNA (5'-D(*TP*GP*CP*GP*CP*TP*TP*CP*GP*CP*GP*CP*T)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)55,45211
Polymers53,8354
Non-polymers1,6177
Water28816
1
A: DNA dC->dU-editing enzyme APOBEC-3A
C: DNA (5'-D(*TP*GP*CP*GP*CP*TP*TP*CP*GP*CP*GP*CP*T)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)27,7736
Polymers26,9182
Non-polymers8564
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1860 Å2
ΔGint-58 kcal/mol
Surface area12000 Å2
MethodPISA
2
B: DNA dC->dU-editing enzyme APOBEC-3A
H: DNA (5'-D(*TP*GP*CP*GP*CP*TP*TP*CP*GP*CP*GP*CP*T)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)27,6785
Polymers26,9182
Non-polymers7613
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1810 Å2
ΔGint-60 kcal/mol
Surface area11870 Å2
MethodPISA
Unit cell
Length a, b, c (Å)53.917, 56.904, 91.549
Angle α, β, γ (deg.)90.000, 103.570, 90.000
Int Tables number4
Space group name H-MP1211
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21B
12H
22C

NCS domain segments:

Component-ID: _ / Refine code: _

Dom-IDEns-IDBeg auth comp-IDBeg label comp-IDEnd auth comp-IDEnd label comp-IDAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11ARGARGASNASNAA10 - 19610 - 196
21ARGARGASNASNBB10 - 19610 - 196
12DTDTDTDTHC-7 - 51 - 13
22DTDTDTDTCD-7 - 51 - 13

NCS ensembles :
ID
1
2

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Components

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Protein / DNA chain , 2 types, 4 molecules ABHC

#1: Protein DNA dC->dU-editing enzyme APOBEC-3A / A3A / Phorbolin-1


Mass: 22982.979 Da / Num. of mol.: 2 / Mutation: E72A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: APOBEC3A / Plasmid: pETite / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: P31941, single-stranded DNA cytosine deaminase
#2: DNA chain DNA (5'-D(*TP*GP*CP*GP*CP*TP*TP*CP*GP*CP*GP*CP*T)-3')


Mass: 3934.546 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)

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Non-polymers , 5 types, 23 molecules

#3: Chemical ChemComp-IHP / INOSITOL HEXAKISPHOSPHATE / MYO-INOSITOL HEXAKISPHOSPHATE / INOSITOL 1,2,3,4,5,6-HEXAKISPHOSPHATE


Mass: 660.035 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C6H18O24P6
#4: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
#5: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Cl
#6: Chemical ChemComp-PO4 / PHOSPHATE ION


Mass: 94.971 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: PO4
#7: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 16 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.51 Å3/Da / Density % sol: 56.3 % / Description: Tiny flattened needle
Crystal growTemperature: 285 K / Method: vapor diffusion, hanging drop / pH: 6.6
Details: A3A-E72A (50 mM MES pH 6.0, 100 mM NaCl, 1 mM TCEP, 0.2 mM EDTA) was mixed with oligonucleotides (10 mM Tris/HCl pH 7.9, 1 mM EDTA) at 0.85 mM and 1.7 mM respectively. Dilution was done with ...Details: A3A-E72A (50 mM MES pH 6.0, 100 mM NaCl, 1 mM TCEP, 0.2 mM EDTA) was mixed with oligonucleotides (10 mM Tris/HCl pH 7.9, 1 mM EDTA) at 0.85 mM and 1.7 mM respectively. Dilution was done with protein buffer. The mixture was added to crystallization liquid 1 to 1 and the mixture was pipetted on siliconized glass disks and sealed on top of a reservoir of crystallization liquid for hanging drop crystallization at 12 degrees Celsius. The crystallization liquid has the following composition: 100 mM Bicine at pH 6.6, 200 mM NaCl, 20 mM putrescine, 1 mM TCEP, 1 mM inositol hexaphosphate (phytic acid) and 45 % pentaerythritol propoxylate (5/4 PO/OH)

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Australian Synchrotron / Beamline: MX2 / Wavelength: 0.953739 Å
DetectorType: DECTRIS EIGER X 9M / Detector: PIXEL / Date: Jun 22, 2021 / Details: 1 vertical and 2 horizontal focussing mirrors
RadiationMonochromator: Si(111) double crystal monochromator / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.953739 Å / Relative weight: 1
ReflectionResolution: 2.22→47.99 Å / Num. obs: 26243 / % possible obs: 97.3 % / Redundancy: 3.4 % / Biso Wilson estimate: 51 Å2 / CC1/2: 0.995 / Rmerge(I) obs: 0.092 / Rpim(I) all: 0.059 / Rrim(I) all: 0.11 / Χ2: 0.46 / Net I/σ(I): 6.2
Reflection shellResolution: 2.22→2.29 Å / Redundancy: 3.2 % / Rmerge(I) obs: 1.535 / Mean I/σ(I) obs: 0.6 / Num. unique obs: 2098 / CC1/2: 0.332 / Rpim(I) all: 0.979 / Rrim(I) all: 1.826 / Χ2: 0.31 / % possible all: 97.9

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Processing

Software
NameVersionClassification
JBluIce-EPICSdata collection
XDSdata reduction
Aimlessdata scaling
pointlessdata scaling
MOLREPphasing
REFMAC5.8.0267refinement
Coot0.8.9.2model building
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5KEG

5keg


Resolution: 2.22→47.99 Å / Cor.coef. Fo:Fc: 0.937 / Cor.coef. Fo:Fc free: 0.935 / SU B: 27.009 / SU ML: 0.299 / Cross valid method: FREE R-VALUE / σ(F): 0 / ESU R: 0.322 / ESU R Free: 0.242 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : WITH TLS ADDED
RfactorNum. reflection% reflectionSelection details
Rfree0.2772 1309 5 %RANDOM
Rwork0.2376 ---
obs0.2396 24929 97.2 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso max: 162.28 Å2 / Biso mean: 62.63 Å2 / Biso min: 38.98 Å2
Baniso -1Baniso -2Baniso -3
1--1.59 Å2-0 Å21.65 Å2
2--1.02 Å2-0 Å2
3----0.21 Å2
Refinement stepCycle: final / Resolution: 2.22→47.99 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3026 520 81 16 3643
Biso mean--112.97 49.14 -
Num. residues----400
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0080.0133789
X-RAY DIFFRACTIONr_bond_other_d0.0020.0153076
X-RAY DIFFRACTIONr_angle_refined_deg1.871.6615272
X-RAY DIFFRACTIONr_angle_other_deg1.3191.5777056
X-RAY DIFFRACTIONr_dihedral_angle_1_deg7.3645374
X-RAY DIFFRACTIONr_dihedral_angle_2_deg32.70421.071196
X-RAY DIFFRACTIONr_dihedral_angle_3_deg16.25615472
X-RAY DIFFRACTIONr_dihedral_angle_4_deg20.661526
X-RAY DIFFRACTIONr_chiral_restr0.1110.2483
X-RAY DIFFRACTIONr_gen_planes_refined0.0110.023952
X-RAY DIFFRACTIONr_gen_planes_other0.0030.02938
Refine LS restraints NCS

Refine-ID: X-RAY DIFFRACTION / Type: interatomic distance / Weight position: 0.05

Ens-IDDom-IDAuth asym-IDNumberRms dev position (Å)
11A59400.11
12B59400.11
21H11600.06
22C11600.06
LS refinement shellResolution: 2.22→2.276 Å / Rfactor Rfree error: 0
RfactorNum. reflection% reflection
Rfree0.419 75 -
Rwork0.372 1560 -
obs--81.63 %
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
11.6446-0.02850.05230.89380.19431.1965-0.0166-0.0365-0.0321-0.09360.11350.0097-0.02120.1375-0.09680.6446-0.0297-0.16040.0291-0.02950.3231.418-0.0142.32
20.9950.0421-0.35441.7657-0.66033.57510.047-0.1858-0.0458-0.02420.0653-0.0442-0.25720.3207-0.11230.6749-0.0299-0.1730.062400.290626.75-10.88112.38
30.6661-0.34160.97785.6585-1.26062.51550.03280.30360.2509-0.20330.26650.10270.01850.2121-0.29930.7705-0.15490.00680.30560.21130.54291.3611.92821.308
43.34972.2688-0.21014.63850.91570.9738-0.2837-0.31220.2095-0.04550.32620.69740.367-0.1346-0.04250.7737-0.1194-0.03950.30940.23390.5556.301-23.71815.258
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A10 - 196
2X-RAY DIFFRACTION2B10 - 196
3X-RAY DIFFRACTION3C-7 - 5
4X-RAY DIFFRACTION4H-7 - 5

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