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- PDB-8fil: Zinc-free APOBEC3A (inactive E72A mutant) in complex with TTC-hai... -

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Basic information

Entry
Database: PDB / ID: 8fil
TitleZinc-free APOBEC3A (inactive E72A 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 / DNA cytosine deamination / : / cytidine to uridine editing / cytidine deaminase activity / clearance of foreign intracellular DNA / negative regulation of single stranded viral RNA replication via double stranded DNA intermediate / : ...mRNA Editing: C to U Conversion / Formation of the Editosome / single-stranded DNA cytosine deaminase / DNA cytosine deamination / : / cytidine to uridine editing / cytidine deaminase activity / clearance of foreign intracellular DNA / negative regulation of single stranded viral RNA replication via double stranded DNA intermediate / : / positive regulation of gene expression via chromosomal CpG island demethylation / retrotransposon silencing / 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 / DNA / DNA (> 10) / DNA dC->dU-editing enzyme APOBEC-3A
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.01 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: To Be Published
Title: DNA-based inhibitors restrict mutagenic activity of APOBEC3 in cells
Authors: Harjes, S. / Kurup, H.M. / Filichev, V. / Harjes, E. / Jameson, G.B.
History
DepositionDec 16, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 6, 2023Provider: repository / Type: Initial release

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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
D: DNA (5'-D(*TP*GP*CP*GP*CP*TP*TP*CP*GP*CP*GP*CP*T)-3')
E: 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)55,2268
Polymers53,8354
Non-polymers1,3914
Water1,45981
1
A: DNA dC->dU-editing enzyme APOBEC-3A
D: 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)27,6134
Polymers26,9182
Non-polymers6952
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1760 Å2
ΔGint-20 kcal/mol
Surface area11830 Å2
MethodPISA
2
B: DNA dC->dU-editing enzyme APOBEC-3A
E: 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)27,6134
Polymers26,9182
Non-polymers6952
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1740 Å2
ΔGint-21 kcal/mol
Surface area11930 Å2
MethodPISA
Unit cell
Length a, b, c (Å)54.750, 57.352, 93.739
Angle α, β, γ (deg.)90.00, 105.78, 90.00
Int Tables number4
Space group name H-MP1211
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-ID
11
22
/ NCS ensembles :
ID
1
2

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Components

#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)
#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-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Cl
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 81 / Source method: isolated from a natural source / Formula: H2O
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: 2.6 Å3/Da / Density % sol: 52.81 % / Description: Thin plate
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 16M / Detector: PIXEL / Date: Sep 28, 2022 / 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: 2.01→48.4 Å / Num. obs: 36510 / % possible obs: 97.8 % / Redundancy: 2.8 % / Biso Wilson estimate: 38.2 Å2 / CC1/2: 0.992 / Rmerge(I) obs: 0.113 / Rpim(I) all: 0.081 / Net I/σ(I): 6.1
Reflection shellResolution: 2.01→2.15 Å / Redundancy: 2.6 % / Rmerge(I) obs: 1.294 / Mean I/σ(I) obs: 0.7 / Num. unique obs: 2435 / CC1/2: 0.188 / Rpim(I) all: 0.955 / % possible all: 88.3

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Processing

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

5keg


Resolution: 2.01→40.94 Å / Cor.coef. Fo:Fc: 0.943 / Cor.coef. Fo:Fc free: 0.926 / SU B: 17.319 / SU ML: 0.202 / Cross valid method: THROUGHOUT / ESU R: 0.207 / ESU R Free: 0.18 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.26586 1827 5 %RANDOM
Rwork0.23118 ---
obs0.23292 34669 97.58 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 43.944 Å2
Baniso -1Baniso -2Baniso -3
1-2.5 Å2-0 Å21.66 Å2
2---2.78 Å2-0 Å2
3----0.57 Å2
Refinement stepCycle: 1 / Resolution: 2.01→40.94 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2992 520 74 81 3667
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0080.0133789
X-RAY DIFFRACTIONr_bond_other_d0.0010.0153111
X-RAY DIFFRACTIONr_angle_refined_deg1.9761.6615275
X-RAY DIFFRACTIONr_angle_other_deg1.3571.5767141
X-RAY DIFFRACTIONr_dihedral_angle_1_deg7.0175375
X-RAY DIFFRACTIONr_dihedral_angle_2_deg30.95120.722194
X-RAY DIFFRACTIONr_dihedral_angle_3_deg15.10915479
X-RAY DIFFRACTIONr_dihedral_angle_4_deg22.5431527
X-RAY DIFFRACTIONr_chiral_restr0.1160.2481
X-RAY DIFFRACTIONr_gen_planes_refined0.0110.023949
X-RAY DIFFRACTIONr_gen_planes_other0.0020.02943
X-RAY DIFFRACTIONr_nbd_refined
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it2.3893.2051494
X-RAY DIFFRACTIONr_mcbond_other2.3893.2031493
X-RAY DIFFRACTIONr_mcangle_it3.8134.781871
X-RAY DIFFRACTIONr_mcangle_other3.8134.7821872
X-RAY DIFFRACTIONr_scbond_it3.1783.6262295
X-RAY DIFFRACTIONr_scbond_other3.1783.6262296
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other5.135.4063405
X-RAY DIFFRACTIONr_long_range_B_refined7.49335.5184263
X-RAY DIFFRACTIONr_long_range_B_other7.48335.4774257
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
Refine LS restraints NCS

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

Ens-IDDom-IDAuth asym-IDNumberRms dev position (Å)
11A58120.11
12B58120.11
21D11260.08
22E11260.08
LS refinement shellResolution: 2.012→2.065 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.365 114 -
Rwork0.36 2317 -
obs--88.08 %
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)
10.6870.1476-0.00941.64210.2381.0035-0.0596-0.0414-0.0685-0.04790.058-0.0281-0.04980.11560.00160.0467-0.0268-0.01330.1075-0.00760.0446-0.29030.253342.5769
21.34450.1916-0.62381.9379-0.72561.52680.1269-0.17690.01340.0094-0.057-0.0761-0.20860.2347-0.06990.1337-0.0480.00710.1069-0.01280.011726.2076-11.589712.6195
31.20030.48320.46145.8739-2.12531.6173-0.0410.25750.1682-0.41120.19940.18670.18770.1843-0.15840.2247-0.07940.01890.1620.0330.16770.125911.990421.7742
42.62743.0407-0.55615.8922-0.43491.7972-0.1977-0.20070.2079-0.06230.28420.48720.2722-0.2511-0.08660.2269-0.04070.03240.22560.08950.21035.7307-24.239415.0009
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A10 - 196
2X-RAY DIFFRACTION2B10 - 196
3X-RAY DIFFRACTION3D-7 - 5
4X-RAY DIFFRACTION4E-7 - 5

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