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- PDB-7bep: Crystal structure of the receptor binding domain of SARS-CoV-2 Sp... -

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Basic information

Entry
Database: PDB / ID: 7bep
TitleCrystal structure of the receptor binding domain of SARS-CoV-2 Spike glycoprotein in a ternary complex with COVOX-384 and S309 Fabs
Components
  • COVOX-384 heavy chain
  • COVOX-384 light chain
  • S309 heavy chain
  • S309 light chain
  • Spike glycoproteinSpike protein
KeywordsVIRAL PROTEIN/IMMUNE SYSTEM / SARS-CoV-2 / antibody / germline / V-gene / receptor-binding-domain / spike / neutralisation / protection / glycosylation / valency / VIRAL PROTEIN / IMMUNE SYSTEM / VIRAL PROTEIN-IMMUNE SYSTEM complex
Function / homology
Function and homology information


Maturation of spike protein / viral translation / Translation of Structural Proteins / Virion Assembly and Release / host cell surface / host extracellular space / suppression by virus of host tetherin activity / Induction of Cell-Cell Fusion / structural constituent of virion / host cell endoplasmic reticulum-Golgi intermediate compartment membrane ...Maturation of spike protein / viral translation / Translation of Structural Proteins / Virion Assembly and Release / host cell surface / host extracellular space / suppression by virus of host tetherin activity / Induction of Cell-Cell Fusion / structural constituent of virion / host cell endoplasmic reticulum-Golgi intermediate compartment membrane / entry receptor-mediated virion attachment to host cell / receptor-mediated endocytosis of virus by host cell / Attachment and Entry / membrane fusion / positive regulation of viral entry into host cell / receptor-mediated virion attachment to host cell / receptor ligand activity / host cell surface receptor binding / fusion of virus membrane with host plasma membrane / fusion of virus membrane with host endosome membrane / viral envelope / symbiont-mediated suppression of host type I interferon-mediated signaling pathway / virion attachment to host cell / SARS-CoV-2 activates/modulates innate and adaptive immune responses / host cell plasma membrane / virion membrane / membrane / identical protein binding / plasma membrane
Similarity search - Function
Spike (S) protein S1 subunit, receptor-binding domain, SARS-CoV-2 / Spike (S) protein S1 subunit, N-terminal domain, SARS-CoV-like / Betacoronavirus spike (S) glycoprotein S1 subunit N-terminal (NTD) domain profile. / Spike glycoprotein, N-terminal domain superfamily / Betacoronavirus spike (S) glycoprotein S1 subunit C-terminal (CTD) domain profile. / Spike glycoprotein, betacoronavirus / Spike (S) protein S1 subunit, receptor-binding domain, betacoronavirus / Spike S1 subunit, receptor binding domain superfamily, betacoronavirus / Betacoronavirus spike glycoprotein S1, receptor binding / Spike glycoprotein S1, N-terminal domain, betacoronavirus-like ...Spike (S) protein S1 subunit, receptor-binding domain, SARS-CoV-2 / Spike (S) protein S1 subunit, N-terminal domain, SARS-CoV-like / Betacoronavirus spike (S) glycoprotein S1 subunit N-terminal (NTD) domain profile. / Spike glycoprotein, N-terminal domain superfamily / Betacoronavirus spike (S) glycoprotein S1 subunit C-terminal (CTD) domain profile. / Spike glycoprotein, betacoronavirus / Spike (S) protein S1 subunit, receptor-binding domain, betacoronavirus / Spike S1 subunit, receptor binding domain superfamily, betacoronavirus / Betacoronavirus spike glycoprotein S1, receptor binding / Spike glycoprotein S1, N-terminal domain, betacoronavirus-like / Betacoronavirus-like spike glycoprotein S1, N-terminal / Spike glycoprotein S2, coronavirus, heptad repeat 1 / Spike glycoprotein S2, coronavirus, heptad repeat 2 / Coronavirus spike (S) glycoprotein S2 subunit heptad repeat 2 (HR2) region profile. / Coronavirus spike (S) glycoprotein S2 subunit heptad repeat 1 (HR1) region profile. / Spike glycoprotein S2 superfamily, coronavirus / Spike glycoprotein S2, coronavirus / Coronavirus spike glycoprotein S2 / Coronavirus spike glycoprotein S1, C-terminal / Coronavirus spike glycoprotein S1, C-terminal
Similarity search - Domain/homology
GLUTAMIC ACID / GLYCINE / IMIDAZOLE / DI(HYDROXYETHYL)ETHER / TRIETHYLENE GLYCOL / Spike glycoprotein
Similarity search - Component
Biological speciesSevere acute respiratory syndrome coronavirus 2
Homo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.61 Å
AuthorsZhou, D. / Zhao, Y. / Ren, J. / Stuart, D.
Funding support United Kingdom, China, 2items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MR/N00065X/1 United Kingdom
CAMS Innovation Fund for Medical Sciences (CIFMS)2018-I2M-2-002 China
CitationJournal: Cell / Year: 2021
Title: The antigenic anatomy of SARS-CoV-2 receptor binding domain.
Authors: Wanwisa Dejnirattisai / Daming Zhou / Helen M Ginn / Helen M E Duyvesteyn / Piyada Supasa / James Brett Case / Yuguang Zhao / Thomas S Walter / Alexander J Mentzer / Chang Liu / Beibei Wang ...Authors: Wanwisa Dejnirattisai / Daming Zhou / Helen M Ginn / Helen M E Duyvesteyn / Piyada Supasa / James Brett Case / Yuguang Zhao / Thomas S Walter / Alexander J Mentzer / Chang Liu / Beibei Wang / Guido C Paesen / Jose Slon-Campos / César López-Camacho / Natasha M Kafai / Adam L Bailey / Rita E Chen / Baoling Ying / Craig Thompson / Jai Bolton / Alex Fyfe / Sunetra Gupta / Tiong Kit Tan / Javier Gilbert-Jaramillo / William James / Michael Knight / Miles W Carroll / Donal Skelly / Christina Dold / Yanchun Peng / Robert Levin / Tao Dong / Andrew J Pollard / Julian C Knight / Paul Klenerman / Nigel Temperton / David R Hall / Mark A Williams / Neil G Paterson / Felicity K R Bertram / C Alistair Siebert / Daniel K Clare / Andrew Howe / Julika Radecke / Yun Song / Alain R Townsend / Kuan-Ying A Huang / Elizabeth E Fry / Juthathip Mongkolsapaya / Michael S Diamond / Jingshan Ren / David I Stuart / Gavin R Screaton /
Abstract: Antibodies are crucial to immune protection against SARS-CoV-2, with some in emergency use as therapeutics. Here, we identify 377 human monoclonal antibodies (mAbs) recognizing the virus spike and ...Antibodies are crucial to immune protection against SARS-CoV-2, with some in emergency use as therapeutics. Here, we identify 377 human monoclonal antibodies (mAbs) recognizing the virus spike and focus mainly on 80 that bind the receptor binding domain (RBD). We devise a competition data-driven method to map RBD binding sites. We find that although antibody binding sites are widely dispersed, neutralizing antibody binding is focused, with nearly all highly inhibitory mAbs (IC < 0.1 μg/mL) blocking receptor interaction, except for one that binds a unique epitope in the N-terminal domain. Many of these neutralizing mAbs use public V-genes and are close to germline. We dissect the structural basis of recognition for this large panel of antibodies through X-ray crystallography and cryoelectron microscopy of 19 Fab-antigen structures. We find novel binding modes for some potently inhibitory antibodies and demonstrate that strongly neutralizing mAbs protect, prophylactically or therapeutically, in animal models.
History
DepositionDec 24, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 3, 2021Provider: repository / Type: Initial release
Revision 1.1Apr 7, 2021Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name
Revision 1.2Apr 28, 2021Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first
Revision 1.3Jan 31, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_entity_branch_descriptor / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
E: Spike glycoprotein
A: COVOX-384 heavy chain
B: COVOX-384 light chain
H: S309 heavy chain
L: S309 light chain
C: Spike glycoprotein
D: COVOX-384 heavy chain
F: COVOX-384 light chain
G: S309 heavy chain
I: S309 light chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)248,15043
Polymers243,57810
Non-polymers4,57233
Water32418
1
E: Spike glycoprotein
A: COVOX-384 heavy chain
B: COVOX-384 light chain
H: S309 heavy chain
L: S309 light chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)124,03921
Polymers121,7895
Non-polymers2,25016
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
C: Spike glycoprotein
D: COVOX-384 heavy chain
F: COVOX-384 light chain
G: S309 heavy chain
I: S309 light chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)124,11122
Polymers121,7895
Non-polymers2,32117
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)108.760, 113.221, 302.773
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121
Space group name HallP2ac2ab
Symmetry operation#1: x,y,z
#2: x+1/2,-y+1/2,-z
#3: -x,y+1/2,-z+1/2
#4: -x+1/2,-y,z+1/2

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Components

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Antibody , 4 types, 8 molecules ADBFHGLI

#2: Antibody COVOX-384 heavy chain


Mass: 26089.506 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Homo sapiens (human)
#3: Antibody COVOX-384 light chain


Mass: 24526.352 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Homo sapiens (human)
#4: Antibody S309 heavy chain


Mass: 24817.738 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Homo sapiens (human)
#5: Antibody S309 light chain


Mass: 23204.697 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Homo sapiens (human)

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Protein / Sugars , 2 types, 4 molecules EC

#1: Protein Spike glycoprotein / Spike protein / S glycoprotein / E2 / Peplomer protein


Mass: 23150.891 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Severe acute respiratory syndrome coronavirus 2
Gene: S, 2 / Production host: Homo sapiens (human) / References: UniProt: P0DTC2
#6: Polysaccharide alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]beta-D-mannopyranose-(1-4)-2-acetamido-2- ...alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-[alpha-L-fucopyranose-(1-6)]2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 1056.964 Da / Num. of mol.: 2 / Source method: obtained synthetically
DescriptorTypeProgram
DManpa1-3[DManpa1-6]DManpb1-4DGlcpNAcb1-4[LFucpa1-6]DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/4,6,5/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1122h-1a_1-5][a1221m-1a_1-5]/1-1-2-3-3-4/a4-b1_a6-f1_b4-c1_c3-d1_c6-e1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(3+1)][a-D-Manp]{}[(6+1)][a-D-Manp]{}}}[(6+1)][a-L-Fucp]{}}}LINUCSPDB-CARE

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Non-polymers , 9 types, 49 molecules

#7: Chemical
ChemComp-IMD / IMIDAZOLE / Imidazole


Mass: 69.085 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C3H5N2
#8: Chemical
ChemComp-CL / CHLORIDE ION / Chloride


Mass: 35.453 Da / Num. of mol.: 10 / Source method: obtained synthetically / Formula: Cl
#9: Chemical ChemComp-GLU / GLUTAMIC ACID / Glutamic acid


Type: L-peptide linking / Mass: 147.129 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C5H9NO4
#10: Chemical
ChemComp-GLY / GLYCINE / Glycine


Type: peptide linking / Mass: 75.067 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C2H5NO2
#11: Chemical ChemComp-PGE / TRIETHYLENE GLYCOL / Polyethylene glycol


Mass: 150.173 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H14O4
#12: Chemical
ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL / Glycerol


Mass: 92.094 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C3H8O3
#13: Chemical ChemComp-PG4 / TETRAETHYLENE GLYCOL / Polyethylene glycol


Mass: 194.226 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Formula: C8H18O5 / Comment: precipitant*YM
#14: Chemical ChemComp-PEG / DI(HYDROXYETHYL)ETHER / Diethylene glycol


Mass: 106.120 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C4H10O3
#15: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 18 / 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: 3.94 Å3/Da / Density % sol: 68.8 %
Crystal growTemperature: 294 K / Method: vapor diffusion, sitting drop / pH: 6.5
Details: containing 0.1 M amino acids (Glu, Ala, Gly, Lys, Ser), 0.1 M MES/imidazole/ pH 6.5, 10% (w/v) PEG 20000 and 20% (w/v) PEG MME 550.

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I03 / Wavelength: 0.97625 Å
DetectorType: DECTRIS EIGER2 XE 16M / Detector: PIXEL / Date: Sep 9, 2020
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97625 Å / Relative weight: 1
ReflectionResolution: 2.61→63 Å / Num. obs: 114437 / % possible obs: 100 % / Redundancy: 13.1 % / Biso Wilson estimate: 79.54 Å2 / CC1/2: 0.998 / Rmerge(I) obs: 0.112 / Rpim(I) all: 0.044 / Net I/σ(I): 13.3
Reflection shellResolution: 2.61→2.65 Å / Mean I/σ(I) obs: 0.5 / Num. unique obs: 5600 / CC1/2: 0.344

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Processing

Software
NameVersionClassification
GDA1.18.1_3865data collection
PHENIX1.18.1_3865refinement
xia2data reduction
xia2data scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 7BEK
Resolution: 2.61→62.93 Å / SU ML: 0.5916 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 33.7863
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2408 5577 4.89 %
Rwork0.2025 108563 -
obs0.2043 114140 99.78 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 99.07 Å2
Refinement stepCycle: LAST / Resolution: 2.61→62.93 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms16614 0 294 18 16926
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.003217313
X-RAY DIFFRACTIONf_angle_d0.631823516
X-RAY DIFFRACTIONf_chiral_restr0.04462657
X-RAY DIFFRACTIONf_plane_restr0.00442997
X-RAY DIFFRACTIONf_dihedral_angle_d18.11376241
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.61-2.640.47311840.44393449X-RAY DIFFRACTION97.24
2.64-2.670.42852100.433559X-RAY DIFFRACTION99.37
2.67-2.70.3891690.42093596X-RAY DIFFRACTION99.26
2.7-2.740.45671950.41363490X-RAY DIFFRACTION99.49
2.74-2.770.43881860.39663573X-RAY DIFFRACTION99.55
2.77-2.810.45361800.42193595X-RAY DIFFRACTION99.81
2.81-2.850.39911710.40163624X-RAY DIFFRACTION99.82
2.85-2.890.3961720.37263588X-RAY DIFFRACTION100
2.89-2.940.37111910.35013572X-RAY DIFFRACTION99.89
2.94-2.990.40331660.33353604X-RAY DIFFRACTION99.92
2.99-3.040.35432320.30843556X-RAY DIFFRACTION100
3.04-3.090.35431880.29413606X-RAY DIFFRACTION99.97
3.09-3.150.34171760.28683596X-RAY DIFFRACTION99.95
3.15-3.220.30861790.2693599X-RAY DIFFRACTION99.97
3.22-3.290.29861850.26573585X-RAY DIFFRACTION100
3.29-3.360.35411850.26833626X-RAY DIFFRACTION100
3.36-3.450.31121830.25573617X-RAY DIFFRACTION99.97
3.45-3.540.26231880.22813606X-RAY DIFFRACTION99.95
3.54-3.650.25332070.21443611X-RAY DIFFRACTION99.97
3.65-3.760.2671840.20913594X-RAY DIFFRACTION99.95
3.76-3.90.21181690.20173662X-RAY DIFFRACTION100
3.9-4.050.23411790.17993634X-RAY DIFFRACTION100
4.05-4.240.19991450.16423678X-RAY DIFFRACTION100
4.24-4.460.18611690.1443639X-RAY DIFFRACTION100
4.46-4.740.16571840.13193691X-RAY DIFFRACTION99.97
4.74-5.110.16831650.13713664X-RAY DIFFRACTION100
5.11-5.620.17811900.14953672X-RAY DIFFRACTION100
5.62-6.430.20082170.1743684X-RAY DIFFRACTION100
6.43-8.10.22032460.17723696X-RAY DIFFRACTION100
8.1-62.930.19031820.15963897X-RAY DIFFRACTION99.25
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)
18.030506857324.00270235298-1.069571046492.21894010466-0.6057777057180.1608288530620.63710567131-1.348625496020.4890304677231.27356187928-1.118483011280.01470418739070.350071483818-1.086037563440.8515585637381.034866757780.153008526067-0.06646711929541.6677314879-0.2266939124370.9963409596938.51209295027-15.970689687836.1727109995
22.592610684071.3666818272.627986400024.952400286653.124724383915.82648285762-0.0096422041309-0.804866929844-0.08464237283480.9320338936550.0994807072215-0.3253291900060.1862439382740.0687850511032-0.03249767318590.7896125389230.1021820233520.03848605034751.31316487386-0.04449809128650.6524791765411.929029772-22.274428322929.3979318866
31.489820405451.862866534211.014746739115.963533337141.704023098616.715154715730.406136692228-0.554866354290.1626019394070.1290162109430.0922582427678-0.1853591764830.2555461948850.158841858785-0.4237729633120.5464055258540.211377096420.05196209624570.9262412207480.019606751680.5832059668497.71182564236-25.224558665916.5546261242
42.23632210754-0.5511721071551.06981289840.0359738210186-0.8791233941843.06911729514-0.148589053538-0.3172591602270.2118346648390.01901454110080.0220960484278-0.316561318111-0.3900109133320.1335056233270.1075681432760.630640768510.1321309628090.08130485668390.961223472146-0.06103724921110.7300436310917.73058810233-19.26490689469.76178573374
54.59626541667-1.84663281990.7455420159535.53054035871-0.5944875750660.880281126853-0.273240335362-0.8200579232440.04458010602760.392812848010.5461013723860.442487886653-0.0130909794363-0.0649852872185-0.09019643306560.688955324030.1087697528320.2329068055530.7161697332540.03588488143620.719724877908-23.1752145168-12.7729796684-2.6988575999
65.14238989486-0.5428117224150.3535559707841.55183727760.1336435305131.47407855737-0.185862283348-0.3743185975510.2500032794630.2305305028830.2756688767660.042056001765-0.0009631083507220.0778598464096-0.09159518233010.7045374700930.1121829202840.1946269081990.6608310588650.01293965913040.68675971518-19.1604443533-8.78651093114-7.21132162889
77.08050145451-1.81057838604-3.05031294172.29082385462-0.03039447198765.23619991822-0.3058120312011.459708706931.00297495025-1.82333129974-0.08151997521740.350143975958-0.262236509053-2.595107342080.296510864920.913805652724-0.00563002419313-0.07567956498121.480885967920.4609075304961.1931971786-52.7117812687-5.87262843916-27.47586279
82.515821805260.6449769790050.2095623715725.4509558422.025777822383.439389861790.08374489175640.6942718754690.748523786077-0.3709741254230.2248731730780.21408319652-0.244044848647-1.10750194568-0.2657216052510.5637757342260.03216937490930.06034408558371.042134494340.2704829353540.914588586612-47.3168659687-7.48066927567-19.1868451075
90.7018164035960.45817287643-0.387681655020.728001985289-0.3802983093430.221506808841-0.1673980928890.1388612680340.98427518909-0.072182519111-0.360059997631-0.530371790476-1.062793572960.06376099425060.02517851232950.8765703432920.03615780462860.02874788045390.8668445037270.05009092905080.910120014619-6.05347900742-5.46038573365-20.0258271667
104.054730295380.4337651803171.898798602161.94495793751-1.05004815735.25793993346-0.3556571694240.893849162846-0.195442088498-0.4642519155020.435822947619-0.0988653339850.3187986413640.0699791283918-0.04141304402650.672548966778-0.03952109884180.1675594455870.670302539513-0.02491087938570.619100788461-17.0969610578-16.4936754643-27.6308573815
113.291091026820.4194634082-0.3468949526551.47551073845-0.02307775865992.603427518190.1435213906571.544184465971.78825165614-0.4804949280650.3044841893390.279629302486-0.466721052246-1.17635653299-0.3010702649470.9696913335420.1586584739780.005037336747071.649494979590.7860641936741.50188864594-47.84341380762.42129615218-33.0242015972
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