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Exploring Cooperative Domain Dynamics in Thermus thermophilus Leucyl-tRNA Synthetase Using...
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Exploring Cooperative Domain Dynamics in Thermus thermophilus Leucyl- tRNA Synthetase Using Low-frequency Normal Mode Calculations and Statistical Coupling Analysis Kristina Weimer , Brianne Shane , Michael Brunetto, Sudeep Bhattacharyay, and Sanchita Hati Department of Chemistry, University of Wisconsin–Eau Claire, WI-54702 Acknowledgements: Research Corporation Cottrell College Science Award UWEC-Office of Research and Sponsored Programs 7.46 Å 5.12 Å Three lowest-frequency normal modes obtained from ANM calculations using open-form structure of Tt LeuRS References 1. Cusack, S., Yaremchuk, A., and Tukalo, M. (2000) EMBO J. 19, 2351-2361. 2. Tukalo, M., Yaremchuk, A., Fukunaga, R., Yokoyama, S., and Cusack, S. (2005) Nat. Struct. Mol. Biol. 12, 923-930. 3. Tirion, M. M. (1996) Phys. Rev. Lett. 77, 1905- 1908. 4. Eyal, E., Yang, L. W., and Bahar, I. (2006) Bioinformatics 22, 2619-2627. 5. Lockless, S. W., and Ranganathan, R. (1999) Science 286, 295-299. 6. Lue, S. W. (2007) Biochemistry 46, 2266-4472. 7. Betha, A. K., Williams, A. M., Martinis, S. A. (2007) Biochemistry 46,6258-6267. 8.Williams, A.M., and Martinis, S.A. (2006) PNAS 103, 3586-3591. 9.Du, X., and Wang, E. (2002) Biochemistry 41, 10623- 10628. Abstract Leucyl-tRNA synthetases are class I synthetases that catalyze the covalent attachment of leucine to the tRNA Leu . The three-dimensional crystal structure of Thermus thermophilus leucyl-tRNA synthetase (Tt LeuRS) demonstrates a complex modular architecture where three flexible domains [the conserved connective polypeptide 1 (CP1) domain (residues 224-417), the leucine-specific (LS) domain (residues 577-634), and the zinc-1 (ZN-1) binding domain (residues 154-189)] are inserted into the central catalytic domain (1). The crystal structure of the Tt LeuRS-tRNA Leu complex (in post-transfer-editing conformation) demonstrated that the CP1 domain undergoes a rotation of 35 from the position observed in the tRNA unbound form. The LS domain, which is critical for aminoacylation, undergoes a rigid-body rotation of 19(2). Various structural elements (including catalytically important H 49 MGH and V 638 MSKS loops) in the central catalytic core also undergo considerable conformational changes due to leucyl-adenylate binding (1). These substrate induced conformational rearrangements of various structural elements of Tt LeuRS suggest that cooperative domain dynamics play an important role in the enzyme function. In the present work, we have investigated the collective motion of various structural elements in Tt LeuRS using normal mode calculations. In addition, statistical coupling analysis has been performed to examine if the evolutionarily coupled networks of residues have significant contributions to these concerted domain motions. Taken together, these studies demonstrate that domain motions in Tt LeuRS are indeed cooperative in nature and lead to the identification of the network of residues that propagate long-range interdomain communications in this enzyme. Statistical Coupling Analysis (SCA) SCA is based upon the assumption that “coupling of two sites in a protein, whether for structural or functional reasons, should cause those two positions to co-evolve” (5). The overall evolutionarily conservation parameter at a position i in the sequence of the chosen protein family is calculated and expressed as where kT* is an arbitrary energy unit, P i x is the probability of any amino acid x at site i, and P MSA x is the probability of x in the MSA. The coupling of site i with site j is calculated and expressed as where P i x | j is the probability of x at site i dependent on perturbation at site j. We performed SCA on an alignment of 484 protein sequences of LeuRS family. The SCA was performed by systematically perturbing each position where a specific amino acid was present in at least 50% of the sequences in the alignment. The initial clustering Properties Mode 1 Mode 2 Mode 3 Overlap (direction of motion) 0.72 0.60 0.71 Correlation (magnitude of motion) 0.51 0.56 0.31 Collectivity 0.37 0.20 0.42 2 stat ] ) [ln( * x x MSA x i i P P kT G 2 , )] / ln( ) [ln( * x MSA x i x j x MSA j x i stat j i P P P P kT G a) A cartoon diagram of the structure of T. Thermophilus LeuRS. b) tRNA Leu unbound (1h3n, magenta) and bound (2byt, cyan) structures were superimposed. To understand the domain dynamics of Tt LeuRS at the molecular level and to identify residue networks that mediate domain-domain communications in this enzyme. Objectives 35° 19° Editing Domain Leucine- Specific Domain C- terminus MSKS motif Catalyti c Domain Zinc-1 Binding Domain Normal Mode Analysis (NMA) The collective motion of the structural elements of a large biomolecule can be represented by normal modes. It is believed that the lowest frequency (large-amplitude) normal modes of a multi-domain enzyme describe the functionally relevant motions. Normal mode calculation is based on the harmonic approximation of the potential energy function around a minimum energy conformation. In this work NMA was carried out using the elastic network model (3). In the elastic network model, protein residues are represented by only their C α atoms. The C α atoms on a protein backbone are considered to be connected by uniform springs and the harmonic potential is given by: where d pq is the distance between atoms p and q, d pq 0 is the distance between these two atoms in the given crystallographic structure, C is the strength of the potential, and R c is an arbitrary cut-off parameter which defines the maximum interaction range between C α atoms. In this work Anisotropic Network Model (ANM) is used to describe functionally important collective motions of Tt LeuRS . In ANM the fluctuations are anisotropic and the overall potential of the system is a sum of the harmonic potentials given by: where represents the uniform spring constant, R pq 0 and R pq are the original and instantaneous distance vectors c pq R d pq pq p d d C E o o 2 ) ( p q q pq pq pq R R V , 2 o ANM ) ( 2 Overlap, Correlation , and collectivity value of the three lowest-frequency modes involved in the conformational change Collective Domain Motions in Tt LeuRS is Best Described by Mode 1 Mode 1 Mode 2 Mode 3 NMA Study Revealed Anticorrelation Between the Fluctuations of Structural Elements in LS and CP1 Domains a) Cross-correlations map for residue fluctuations in mode 1 that is most involved in the conformational change. Correlated motion is shown by positive values up to 1 (green to red) and anticorrelated motion is shown by negative values down to -1 (cyan to blue); b) Protein segments engaged in correlated and anticorrelated motion with respect to LS domain (green) are colored in red and blue, respectively. a) b) a) b) SCA of LeuRS family. a) the color scale linearly maps the data from 0 kT * (blue) to 1 kT* (red); b) the unclustered matrix; c) statistical coupling matrix where rows represent positions (N to C terminus, top to bottom) and columns represent perturbations (N to C terminus, left to right); d-f) Two dimensional clustering showing three separate co-evolving networks. Stage-2: Re-clustering Stage-1: Clustering 644 641 640 638 639 648 637 684 286 837 556 770 789 89 100 216 108 543 534 126 283 53 663 54 577 61 177 202 491 446 454 266 128 55 496 500 499 170 501 152 755 185 723 354 109 642 647 595 102 287 423 185 61 330 539 162 313 6 675 681 722 59 126 543 327 13 268 295 75 189 254 88 454 152 534 552 266 446 491 50 54 663 577 53 89 216 283 326 354 55 496 499 500 170 108 128 501 100 177 644 641 640 638 639 648 637 684 286 837 556 770 789 89 100 216 108 543 534 126 283 53 663 54 577 61 177 202 491 446 454 266 128 55 496 500 499 170 501 152 755 185 723 354 109 642 647 595 102 287 423 185 61 330 539 162 313 6 675 681 722 59 126 543 327 13 268 295 75 189 254 88 454 152 534 552 266 446 491 50 54 663 577 53 89 216 283 326 354 55 496 499 500 170 108 128 501 100 177 499 500 496 53 89 216 55 170 501 577 663 283 13 75 50 54 108 128 162 595 326 287 330 423 313 37 189 520 254 61 266 539 295 354 102 642 647 188 263 606 268 299 598 100 177 572 674 557 162 647 539 642 675 6 722 681 50 330 326 268 287 299 188 423 102 313 811 58 528 499 500 496 53 89 216 55 170 501 577 663 283 13 75 50 54 108 128 162 595 326 287 330 423 313 37 189 520 254 61 266 539 295 354 102 642 647 188 263 606 268 299 598 100 177 572 674 557 162 647 539 642 675 6 722 681 50 330 326 268 287 299 188 423 102 313 811 58 528 162 647 642 330 326 675 539 50 6 268 299 287 423 313 188 102 528 811 722 681 58 674 557 572 190 649 643 93 446 491 6 152 454 534 552 557 681 675 722 66 464 770 723 136 788 298 300 583 202 273 766 398 402 107 667 738 498 528 39 645 674 811 58 572 162 647 642 330 326 675 539 50 6 268 299 287 423 313 188 102 528 811 722 681 58 674 557 572 190 649 643 93 446 491 6 152 454 534 552 557 681 675 722 66 464 770 723 136 788 298 300 583 202 273 766 398 402 107 667 738 498 528 39 645 674 811 58 572 a) b) c) d) e) f) Statistically Coupled Residues in Tt LeuRS Conclusions Cooperative Domain Dynamics: The NMA study demonstrates that mode 1 adequately describes the conformational change in the Tt LeuRS. Analysis of the motion indicates that the LS and CP1 domains are engaged in anticorrelated motion. Coevolved Residue Network: SCA has identified a core set of residues which are evolutionarily coupled and reside at the domain interface. They form a sparse but contiguous network of interactions between the domains. Identifying a Functionally Relevant Network: Combined results of the NMA and SCA have produced a subset of residues which are not only correlated by evolution but also are coupled by thermal motions. These residues are within the van der Waals contact and appear to be critical for maintaining key structural scaffolds and domain dynamics in Tt LeuRS. Mutational Data •Existing E. coli (Ec) LeuRS mutational data demonstrates that mutation of some of these evolutionarily and thermally coupled residues have a strong impact on enzyme function. For example, mutation of a single residue at the interface of LS and catalytic domains (position 577) alters amino acid discrimination and tRNA aminoacylation (6). •Mutation of positions 292 and 188 have significant effect on enzyme catalysis. •Various constructs of Ec LeuRS, obtained by deleting the “hinge” regions, have a profound effect on editing reaction. Some of these hydrophobic “hinge” residues (position 423 and 216) are thermally and evolutionarily coupled with the main body of the enzyme (7). •These mutational results support the validity of this combined NMA-SCA approach to identify the important residues which are involved in maintaining the cooperative domain dynamics. Future work: Mutational studies to further explore the role of networking residues (identified in this work) that Mapping of the thermally and evolutionarily coupled residue network on the 3D structure of Tt LeuRS. Out of 876 residues of Tt LeuRS, only 58 residues were identified which exhibit strong co-evolutionary pattern of variations as well as coupled dynamics. SCA NMA Thermally Coupled and Coevolved Residues 55 58 102 126 162 423 539 543 556 557 572 637 639 640 642 647 663 674 37 39 50 53 54 61 75 89 93 100 107 108 128 156 170 177 188 189 216 292 498 499 500 501 520 534 552 577 598 602 638 641 643 644 645 648 667 675 681 738 0.9 0.0 0.1 0.2 0.3 0.4 0.8 0.7 0.6 0.5 1.0 55 58 102 126 162 423 539 543 556 557 572 637 639 640 642 647 663 674 37 39 50 53 54 61 75 89 93 100 107 108 128 156 170 177 188 189 216 292 498 499 500 501 520 534 552 577 598 602 638 641 643 644 645 648 667 675 681 738 55 58 102 126 162 423 539 543 556 557 572 637 639 640 642 647 663 674 37 39 50 53 54 61 75 89 93 100 107 108 128 156 170 177 188 189 216 292 498 499 500 501 520 534 552 577 598 602 638 641 643 644 645 648 667 675 681 738 0.9 0.0 0.1 0.2 0.3 0.4 0.8 0.7 0.6 0.5 1.0 0.9 0.0 0.1 0.2 0.3 0.4 0.8 0.7 0.6 0.5 1.0 55 58 102 126 162 423 539 543 556 557 572 637 639 640 642 647 663 674 37 39 50 53 54 61 75 89 93 100 107 108 128 156 170 177 188 189 216 292 498 499 500 501 520 534 552 577 598 602 638 641 643 644 645 648 667 675 681 738 0.8 1.0 0.8 0.6 0.4 0.2 0.6 0.4 0.2 0.0 1.0 55 58 102 126 162 423 539 543 556 557 572 637 639 640 642 647 663 674 37 39 50 53 54 61 75 89 93 100 107 108 128 156 170 177 188 189 216 292 498 499 500 501 520 534 552 577 598 602 638 641 643 644 645 648 667 675 681 738 0.8 1.0 0.8 0.6 0.4 0.2 0.6 0.4 0.2 0.0 1.0 55 58 102 126 162 423 539 543 556 557 572 637 639 640 642 647 663 674 37 39 50 53 54 61 75 89 93 100 107 108 128 156 170 177 188 189 216 292 498 499 500 501 520 534 552 577 598 602 638 641 643 644 645 648 667 675 681 738 55 58 102 126 162 423 539 543 556 557 572 637 639 640 642 647 663 674 37 39 50 53 54 61 75 89 93 100 107 108 128 156 170 177 188 189 216 292 498 499 500 501 520 534 552 577 598 602 638 641 643 644 645 648 667 675 681 738 0.8 1.0 0.8 0.6 0.4 0.2 0.6 0.4 0.2 0.0 1.0 0.8 1.0 0.8 0.6 0.4 0.2 0.6 0.4 0.2 0.0 1.0
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Transcript of Exploring Cooperative Domain Dynamics in Thermus thermophilus Leucyl-tRNA Synthetase Using...
Exploring Cooperative Domain Dynamics in Thermus thermophilus Leucyl-tRNA Synthetase Using Low-frequency Normal Mode Calculations and Statistical Coupling Analysis
Kristina Weimer, Brianne Shane, Michael Brunetto, Sudeep Bhattacharyay, and Sanchita HatiDepartment of Chemistry, University of Wisconsin–Eau Claire, WI-54702
Acknowledgements: Research Corporation Cottrell College Science Award UWEC-Office of Research and Sponsored Programs
7.46 Å
5.12 Å
Three lowest-frequency normal modes obtained from ANM calculations using open-form structure of Tt LeuRS
References1. Cusack, S., Yaremchuk, A., and Tukalo, M. (2000) EMBO J. 19, 2351-2361.2. Tukalo, M., Yaremchuk, A., Fukunaga, R., Yokoyama, S., and Cusack, S. (2005) Nat. Struct. Mol. Biol. 12, 923-930.3. Tirion, M. M. (1996) Phys. Rev. Lett. 77, 1905-1908.4. Eyal, E., Yang, L. W., and Bahar, I. (2006) Bioinformatics 22, 2619-2627.
5. Lockless, S. W., and Ranganathan, R. (1999) Science 286, 295-299.6. Lue, S. W. (2007) Biochemistry 46, 2266-4472.7. Betha, A. K., Williams, A. M., Martinis, S. A. (2007) Biochemistry 46,6258-6267.8.Williams, A.M., and Martinis, S.A. (2006) PNAS 103, 3586-3591.9.Du, X., and Wang, E. (2002) Biochemistry 41, 10623-10628.
Abstract
Leucyl-tRNA synthetases are class I synthetases that catalyze the covalent attachment of leucine to the tRNALeu. The three-dimensional crystal structure of Thermus thermophilus leucyl-tRNA synthetase (Tt LeuRS) demonstrates a complex modular architecture where three flexible domains [the conserved connective polypeptide 1 (CP1) domain (residues 224-417), the leucine-specific (LS) domain (residues 577-634), and the zinc-1 (ZN-1) binding domain (residues 154-189)] are inserted into the central catalytic domain (1). The crystal structure of the Tt LeuRS-tRNALeu complex (in post-transfer-editing conformation) demonstrated that the CP1 domain undergoes a rotation of 35 from the position observed in the tRNA unbound form. The LS domain, which is critical for aminoacylation, undergoes a rigid-body rotation of 19(2). Various structural elements (including catalytically important H49MGH and V638MSKS loops) in the central catalytic core also undergo considerable conformational changes due to leucyl-adenylate binding (1). These substrate induced conformational rearrangements of various structural elements of Tt LeuRS suggest that cooperative domain dynamics play an important role in the enzyme function. In the present work, we have investigated the collective motion of various structural elements in Tt LeuRS using normal mode calculations. In addition, statistical coupling analysis has been performed to examine if the evolutionarily coupled networks of residues have significant contributions to these concerted domain motions. Taken together, these studies demonstrate that domain motions in Tt LeuRS are indeed cooperative in nature and lead to the identification of the network of residues that propagate long-range interdomain communications in this enzyme.
Statistical Coupling Analysis (SCA)
SCA is based upon the assumption that “coupling of two sites in a protein, whether for structural or functional reasons, should cause those two positions to co-evolve” (5). The overall evolutionarily conservation parameter at a position i in the sequence of the chosen protein family is calculated and expressed as
where kT* is an arbitrary energy unit, Pix is the probability of any amino acid x at site
i, and PMSAx is the probability of x in the MSA. The coupling of site i with site j is
calculated and expressed as
where Pix |j is the probability of x at site i dependent on perturbation at site j.
We performed SCA on an alignment of 484 protein sequences of LeuRS family. The SCA was performed by systematically perturbing each position where a specific amino acid was present in at least 50% of the sequences in the alignment. The initial clustering resulted in a matrix with 876 (residue number) 216 (perturbation site) matrix elements representing the coupling between residues. The SCA on the LeuRS family demonstrates a group of residues which have coevolved in the Tt LeuRS.
Properties Mode 1 Mode 2 Mode 3
Overlap (direction of motion)
0.72 0.60 0.71
Correlation (magnitude of motion)
0.51 0.56 0.31
Collectivity 0.37 0.20 0.42
2stat ])[ln(* x
xMSA
xii PPkTG
2, )]/ln()[ln(* x
MSAxi
xj
xMSAj
xi
statji PPPPkTG
a) A cartoon diagram of the structure of T. Thermophilus LeuRS. b) tRNALeu unbound (1h3n, magenta) and bound (2byt, cyan) structures were superimposed.
To understand the domain dynamics of Tt LeuRS at the molecular level and to identify residue networks that mediate domain-domain communications in this enzyme.
Objectives
35°
19°
Editing Domain
Leucine-Specific Domain
C-terminus
MSKS motif
Catalytic Domain
Zinc-1 Binding Domain
Normal Mode Analysis (NMA)
The collective motion of the structural elements of a large biomolecule can be represented by normal modes. It is believed that the lowest frequency (large-amplitude) normal modes of a multi-domain enzyme describe the functionally relevant motions. Normal mode calculation is based on the harmonic approximation of the potential energy function around a minimum energy conformation. In this work NMA was carried out using the elastic network model (3). In the elastic network model, protein residues are represented by only their Cα atoms. The Cα atoms on a protein backbone are considered to be connected by uniform springs and the harmonic potential is given by:
where dpq is the distance between atoms p and q, dpq0 is the distance between these
two atoms in the given crystallographic structure, C is the strength of the potential, and Rc is an arbitrary cut-off parameter which defines the maximum interaction range between Cα atoms.
In this work Anisotropic Network Model (ANM) is used to describe functionally important collective motions of Tt LeuRS . In ANM the fluctuations are anisotropic and the overall potential of the system is a sum of the harmonic potentials given by:
where represents the uniform spring constant, Rpq0 and Rpq are the original and
instantaneous distance vectors between residues p and q, Γpq is the pq-th element of the connectivity matrix of inter-residue contacts. Based on an interaction cut-off distance of Rc, Γpq is equal to 1 if Rpq
0 < Rc and zero otherwise (4). The online server http://ignmtest.ccbb.pitt.edu/cgi-bin/anm/anm1.cgi was used to analyze the functional motion of Tt LeuRS. The optimal cut-off interactions between Cα atoms was kept at 15Å.
cpq Rd
pqpqp ddCEo
o 2)(
pqq
pqpqpq RRV,
2oANM )(
2
Overlap, Correlation , and collectivity value of the three lowest-frequency modes involved in the conformational change
Collective Domain Motions in Tt LeuRS is Best Described by Mode 1
Mode 1 Mode 2 Mode 3
NMA Study Revealed Anticorrelation Between the Fluctuations of Structural Elements in LS and CP1 Domains
a) Cross-correlations map for residue fluctuations in mode 1 that is most involved in the conformational change. Correlated motion is shown by positive values up to 1 (green to red) and anticorrelated motion is shown by negative values down to -1 (cyan to blue); b) Protein segments engaged in correlated and anticorrelated motion with respect to LS domain (green) are colored in red and blue, respectively.
a) b)
a) b)
SCA of LeuRS family. a) the color scale linearly maps the data from 0 kT* (blue) to 1 kT* (red); b) the unclustered matrix; c) statistical coupling matrix where rows represent positions (N to C terminus, top to bottom) and columns represent perturbations (N to C terminus, left to right); d-f) Two dimensional clustering showing three separate co-evolving networks.
Stage-2: Re-clusteringStage-1: Clustering
644
641
640
638
639
648
637
684
286
837
556
770
789
89 100
216
108
543
534
126
283
53 663
54 577
61 177
202
491
446
454
266
128
55 496
500
499
170
501
152
755
185
723
354
109
642647595102287423185
61330539162313
6675681722
59126543327
13268295
75189254
884541525345522664464915054
6635775389
21628332635455
496499500170108128501100177
644
641
640
638
639
648
637
684
286
837
556
770
789
89 100
216
108
543
534
126
283
53 663
54 577
61 177
202
491
446
454
266
128
55 496
500
499
170
501
152
755
185
723
354
109
642647595102287423185
61330539162313
6675681722
59126543327
13268295
75189254
884541525345522664464915054
6635775389
21628332635455
496499500170108128501100177
499500496
5389
21655
170501577663283
13755054
108128162595326287330423313
37189520254
61266539295354102642647188263606268299598100177
572
674
557
162
647
539
642
675
6 722
681
50 330
326
268
287
299
188
423
102
313
811
58 528
499500496
5389
21655
170501577663283
13755054
108128162595326287330423313
37189520254
61266539295354102642647188263606268299598100177
572
674
557
162
647
539
642
675
6 722
681
50 330
326
268
287
299
188
423
102
313
811
58 528
162
647
642
330
326
675
539
50 6 268
299
287
423
313
188
102
528
811
722
681
58 674
557
572
19064964393
446491
615245453455255768167572266
46477072313678829830058320227376639840210766773849852839
64567481158
572
162
647
642
330
326
675
539
50 6 268
299
287
423
313
188
102
528
811
722
681
58 674
557
572
19064964393
446491
615245453455255768167572266
46477072313678829830058320227376639840210766773849852839
64567481158
572
a) b) c) d)
e) f)
Statistically Coupled Residues in Tt LeuRS
ConclusionsCooperative Domain Dynamics: The NMA study demonstrates that mode 1
adequately describes the conformational change in the Tt LeuRS. Analysis of the motion indicates that the LS and CP1 domains are engaged in anticorrelated motion.
Coevolved Residue Network: SCA has identified a core set of residues which are evolutionarily coupled and reside at the domain interface. They form a sparse but contiguous network of interactions between the domains.
Identifying a Functionally Relevant Network: Combined results of the NMA and SCA have produced a subset of residues which are not only correlated by evolution but also are coupled by thermal motions. These residues are within the van der Waals contact and appear to be critical for maintaining key structural scaffolds and domain dynamics in Tt LeuRS.
Mutational Data• Existing E. coli (Ec) LeuRS mutational data demonstrates that mutation of
some of these evolutionarily and thermally coupled residues have a strong impact on enzyme function. For example, mutation of a single residue at the interface of LS and catalytic domains (position 577) alters amino acid discrimination and tRNA aminoacylation (6).
• Mutation of positions 292 and 188 have significant effect on enzyme catalysis.• Various constructs of Ec LeuRS, obtained by deleting the “hinge” regions, have
a profound effect on editing reaction. Some of these hydrophobic “hinge” residues (position 423 and 216) are thermally and evolutionarily coupled with the main body of the enzyme (7).
• These mutational results support the validity of this combined NMA-SCA approach to identify the important residues which are involved in maintaining the cooperative domain dynamics.
Future work: Mutational studies to further explore the role of networking residues (identified in this work) that mediate long-range communications between domains.
Mapping of the thermally and evolutionarily coupled residue network on the 3D structure of Tt LeuRS. Out of 876 residues of Tt LeuRS, only 58 residues were identified which exhibit strong co-evolutionary pattern of variations as well as coupled dynamics.
SCA NMA
Thermally Coupled and Coevolved Residues
55 58 102
126
162
423
539
543
556
557
572
637
639
640
642
647
663
674
373950535461758993
100107108128156170177188189216292498499500501520534552577598602638641643644645648667675681738
0.9
0.0
0.1
0.2
0.3
0.4
0.8
0.7
0.6
0.5
1.0
55 58 102
126
162
423
539
543
556
557
572
637
639
640
642
647
663
674
373950535461758993
100107108128156170177188189216292498499500501520534552577598602638641643644645648667675681738
55 58 102
126
162
423
539
543
556
557
572
637
639
640
642
647
663
674
373950535461758993
100107108128156170177188189216292498499500501520534552577598602638641643644645648667675681738
0.9
0.0
0.1
0.2
0.3
0.4
0.8
0.7
0.6
0.5
1.0
0.9
0.0
0.1
0.2
0.3
0.4
0.8
0.7
0.6
0.5
1.0
55 58 102
126
162
423
539
543
556
557
572
637
639
640
642
647
663
674
373950535461758993
100107108128156170177188189216292498499500501520534552577598602638641643644645648667675681738
0.8
1.0
0.8
0.6
0.4
0.2
0.6
0.4
0.2
0.0
1.0
55 58 102
126
162
423
539
543
556
557
572
637
639
640
642
647
663
674
373950535461758993
100107108128156170177188189216292498499500501520534552577598602638641643644645648667675681738
0.8
1.0
0.8
0.6
0.4
0.2
0.6
0.4
0.2
0.0
1.0
55 58 102
126
162
423
539
543
556
557
572
637
639
640
642
647
663
674
373950535461758993
100107108128156170177188189216292498499500501520534552577598602638641643644645648667675681738
55 58 102
126
162
423
539
543
556
557
572
637
639
640
642
647
663
674
373950535461758993
100107108128156170177188189216292498499500501520534552577598602638641643644645648667675681738
0.8
1.0
0.8
0.6
0.4
0.2
0.6
0.4
0.2
0.0
1.0
0.8
1.0
0.8
0.6
0.4
0.2
0.6
0.4
0.2
0.0
1.0
