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- PDB-9h38: C-terminal domain of the F-ENA tip fibrillum F-BclA from Bacillus... -

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Basic information

Entry
Database: PDB / ID: 9h38
TitleC-terminal domain of the F-ENA tip fibrillum F-BclA from Bacillus thuringiensis
ComponentsFlagellar hook-length control protein FliK
KeywordsSTRUCTURAL PROTEIN / Spore appendage / ENA / BclA / F-ENA / Bacillus thuringiensis
Function / homologyTumour necrosis factor-like domain superfamily / Collagen triple helix repeat / Collagen triple helix repeat (20 copies) / Flagellar hook-length control protein FliK
Function and homology information
Biological speciesBacillus thuringiensis (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.29 Å
AuthorsSogues, A. / Sleutel, M. / Remaut, H.
Funding supportEuropean Union, 2items
OrganizationGrant numberCountry
European Molecular Biology Organization (EMBO)ALTF 709-2021European Union
H2020 Marie Curie Actions of the European CommissionSLYDIVEuropean Union
CitationJournal: bioRxiv / Year: 2025
Title: Cryo-EM analysis of the extrasporal matrix identifies F-ENA as a widespread family of endospore appendages across the Firmicutes phylum.
Authors: Mike Sleutel / Adrià Sogues / Nani Van Gerven / Unni Lise Jonsmoen / Inge Van Molle / Marcus Fislage / Laurent Dirk Theunissen / Nathan F Bellis / Diana P Baquero / Edward H Egelman / Mart ...Authors: Mike Sleutel / Adrià Sogues / Nani Van Gerven / Unni Lise Jonsmoen / Inge Van Molle / Marcus Fislage / Laurent Dirk Theunissen / Nathan F Bellis / Diana P Baquero / Edward H Egelman / Mart Krupovic / Fengbin Wang / Marina Aspholm / Han Remaut /
Abstract: For over 100 years, (Bt) has been used as an agricultural biopesticide to control pests caused by insect species in the orders of Lepidoptera, Diptera and Coleoptera. Under nutrient starvation, Bt ...For over 100 years, (Bt) has been used as an agricultural biopesticide to control pests caused by insect species in the orders of Lepidoptera, Diptera and Coleoptera. Under nutrient starvation, Bt cells differentiate into spores and associated toxin crystals that can adopt biofilm-like aggregates. We reveal that such Bt spore/toxin biofilms are embedded in a fibrous extrasporal matrix (ESM), and using cryoID, we resolved the structure and molecular identity of an uncharacterized type of pili, referred to here as Fibrillar ENdospore Appendages or 'F-ENA'. F-ENA are monomolecular protein polymers tethered to the exosporium of Bt and are decorated with a flexible tip fibrillum. Phylogenetic analysis reveals that F-ENA is widespread not only in the class Bacilli, but also in the class Clostridia, and the cryoEM structures of F-ENA filaments from and reveal subunits with a generic head-neck domain structure, where the β-barrel neck of variable length latch onto a preceding head domain through short N-terminal hook peptides. In , two collagen-like proteins (CLP) respectively tether F-ENA to the exosporium (F-Anchor), or constitute the tip fibrillum at the distal terminus of F-ENA (F-BclA). Sedimentation assays point towards F-ENA involvement in spore-spore clustering, likely mediated via F-BclA contacts and F-ENA bundling through the antiparallel interlocking of the head-neck units.
History
DepositionOct 16, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 12, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Flagellar hook-length control protein FliK
B: Flagellar hook-length control protein FliK
C: Flagellar hook-length control protein FliK
D: Flagellar hook-length control protein FliK
E: Flagellar hook-length control protein FliK
F: Flagellar hook-length control protein FliK
G: Flagellar hook-length control protein FliK
H: Flagellar hook-length control protein FliK
I: Flagellar hook-length control protein FliK
J: Flagellar hook-length control protein FliK
K: Flagellar hook-length control protein FliK
L: Flagellar hook-length control protein FliK
M: Flagellar hook-length control protein FliK
N: Flagellar hook-length control protein FliK
O: Flagellar hook-length control protein FliK
P: Flagellar hook-length control protein FliK
Q: Flagellar hook-length control protein FliK
R: Flagellar hook-length control protein FliK
S: Flagellar hook-length control protein FliK
T: Flagellar hook-length control protein FliK
U: Flagellar hook-length control protein FliK
hetero molecules


Theoretical massNumber of molelcules
Total (without water)323,43330
Polymers322,72521
Non-polymers7099
Water32,7151816
1
A: Flagellar hook-length control protein FliK
E: Flagellar hook-length control protein FliK
F: Flagellar hook-length control protein FliK
hetero molecules


Theoretical massNumber of molelcules
Total (without water)46,3206
Polymers46,1043
Non-polymers2163
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area6480 Å2
ΔGint-21 kcal/mol
Surface area12820 Å2
MethodPISA
2
B: Flagellar hook-length control protein FliK
C: Flagellar hook-length control protein FliK
D: Flagellar hook-length control protein FliK
hetero molecules


Theoretical massNumber of molelcules
Total (without water)46,2585
Polymers46,1043
Non-polymers1542
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area6320 Å2
ΔGint-24 kcal/mol
Surface area12460 Å2
MethodPISA
3
G: Flagellar hook-length control protein FliK
H: Flagellar hook-length control protein FliK
I: Flagellar hook-length control protein FliK
hetero molecules


Theoretical massNumber of molelcules
Total (without water)46,2885
Polymers46,1043
Non-polymers1842
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area6350 Å2
ΔGint-29 kcal/mol
Surface area12390 Å2
MethodPISA
4
J: Flagellar hook-length control protein FliK
K: Flagellar hook-length control protein FliK
L: Flagellar hook-length control protein FliK
hetero molecules


Theoretical massNumber of molelcules
Total (without water)46,1964
Polymers46,1043
Non-polymers921
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area6610 Å2
ΔGint-36 kcal/mol
Surface area12630 Å2
MethodPISA
5
M: Flagellar hook-length control protein FliK
N: Flagellar hook-length control protein FliK
O: Flagellar hook-length control protein FliK
hetero molecules


Theoretical massNumber of molelcules
Total (without water)46,1664
Polymers46,1043
Non-polymers621
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area5960 Å2
ΔGint-27 kcal/mol
Surface area12540 Å2
MethodPISA
6
P: Flagellar hook-length control protein FliK
Q: Flagellar hook-length control protein FliK
R: Flagellar hook-length control protein FliK


Theoretical massNumber of molelcules
Total (without water)46,1043
Polymers46,1043
Non-polymers00
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area6380 Å2
ΔGint-38 kcal/mol
Surface area13070 Å2
MethodPISA
7
S: Flagellar hook-length control protein FliK
T: Flagellar hook-length control protein FliK
U: Flagellar hook-length control protein FliK


Theoretical massNumber of molelcules
Total (without water)46,1043
Polymers46,1043
Non-polymers00
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area6040 Å2
ΔGint-35 kcal/mol
Surface area12670 Å2
MethodPISA
Unit cell
Length a, b, c (Å)71.826, 113.486, 113.599
Angle α, β, γ (deg.)60.110, 89.190, 86.360
Int Tables number1
Space group name H-MP1
Space group name HallP1
Symmetry operation#1: x,y,z

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Components

#1: Protein ...
Flagellar hook-length control protein FliK


Mass: 15367.842 Da / Num. of mol.: 21
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus thuringiensis (bacteria) / Gene: BK775_04930 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A9X6QBI1
#2: Chemical
ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C3H8O3 / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical
ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C2H6O2 / Feature type: SUBJECT OF INVESTIGATION
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 1816 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY
Has protein modificationN

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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: 2.48 Å3/Da / Density % sol: 50.44 %
Crystal growTemperature: 294 K / Method: vapor diffusion, sitting drop
Details: 6% w/v PEG3350, 0.15 M Sodium Chloride and 4M Potassium iodide

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SOLEIL / Beamline: PROXIMA 2 / Wavelength: 0.98011 Å
DetectorType: DECTRIS EIGER X 9M / Detector: PIXEL / Date: Mar 30, 2023
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.98011 Å / Relative weight: 1
ReflectionResolution: 2.29→49.24 Å / Num. obs: 128674 / % possible obs: 97.8 % / Redundancy: 3.3 % / Biso Wilson estimate: 12.2 Å2 / Rmerge(I) obs: 0.148 / Net I/σ(I): 4.9
Reflection shellResolution: 2.292→2.292 Å / Num. unique obs: 8218 / Rpim(I) all: 0.423 / Rrim(I) all: 0.617

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Processing

Software
NameVersionClassification
PHENIX1.20_4459refinement
autoPROCdata processing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.29→49.24 Å / SU ML: 0.2864 / Cross valid method: FREE R-VALUE / σ(F): 1.96 / Phase error: 24.5101
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2418 6370 4.95 %
Rwork0.1854 122290 -
obs0.1882 128660 92.29 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 17.25 Å2
Refinement stepCycle: LAST / Resolution: 2.29→49.24 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms20903 0 46 1816 22765
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00721294
X-RAY DIFFRACTIONf_angle_d0.861429158
X-RAY DIFFRACTIONf_chiral_restr0.0553685
X-RAY DIFFRACTIONf_plane_restr0.00573781
X-RAY DIFFRACTIONf_dihedral_angle_d6.76413000
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.29-2.320.3142900.24122026X-RAY DIFFRACTION44.98
2.32-2.340.2571450.20542721X-RAY DIFFRACTION62.43
2.34-2.370.30891780.21353057X-RAY DIFFRACTION68.9
2.37-2.40.26811640.21483329X-RAY DIFFRACTION75.95
2.4-2.440.28721700.21883593X-RAY DIFFRACTION81.79
2.44-2.470.30171680.21913940X-RAY DIFFRACTION86.32
2.47-2.50.27281600.21553959X-RAY DIFFRACTION90.37
2.5-2.540.3021600.20394172X-RAY DIFFRACTION92.96
2.54-2.580.26541890.20654195X-RAY DIFFRACTION93.88
2.58-2.620.2672240.2074219X-RAY DIFFRACTION96.34
2.62-2.670.2742360.21354347X-RAY DIFFRACTION98.03
2.67-2.720.28272320.22634327X-RAY DIFFRACTION98.19
2.72-2.770.28672700.21484335X-RAY DIFFRACTION97.98
2.77-2.830.29462150.20324282X-RAY DIFFRACTION98.45
2.83-2.890.29622020.20424413X-RAY DIFFRACTION98.48
2.89-2.950.25212380.19964299X-RAY DIFFRACTION98.57
2.95-3.030.26972390.19324357X-RAY DIFFRACTION98.54
3.03-3.110.25962260.18774364X-RAY DIFFRACTION98.58
3.11-3.20.24672090.18824352X-RAY DIFFRACTION98.62
3.2-3.30.25262260.18174384X-RAY DIFFRACTION98.8
3.3-3.420.24762020.17984402X-RAY DIFFRACTION98.59
3.42-3.560.19572220.16374380X-RAY DIFFRACTION98.73
3.56-3.720.23262680.16324300X-RAY DIFFRACTION99.15
3.72-3.920.20813040.16784291X-RAY DIFFRACTION98.48
3.92-4.160.20212400.16284361X-RAY DIFFRACTION99.5
4.16-4.480.1992930.14734342X-RAY DIFFRACTION99.12
4.49-4.940.19821890.1524398X-RAY DIFFRACTION98.94
4.94-5.650.19492420.16534376X-RAY DIFFRACTION99.59
5.65-7.110.21352770.17344352X-RAY DIFFRACTION99.44
7.11-49.240.21961920.17984417X-RAY DIFFRACTION99.5
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)
10.540253199245-0.262939029601-0.1620802469310.753320917132-0.05216392031820.479315404949-0.0166387904975-0.0227642194775-0.05119976402680.0442043153260.007760184503580.0400956771701-0.0481017118294-0.03024317851570.01266356534140.063749558313-0.01305418360370.01775873287640.0110603554841-0.02199834820180.06691270577992.08298289183-33.42843604-16.6830676524
20.255283576905-0.15621201806-0.02387552914380.379377308767-0.08926977924580.482095542805-0.0439347851262-0.03176140469070.0014256281259-0.0405597358834-0.01547675276450.00718553202749-0.01017397278190.02761297788550.03717542915260.0410138207789-0.0308056660984-0.01591018890210.0406023473581-0.004090300337980.03806896777673.749880665-7.17012452827-10.9934440181
30.122422391229-0.108265950066-0.02210362573280.3604775873090.07465794459460.367792161743-0.0321399689097-0.03408963829240.03180826435260.1132618953150.0062397832407-0.0843540095276-0.02314863639510.1195567276820.01867684253570.136081762019-0.0100324445994-0.04821970288760.06646190718550.01490066361620.04707876302267.25486635106-3.0049125844310.2813267603
40.026550182558-0.07904819105940.04690488771630.277375429018-0.003410315120350.543134449856-0.0423902458673-0.06414295084210.07498863108110.1386883442950.0467185413248-0.058543424841-0.1670060477350.01355136356310.006676358943840.1502325229130.0152728177697-0.0525809390350.042968771745-0.01646144583850.0648707890602-0.51287054549712.7462110562-2.86126790555
50.265031241579-0.0594613505425-0.1434995911320.471125680717-0.1750305816940.372499336384-0.010716074783-0.04651709149020.024893679859-0.09378249287570.0150660694752-0.06863037633460.060366955198-0.0137171456502-0.003925192047990.0575870484057-0.01011034451920.02060420394020.041241913004-0.01811601479980.07385356865124.79657785882-54.1028289874-23.272461606
60.09966438476770.0245106874758-0.04532306209410.4241679074020.08507831912750.350656069718-0.0308373751266-0.050157063564-0.08053462582870.1215476088320.02446186465290.08808850933320.0416262620269-0.1020958727750.01648671603570.07372701629840.00479514280130.05686202988190.07555114436250.0007018556050390.113377219746-4.00674237059-49.9361887499-3.6934740978
70.134826067180.0478553221253-0.05034230736340.427907835711-0.0364387138610.196051547155-0.0341241548991-0.02092455974770.03333250620840.0490771551657-0.00140166505239-0.107281722411-0.03865163798940.08711589287650.01636606000180.0278483121522-0.0153882694304-0.01995183742850.1108738324840.006145265379070.054524215353210.8553385995-10.841080363653.7940081888
80.2013016544250.0794744262682-0.05414593971840.3712557098660.03626418069510.370285388139-0.02607552475970.0620169152411-0.0184395708367-0.137958120958-0.00663039156861-0.08259118361090.003955199335980.0648052287940.01959555834070.09365180110990.006112778788740.02947438183040.09539123442690.02069891432540.041701222726510.8708293265-15.559759135132.3838431824
90.209583963331-0.05252125329590.02206081660030.673665556636-0.07670942479770.7096414476380.0227908271369-0.0060261321653-0.00976635447539-0.1428145487980.0109269743902-0.0841826039420.1633706949790.0954228479145-0.02891237062940.09145371036520.0173306874182-0.003564923802060.05995278706930.01051081645620.07250890306559.371247537-31.687311070647.2072641921
100.293936855437-0.0482469335495-0.1516393722410.4090609064270.148368565050.1420963517210.01674959098090.08288209129370.0668330660096-0.09693574373860.03632930163830.0748821380492-0.0500298516935-0.0757456515269-0.01200479020760.04080052667370.0163852577744-0.008693807120810.1042857721820.02136496758220.0901723511991-15.027926734741.3608815063-29.4672701204
110.139931548165-0.0413194874865-0.02764132213540.0799824500236-0.04197452321420.1438883153430.0167640102711-0.02879234757470.01469024356970.02493908423490.02809266341020.129365013836-0.0354220002499-0.135840070883-0.01922187095920.0352334229740.004860325262760.02739743538120.153606607414-0.0303787072480.16625493294-30.512203522438.3490670595-14.6213203794
120.246968106117-0.0170458453727-0.07143544179190.133908755241-0.006859100049570.2193657565020.006465657272080.0662238264819-0.0379874563533-0.061763119351-0.007609101017670.1432392154290.00897323843555-0.107572308092-0.0303239218260.0476805034704-0.0112173889152-0.05037032881190.08598689636610.01842743195730.128066983571-24.331963376522.0800097627-27.5082880492
130.1689432830120.1033876369260.05621099480830.587170665036-0.1205731054310.492167889132-0.04191580834270.0460809161357-0.0665788787889-0.1892900453750.01730322370710.004384781659460.14577289608-0.03225043746050.02570313053260.135437608327-0.0215152442050.01084802531760.07296279678260.01010718215660.04026002623253.88656767911-15.6462168147-36.3984378543
140.1187822903370.108665863038-0.008872507201570.266658101686-0.1974954629710.35404725329-0.03735644848390.0430435781971-0.0270024992946-0.210994799375-0.0123600885703-0.05810330538380.02843408876090.04086516191060.03137822584010.243400832943-0.01784169822480.009434009479250.06591615680270.0189728896420.07184343997678.503931875860.277536111548-50.878208056
150.225505938452-0.0736528592434-0.08277367141490.1783594538170.05053308154150.258973250871-0.03124893652930.140940929973-0.0145564245604-0.182096389456-0.02680630431320.03992124659810.0510842820823-0.0880128556269-0.01545011531270.418473744849-0.0723752917098-0.03326494855890.152371250044-0.01912345052860.0773590025286-1.36658903562-18.0839097639-57.6845022041
160.706351111490.287841403326-0.2196176813940.853776559709-0.04878945629820.5899556207090.0142860569005-0.1366513153750.08037002217780.303995431180.02209672830720.24547088728-0.289915837837-0.332955182877-0.03490433862910.3161699834970.07032248500180.05505425529080.3299311632750.02354631255520.151950549503-21.8562341783-40.315587517432.7645730903
170.501654178457-0.0718050838893-0.02936559017680.052322777890.06077818305870.224771920813-0.002854342764870.064212420025-0.08023103490030.06604163125650.08988378791380.334975553188-0.00121690716945-0.370310155257-0.06169112606950.173885346259-0.0228783785740.04429901390240.4862366158480.03698760486780.378122322705-36.6270865594-50.702490280520.9130123601
180.4581973515910.329243825511-0.1275470319540.7612985417470.3935880692030.568593840786-0.05341915305340.07503394627020.1732704599390.0600700665538-0.01560889678780.285089816342-0.311413304995-0.4091750060180.05115501755810.3309894954310.1550543489230.02518842652580.3742319188980.02597383961770.2069794618-28.7187368931-31.758568890714.228821182
190.03546803481840.0196388922560.01445530507260.20598501673-0.1022001586160.0534211110516-0.00365674649516-0.03576589614340.02596842616350.08907549587270.01798555825720.0718620304278-0.0540017839695-0.0367359682538-0.004286405435240.1461024034760.02385626749890.01174162949720.137235070288-0.01017908840250.147905009127-11.1942467297-2.6611298569913.6533464154
200.335958712129-0.322451269480.1377734283161.53141293348-0.1915029390491.68541960889-0.05498149848340.00816053836447-0.1304100939110.0157619619574-0.05320345551490.5244777750210.217492574518-0.3949476860520.09834298123450.306830113804-0.05587469361880.01471537595830.20397720975-0.06338697023870.299569340064-18.91412428433.2576430495829.4930394135
210.545336123096-0.133678233124-0.02027433606910.9576436728130.2585215440061.46679501973-0.06453518524890.0846454052069-0.00975935177078-0.193585372908-0.1234972538150.647226216244-0.113933536287-0.5164892910410.1555741165090.3735897239130.0800972092706-0.1101560955240.38400125959-0.09627508758090.58464417195-32.630083531119.596636102324.9932748797
Refinement TLS group

Refine-ID: X-RAY DIFFRACTION

IDRefine TLS-IDSelection detailsAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11(chain A and resseq 40:201)AA - B40 - 2011
22(chain B and resseq 43:179)BC43 - 1791 - 137
33(chain C and resseq 43:201)CD - E43 - 2011
44(chain D and resseq 43:201)DF - G43 - 2011
55(chain E and resseq 43:201)EH - I43 - 2011
66(chain F and resseq 43:201)FJ - K43 - 2011
77(chain G and resseq 43:179)GL - N43 - 1791
88(chain H and resseq 43:179)HO43 - 1791 - 137
99(chain I and resseq 43:179)IP43 - 1791 - 137
1010(chain J and resseq 40:179)JQ40 - 1791 - 140
1111(chain K and resseq 41:179)KR41 - 1791 - 139
1212(chain L and resseq 39:179)LS39 - 1791 - 140
1313(chain M and resseq 44:178)MT44 - 1781 - 135
1414(chain N and resseq 43:201)NU - V43 - 2011
1515(chain O and resseq 43:179)OW43 - 1791 - 137
1616(chain P and resseq 40:179)PX40 - 1791 - 140
1717(chain Q and resseq 41:179)QY41 - 1791 - 139
1818(chain R and resseq 39:179)RZ39 - 1791 - 141
1919(chain S and resseq 43:179)SAA - BA43 - 1791
2020(chain T and resseq 42:179)TCA42 - 1791 - 138
2121(chain U and resseq 42:179)UDA42 - 1791 - 138

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