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- PDB-9e8p: Crystal Structure of GABARAP-ATG3 conjugate -

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Basic information

Entry
Database: PDB / ID: 9e8p
TitleCrystal Structure of GABARAP-ATG3 conjugate
Components
  • Gamma-aminobutyric acid receptor-associated protein
  • Ubiquitin-like-conjugating enzyme ATG3
KeywordsLIGASE / Autophagy / E2 / lipidation / ubiquitin-like protein
Function / homology
Function and homology information


Atg12 transferase activity / Atg8-family conjugating enzyme activity / Atg8-family ligase activity / Atg12-Atg5-Atg16 complex / ubiquitin-like protein transferase activity / positive regulation of protein K48-linked ubiquitination / glycophagy / regulation of cilium assembly / regulation of Rac protein signal transduction / nucleophagy ...Atg12 transferase activity / Atg8-family conjugating enzyme activity / Atg8-family ligase activity / Atg12-Atg5-Atg16 complex / ubiquitin-like protein transferase activity / positive regulation of protein K48-linked ubiquitination / glycophagy / regulation of cilium assembly / regulation of Rac protein signal transduction / nucleophagy / mitochondrial fragmentation involved in apoptotic process / GABA receptor binding / autophagy of mitochondrion / phosphatidylethanolamine binding / phagophore assembly site / Transferases; Acyltransferases; Aminoacyltransferases / TBC/RABGAPs / cellular response to nitrogen starvation / protein targeting to membrane / microtubule associated complex / negative regulation of phagocytosis / Macroautophagy / regulation of neurotransmitter receptor localization to postsynaptic specialization membrane / autophagosome membrane / extrinsic apoptotic signaling pathway via death domain receptors / axoneme / autophagosome assembly / autophagosome maturation / beta-tubulin binding / protein targeting / smooth endoplasmic reticulum / mitophagy / sperm midpiece / autophagosome / GABA-ergic synapse / microtubule cytoskeleton organization / positive regulation of proteasomal ubiquitin-dependent protein catabolic process / protein transport / actin cytoskeleton / cytoplasmic vesicle / microtubule binding / chemical synaptic transmission / microtubule / lysosome / protein ubiquitination / Golgi membrane / intracellular membrane-bounded organelle / ubiquitin protein ligase binding / enzyme binding / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Ubiquitin-like-conjugating enzyme Atg3/Atg10 / Autophagocytosis associated protein, active-site domain / Autophagy protein Atg8 ubiquitin-like / Autophagy protein Atg8 ubiquitin like / Ubiquitin-like domain superfamily
Similarity search - Domain/homology
Gamma-aminobutyric acid receptor-associated protein / Ubiquitin-like-conjugating enzyme ATG3
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.7 Å
AuthorsOhashi, K. / Otomo, T.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM092740 United States
CitationJournal: Biochemistry / Year: 2025
Title: Structural Insights into the GABARAP-ATG3 Backside Interaction and Apo ATG3 Conformation.
Authors: Ohashi, K. / Kroon, G.J. / Otomo, T.
History
DepositionNov 5, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 6, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Ubiquitin-like-conjugating enzyme ATG3
B: Ubiquitin-like-conjugating enzyme ATG3
C: Gamma-aminobutyric acid receptor-associated protein
D: Gamma-aminobutyric acid receptor-associated protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)80,2759
Polymers79,9314
Non-polymers3445
Water1629
1
A: Ubiquitin-like-conjugating enzyme ATG3
C: Gamma-aminobutyric acid receptor-associated protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)40,1865
Polymers39,9662
Non-polymers2203
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: Ubiquitin-like-conjugating enzyme ATG3
D: Gamma-aminobutyric acid receptor-associated protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)40,0904
Polymers39,9662
Non-polymers1242
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)97.388, 97.388, 166.774
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number96
Space group name H-MP43212
Space group name HallP4nw2abw
Symmetry operation#1: x,y,z
#2: -y+1/2,x+1/2,z+3/4
#3: y+1/2,-x+1/2,z+1/4
#4: x+1/2,-y+1/2,-z+1/4
#5: -x+1/2,y+1/2,-z+3/4
#6: -x,-y,z+1/2
#7: y,x,-z
#8: -y,-x,-z+1/2

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Components

#1: Protein Ubiquitin-like-conjugating enzyme ATG3 / Autophagy-related protein 3 / APG3-like / hApg3 / Protein PC3-96


Mass: 25911.402 Da / Num. of mol.: 2 / Mutation: C264K
Source method: isolated from a genetically manipulated source
Details: A truncated human ATG3 construct, consisting of an extra glycine at the N-terminus, followed by ATG3 residues 26-91, 148-181, and 191-314, with a point mutation of C264K. The lysine at ...Details: A truncated human ATG3 construct, consisting of an extra glycine at the N-terminus, followed by ATG3 residues 26-91, 148-181, and 191-314, with a point mutation of C264K. The lysine at position 264 is covalently linked to the C-terminal glycine of GABARAP via an isopeptide bond.,A truncated human ATG3 construct, consisting of an extra glycine at the N-terminus, followed by ATG3 residues 26-91, 148-181, and 191-314, with a point mutation of C264K. The lysine at position 264 is covalently linked to the C-terminal glycine of GABARAP via an isopeptide bond.,A truncated human ATG3 construct, consisting of an extra glycine at the N-terminus, followed by ATG3 residues 26-91, 148-181, and 191-314, with a point mutation of C264K. The lysine at position 264 is covalently linked to the C-terminal glycine of GABARAP via an isopeptide bond.
Source: (gene. exp.) Homo sapiens (human) / Gene: ATG3, APG3, APG3L / Production host: Escherichia coli (E. coli)
References: UniProt: Q9NT62, Transferases; Acyltransferases; Aminoacyltransferases
#2: Protein Gamma-aminobutyric acid receptor-associated protein / GABA(A) receptor-associated protein / MM46


Mass: 14054.113 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: A truncated human GABARAP construct, consisting of extra serine and histidine at the N-terminus followed by GABARAP residues 1-116, has its C-terminal glycine covalently linked to the lysine ...Details: A truncated human GABARAP construct, consisting of extra serine and histidine at the N-terminus followed by GABARAP residues 1-116, has its C-terminal glycine covalently linked to the lysine at position 264 of ATG3 via an isopeptide bond.
Source: (gene. exp.) Homo sapiens (human) / Gene: GABARAP, FLC3B, HT004 / Production host: Escherichia coli (E. coli) / References: UniProt: O95166
#3: Chemical ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: SO4
#4: Chemical
ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C2H6O2
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 9 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN
Has protein modificationY

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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: 2.47 Å3/Da / Density % sol: 50.28 %
Crystal growTemperature: 277.15 K / Method: vapor diffusion, sitting drop / pH: 8.5 / Details: 0.1 mM Tris pH8.5, 25%(w/v) PEG3550

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SSRL / Beamline: BL11-1 / Wavelength: 0.9794 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Dec 13, 2012
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9794 Å / Relative weight: 1
ReflectionResolution: 2.7→38.33 Å / Num. obs: 41972 / % possible obs: 99.57 % / Redundancy: 10.1 % / Biso Wilson estimate: 75.8 Å2 / CC1/2: 1 / CC star: 1 / Rmerge(I) obs: 0.06022 / Rpim(I) all: 0.01975 / Net I/σ(I): 24.97
Reflection shellResolution: 2.7→2.796 Å / Redundancy: 9.8 % / Rmerge(I) obs: 1.232 / Mean I/σ(I) obs: 1.95 / Num. unique obs: 4400 / CC1/2: 0.764 / CC star: 0.931 / Rpim(I) all: 0.4076 / % possible all: 99.46

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Processing

Software
NameVersionClassification
PHENIX1.18.2_3874refinement
XDSdata reduction
XSCALEdata scaling
PHENIXphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.7→38.33 Å / SU ML: 0.4196 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 32.3686
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2492 3706 8.85 %
Rwork0.2176 38190 -
obs0.2205 41896 99.63 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 89.21 Å2
Refinement stepCycle: LAST / Resolution: 2.7→38.33 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4846 0 21 9 4876
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00215008
X-RAY DIFFRACTIONf_angle_d0.49286780
X-RAY DIFFRACTIONf_chiral_restr0.0423718
X-RAY DIFFRACTIONf_plane_restr0.0036858
X-RAY DIFFRACTIONf_dihedral_angle_d11.62841884
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.7-2.740.41731370.34831457X-RAY DIFFRACTION99.25
2.74-2.770.34281430.34321468X-RAY DIFFRACTION100
2.77-2.810.37351390.33071450X-RAY DIFFRACTION99.19
2.81-2.850.35431430.33541491X-RAY DIFFRACTION99.82
2.85-2.90.39251400.3071458X-RAY DIFFRACTION99.81
2.9-2.950.29861470.3151493X-RAY DIFFRACTION100
2.95-30.3871370.32241448X-RAY DIFFRACTION99.87
3-3.050.31341460.31091521X-RAY DIFFRACTION100
3.05-3.110.35681450.29641446X-RAY DIFFRACTION99.87
3.11-3.170.2971470.29321460X-RAY DIFFRACTION99.81
3.17-3.240.31441370.2871489X-RAY DIFFRACTION99.88
3.24-3.320.34421440.25411467X-RAY DIFFRACTION100
3.32-3.40.30381470.2471473X-RAY DIFFRACTION99.94
3.4-3.490.27811420.24361451X-RAY DIFFRACTION98.39
3.49-3.60.24041410.23491466X-RAY DIFFRACTION99.81
3.6-3.710.2731400.23141495X-RAY DIFFRACTION99.88
3.71-3.840.24411410.23371453X-RAY DIFFRACTION99.62
3.84-40.22271400.20061459X-RAY DIFFRACTION99.75
4-4.180.25911450.19261486X-RAY DIFFRACTION100
4.18-4.40.20641380.17731499X-RAY DIFFRACTION100
4.4-4.670.16351450.16281453X-RAY DIFFRACTION99.25
4.68-5.040.17651430.17071471X-RAY DIFFRACTION99.88
5.04-5.540.22861450.17721471X-RAY DIFFRACTION100
5.54-6.340.27061510.20931460X-RAY DIFFRACTION100
6.34-7.980.22171420.20861481X-RAY DIFFRACTION99.63
7.98-38.330.22161410.17661424X-RAY DIFFRACTION96.6
Refinement TLS params.

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

IDL112)L122)L132)L222)L232)L332)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T112)T122)T132)T222)T232)T332)Origin x (Å)Origin y (Å)Origin z (Å)
14.23372671542-1.74007569814-1.505471734375.79482876527-1.099790220791.70855879899-0.009585965559760.7127667575990.279541983367-0.265572570013-0.384171590414-0.786198611423-0.4386403297430.7121204637980.3977112777530.5355581331480.0149606679031-0.1084796562711.345685314340.1573987557680.79875094867754.250879060680.5234701599159.578525057
24.4478765712-1.63919999777-0.9241982482382.193490841810.4710158823183.927843958910.0473850893469-0.144864010126-0.715523204585-0.503515482513-0.453773648229-0.5000696330070.1166530094511.166851401170.3866883459630.6378807739880.2965946047020.07546030523461.123924363030.05955332270190.77971266914156.751774342866.5185350395161.518227378
32.39664382592-0.6124494049911.422946087853.57412897672-1.115381819855.592580941270.121698935641-0.452262479128-0.04432237921810.3781405944060.1299708004060.3385322194921.257984780450.772848057217-0.1038704792870.7297513683580.213358566906-0.07140421546620.85962821766-0.03318754615290.58425051555745.355079568168.3593622955180.541977059
43.58060738532-0.148814225138-1.232218323094.25645448478-1.352002815837.27196209244-0.003777665295780.429461849175-0.622753563482-0.300649415762-0.09936385897120.1168329527061.221627762130.5478188107010.0972626752340.5394218246790.169537284733-0.06315346121970.765572806937-0.08733277998350.65775222227445.436513488468.5465127505165.573277504
55.02359003477-0.2133578576410.9031050389446.072439466860.4174224889322.471440273510.877890200730.0504894640119-1.03934123150.900123604764-0.3135929389180.03929061477191.97507025087-0.411804942547-0.3769923639621.021878966350.02685137646960.01741820143020.8197655868880.1651652926260.88613404840139.310372863560.4344379956179.200100131
62.22689515043-1.973915570534.611983859146.55227578569-4.619668757019.59378344627-0.2150828127452.18885727357-0.4727710154110.933060982171-0.5215200592532.67541357908-0.647554148872-2.992998681430.5509370970310.6884430971670.1093843626950.02016993387911.42917326789-0.1168328841061.1442876597830.958398569272.5764342424170.384081969
76.61026255897-3.784326962970.2135165129613.19049914720.1425027218385.506559419610.1349818369251.70905063096-2.50086182624-0.318965756528-0.8080073466070.7944093568321.373771970640.7568103442020.4553407965790.6525159763090.1435155109160.03125560448440.722339144451-0.1213214611580.629841374342.630414062162.4808978554164.345854069
85.18961927453-0.848560999581-1.453270206342.45932882208-0.7646570426337.740611015590.08783538898561.258863926321.06668320978-0.620590972304-0.4348013665310.907138135723-0.838727275510.2923505338930.4501507061140.553786384060.0350392787318-0.05268324119950.7678064555330.1090485024490.57330630283940.942474730986.2126366014160.132892516
93.83088435205-2.84445232466-0.06766155783689.563114009580.536386079084.626573692060.4888334793930.3596549132610.1417409193190.787239155527-0.761315688909-1.35504546571-0.6311463743112.271648963180.01119854051590.742977586955-0.222062804894-0.1953307070351.489736705140.3099636070251.1630578023952.710593267590.0560355079163.049381655
103.30732757373-2.04219038325-0.3409580467667.58670615494-1.953632684586.04450374869-0.5089804304680.2513110295150.6341466027941.241274105560.151741306909-0.471012751273-0.5211523579590.5124848912870.143868546110.4715567892090.0581187902766-0.1049036283830.7349697182750.04004495774150.60949343331845.854239169578.7620008866172.857262757
114.61758110544-0.1847398628254.018123730970.2329524785780.7501281547325.91241498809-0.521540470326-0.271594519871.090507737741.77307151299-0.0227871565059-0.504047238859-1.938889894980.4528341833130.5024936566591.766583846730.029883811155-0.2649582380190.611668427439-0.07255701558480.78756202092134.1118348922102.816381884173.19537725
120.9858225703110.6820334914061.106474408974.382354243743.90891834873.90040410361-1.0079095489-0.1911627651591.406310945880.9095782327680.8438626730821.22673010035-2.87833719374-0.556881971246-1.909862074131.915870182410.9265801529160.2832167873970.170878940898-0.1909889276040.76544587507720.2077685459106.426937332171.837184564
132.751945692020.5808061457710.005969168059952.98592776812-3.985970149755.6854810850.1827524780970.390052216342-0.489733669989-0.2842428969880.4004407037930.02418566981890.112359481595-1.14964801154-0.4013515062850.7450763645790.2967813143980.008906400810450.7636622852050.1120068786390.57451312486620.268667959394.1635878653153.107149713
146.14980980919-0.05678019255021.317102040995.94924182376-0.01731710426573.692086754130.2207395135940.0228917351578-0.6862141244130.6796343921070.3766885697180.7828518810430.0196932442275-1.3376615778-0.5508861194830.6846987153430.1568935330720.06976047075610.6479433616320.04194790886830.63992088898318.221084512491.169217976162.835282039
158.5017200891-0.4358935826150.5419172609985.919039945190.03898934000486.95265815481-0.549393476353-0.388848791502-0.47291457611.089737915510.3738771277330.161031736015-0.672415262493-0.4705621268930.2127833903970.8183030173350.07831719185520.07454006052250.3452111820820.04824994576440.29242576584726.018231206989.6668796308170.835446257
165.86469955306-2.444233754591.125145666692.009205869220.09894731472064.297923143330.1209265034531.064349347080.6042855392010.0200548406626-0.310675824151-0.575666421139-1.08107304090.6760636163290.2358867439070.82734021692-0.00981114390042-0.051364553320.4865688852650.08697570241530.5950298203633.877357830795.7301669385162.58671124
170.6289450998580.106793338779-1.344975199783.74938725281-1.78421521844.66479997516-0.3442884935250.3085135211961.03383797552-1.020760645740.7037176974812.638458255310.16924936736-2.71371513691-0.2366870237470.729309350026-0.00120952566349-0.1130032837621.67954514660.1779830391881.466653399489.5264047474857.874865106149.494166206
185.77473996143-0.7980734181010.1994483031075.7564164388-1.428046777347.75291340421-0.3456265871980.1203485836130.159588647889-0.1303290565390.00633281093653-0.0099381875687-0.0618367315339-0.9615443357230.3026714087930.759483112567-0.0691259362895-0.06720845662060.896693053891-0.2106193060640.6165727926325.533482140955.480617726147.430601373
192.642826909921.970903452780.5446309372836.72287206137-3.023545810877.90601187204-0.241292641782-0.65330598636-0.00986422210727-0.01567566269760.0494164305791-0.281861610459-0.2269935851911.132619481570.200103295270.70781094654-0.0792450089121-0.1137911594850.940631238248-0.1761186665870.66388094481532.54032187258.6669590595153.666559035
205.16601131972-1.33847935639-1.105957494987.84097198622-0.9270492945214.01370403061-0.259762500717-0.895187607441-0.5892929982811.244214648340.21856735031.093788894320.35542370113-0.797437936620.01752114674990.894449887753-0.1544970647760.09128439458531.40208430646-0.1303925252830.66720072223918.638484606255.5135118209156.282620122
217.85636187642-0.6955587985780.2218428930482.381020539930.9638075913013.00593223505-0.8413236696490.7892807220062.4371881781-1.180485436470.04034780869921.884091130651.52994596424-1.941261387290.9005145889480.92050736505-0.0698878680525-0.3176338310581.00880080775-0.1303973860521.0976475027832.036354866669.0518296305155.624353571
224.62452825211.476604652890.2883871452723.72067728229-1.159739742344.467680364140.135319373288-0.193628032879-3.167987091370.6190754198110.00642488799785-1.686496703083.119854774330.2157051615970.05398958159382.11545184864-0.0237433927503-0.06300657249510.8481932335510.07424009954791.6914541458710.634377919659.0665016202185.033508003
236.100049502450.140776694685-0.9064206260726.127005905871.432800201019.863877827460.02398427117580.1397606989360.0494844921495-0.41743619125-0.2651165462660.04450421743920.755506200792-0.1736376980010.266388570080.886499859835-0.08603567453680.1884668990350.7472515580380.08958601864640.5783100419948.3201109580377.2223846798184.365736018
244.98272384977-0.121036903807-0.04416707641955.52198965146-0.4223817324814.36487105122-0.6273635630671.44088859692-0.966310554908-1.172532058320.418753682153-0.1771214512181.44315034257-0.2772169420630.1741962978241.36948264677-0.2287183266910.2424599396620.82289226578-0.09820802884140.6106355526110.632267630370.7181022506177.99872371
Refinement TLS group

Refine-ID: X-RAY DIFFRACTION

IDRefine TLS-IDSelection detailsAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11chain 'A' and (resid 27 through 49 )AA27 - 491 - 23
22chain 'A' and (resid 50 through 72 )AA50 - 7224 - 46
33chain 'A' and (resid 73 through 197 )AA73 - 19747 - 63
44chain 'A' and (resid 198 through 220 )AA198 - 22064 - 86
55chain 'A' and (resid 221 through 235 )AA221 - 23587 - 101
66chain 'A' and (resid 236 through 244 )AA236 - 244102 - 110
77chain 'A' and (resid 245 through 262 )AA245 - 262111 - 128
88chain 'A' and (resid 263 through 279 )AA263 - 279129 - 145
99chain 'A' and (resid 280 through 288 )AA280 - 288146 - 154
1010chain 'A' and (resid 289 through 310 )AA289 - 310155 - 176
1111chain 'B' and (resid 27 through 49 )BB27 - 491 - 23
1212chain 'B' and (resid 50 through 72 )BB50 - 7224 - 46
1313chain 'B' and (resid 73 through 197 )BB73 - 19747 - 63
1414chain 'B' and (resid 198 through 240 )BB198 - 24064 - 106
1515chain 'B' and (resid 241 through 279 )BB241 - 279107 - 145
1616chain 'B' and (resid 280 through 310 )BB280 - 310146 - 176
1717chain 'C' and (resid 2 through 24 )CC2 - 241 - 23
1818chain 'C' and (resid 25 through 68 )CC25 - 6824 - 67
1919chain 'C' and (resid 69 through 90 )CC69 - 9068 - 89
2020chain 'C' and (resid 91 through 110 )CC91 - 11090 - 109
2121chain 'C' and (resid 111 through 116 )CC111 - 116110 - 115
2222chain 'D' and (resid 2 through 24 )DD2 - 241 - 23
2323chain 'D' and (resid 25 through 90 )DD25 - 9024 - 89
2424chain 'D' and (resid 91 through 116 )DD91 - 11690 - 115

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