D-I-TASSER Q66PJ3-D1

D-I-TASSER results for Q66PJ3-D1

[Click result.zip to download all results on this page]

Input Sequence in FASTA format

>Q66PJ3 (421 residues)
MPRCTYQLEQNPGFLPDGPGVHARAHCQDLSGPYGHEFATSESLGGRVGKTRAPQSGARS
RMERAGPAGEEGGAREGRLLPRAPGAWVLRACAERAALEVGAASADTGVRGCGARGPAPL
LASAGGGRARDGTWGVRTKGSGAALPSRPASRAAPRPEASSPPLPLEKARGGLSGPQGGR
ARGAMAHVGSRKRSRSRSRSRGRGSEKRKKKSRKDTSRNCSASTSQGRKASTAPGAEASP
SPCITERSKQKARRRTRSSSSSSSSSSSSSSSSSSSSSSSSSDGRKKRGKYKDKRRKKKK
KRKKLKKKGKEKAEAQQVEALPGPSLDQWHRSAGEEEDGPVLTDEQKSRIQAMKPMTKEE
WDARQSIIRKVVDPETGRTRLIKGDGEVLEEIVTKERHREINKQATRGDCLAFQMRAGLL
P

Predicted Secondary Structure

Sequence	20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| MPRCTYQLEQNPGFLPDGPGVHARAHCQDLSGPYGHEFATSESLGGRVGKTRAPQSGARSRMERAGPAGEEGGAREGRLLPRAPGAWVLRACAERAALEVGAASADTGVRGCGARGPAPLLASAGGGRARDGTWGVRTKGSGAALPSRPASRAAPRPEASSPPLPLEKARGGLSGPQGGRARGAMAHVGSRKRSRSRSRSRGRGSEKRKKKSRKDTSRNCSASTSQGRKASTAPGAEASPSPCITERSKQKARRRTRSSSSSSSSSSSSSSSSSSSSSSSSSDGRKKRGKYKDKRRKKKKKRKKLKKKGKEKAEAQQVEALPGPSLDQWHRSAGEEEDGPVLTDEQKSRIQAMKPMTKEEWDARQSIIRKVVDPETGRTRLIKGDGEVLEEIVTKERHREINKQATRGDCLAFQMRAGLLP
Prediction	CCCCHHHHHCCCCCCCCCCCCCCHHHHHCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCHHHHHHHCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCHHHCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCHHHHCCCCCCCCCCCCCCCCCCCCCCCCCCCCCHHHHHHCCCCCHHHHHHHHHHHHHHHHHHCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCHHHHHCCCCCCCCCHHHHHHHCCSSSSSSCCCCCCSSSSCCCCHHHHHHHHHHHHHHHHHHCCCCCHHHHHHHHCCCC
Confidence	9851344531899899998732011111147875544444424477567777886666676545786533345677888898721234331555555555667777766677899986322467788654567665667888889877776667765567999730002467889862224433233467777654334776653011121001355432223576766677543466776654311134422477889988665555665678877654302333135521122343211112345442124445666678877554444444456777655455421224577661234442041466644666734565077167888874666777642111233477776416899
	H:Helix; S:Strand; C:Coil

Predicted Solvent Accessibility

Sequence	20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| \| MPRCTYQLEQNPGFLPDGPGVHARAHCQDLSGPYGHEFATSESLGGRVGKTRAPQSGARSRMERAGPAGEEGGAREGRLLPRAPGAWVLRACAERAALEVGAASADTGVRGCGARGPAPLLASAGGGRARDGTWGVRTKGSGAALPSRPASRAAPRPEASSPPLPLEKARGGLSGPQGGRARGAMAHVGSRKRSRSRSRSRGRGSEKRKKKSRKDTSRNCSASTSQGRKASTAPGAEASPSPCITERSKQKARRRTRSSSSSSSSSSSSSSSSSSSSSSSSSDGRKKRGKYKDKRRKKKKKRKKLKKKGKEKAEAQQVEALPGPSLDQWHRSAGEEEDGPVLTDEQKSRIQAMKPMTKEEWDARQSIIRKVVDPETGRTRLIKGDGEVLEEIVTKERHREINKQATRGDCLAFQMRAGLLP
Prediction	6540354047424112724222031205415232224103454133434444334554444255344345534545444245323331234235433364344444433444455332432445534444644444434544441344425544435455545444545545455554542443445436564563556455555565445545556544553655645645465465545654664556535546555455545545554565465555565555565545555555546654455556555545546544545454344554646634433544455444345445652544443034213563233211415140043014463145035413432142034425237
	Values range from 0 (buried residue) to 8 (highly exposed residue)

Predicted Contact, Hydrogen and Distance Map Used in D-I-TASSER simulation

Contact Map

Distance Map

Hydrogen bond networks

D-I-TASSER simulation is guided by the consensus contact map (left figure), distance map (middle figure) and Hydrogen bond network (right figure) derived based on confidence scores of AttentionPotential. In the contact, distance map and hydrogen bond networks, the axes mark the residue index along the sequence. For the contact map, each dot represents a residue pair with predicted contact, while for the distance map and hydrogen bond network, a color scale represents a distance of 1-20+ angstroms or a angle of 0-180 degree.

Top 10 threading templates used by D-I-TASSER

Rank

PDB
hit

ID1

ID2

Cov

Norm.
Zscore

Download
alignment

                  20                  40                  60                  80                 100                 120                 140                 160                 180                 200                 220                 240                 260                 280                 300                 320                 340                 360                 380                 400                 420

                   |                   |                   |                   |                   |                   |                   |                   |                   |                   |                   |                   |                   |                   |                   |                   |                   |                   |                   |                   |                   |

SS
Seq

CCCCHHHHHCCCCCCCCCCCCCCHHHHHCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCHHHHHHHCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCHHHCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCHHHHCCCCCCCCCCCCCCCCCCCCCCCCCCCCCHHHHHHCCCCCHHHHHHHHHHHHHHHHHHCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCHHHHHCCCCCCCCCHHHHHHHCCSSSSSSCCCCCCSSSSCCCCHHHHHHHHHHHHHHHHHHCCCCCHHHHHHHHCCCC
MPRCTYQLEQNPGFLPDGPGVHARAHCQDLSGPYGHEFATSESLGGRVGKTRAPQSGARSRMERAGPAGEEGGAREGRLLPRAPGAWVLRACAERAALEVGAASADTGVRGCGARGPAPLLASAGGGRARDGTWGVRTKGSGAALPSRPASRAAPRPEASSPPLPLEKARGGLSGPQGGRARGAMAHVGSRKRSRSRSRSRGRGSEKRKKKSRKDTSRNCSASTSQGRKASTAPGAEASPSPCITERSKQKARRRTRSSSSSSSSSSSSSSSSSSSSSSSSSDGRKKRGKYKDKRRKKKKKRKKLKKKGKEKAEAQQVEALPGPSLDQWHRSAGEEEDGPVLTDEQKSRIQAMKPMTKEEWDARQSIIRKVVDPETGRTRLIKGDGEVLEEIVTKERHREINKQATRGDCLAFQMRAGLLP

5wznA

0.08

3.11

0.52

CEthreader

AGAATVTHERIETGTGVGVRSVFAGFAGADYAFETYIWIERSSGDVLCEWVPLRECRIDRVLWPAPLSFDRADAHDVTLITHEQGVMIPNSWPTEVGTDAVSFGGRFETAGGYMPWFAQLRSDGHAYIATPWNAGYDIDHPAGGPYTHVGMWFEPSLGRMDYRRVVRYRLLDHADHTAICKTYRAYVNERGRLRTLAEKAARNPSVRDLLGRSWVHVGIKTNVQPDSSFYDPAQPGKNDSLVTFAQRERQMRTLHEMGAGRLYLHLDGWAQPGYDNGHPDYLPACREAGGWKGMKSLIDACHEQGDLFGTHDQYRDYYFAARTFDPRNAIRLADGTMPEHAMWAGGRQTYLCAE-LAPDYVRRNFSEIATHGIVLDCAYLDVFTCNEGDECSHPEHRMTRRECYERRAECFEYLLAHGILT

2xpiA

0.06

0.05

2.47

0.85

EigenThreader

REDYLRLWRHDALMQQQYKCAAFVGEKVLDITGNPNDAFWLAQVYCCTGDDLYNRSSACRYLAAFCLVKLYDWQGALNLLGETNPLRGQVYTNLRAKECYKEALMVDAKCYEAFDQLVSNHLLTKLNYAAFLRSLYMLKLNKTSHDYLSSINGLEKSSDLLLCKADTLFVRSRFIDVLAITTKILEIDPYNLDVHLASLHESGEKNKLYLISNDLVDRHPEKAVTWLAVGIYYLCV----NKISEARRYFSKSSTMDPQFGPAWIGFAHSFAIEGEHDQAISAYTTAARLPYLFLGMQHMQLGLLANEYLQSSYALFQYDPLLLNELGVVAFNKSDMQTAINHFQNALLLVKKTQSNEKPWAATWANLGHAYRKLKMYDAAIDALNQGLLLSDANVHTAIALVYLHKKIPGLAITHLHESL

2akhY

0.10

0.08

3.09

0.61

FFAS-3D

-------------MAK-QPGLDFQSAKGGLGELKRRLLFVIGALIGSFISIFAPYISASIIIQLLTVVHPTLAEIK---KEGESGRRKISQYTRYGTLVLAIFQSIGIATGLPNMPGMQGLVINPGFAFYFTERGI---GNGISIPPAIAHTIEQARQGDLHFLVLLLVA---VLVFAVTFFVVFVERGQRRIVVNYAKRQQGRR---------------VYAAQSTHLPLKVNMAGVIPAIFASSIILFPATIASWFGGGTGWNWLTTISLYLQPGQP-------------------LYVLLYASAIIFFCFFYTALVFNPRETADNLKKSGAFVRPGEQ-TAKYIDKVMTRLTLVGALYITFICLIPEFMRDAMKVPFYFGGTSLLIVVVVIMDFMAQVQTLMMS------SQYESAL-

6zywY

0.09

3.23

1.49

SPARKS-K

KLSAYKNLYEQMQISQAITPVENHIGVILVNGSYCRKFAENIRFGSDNNLRLHLYKFDLNEMSELTEKSEKKIQNTDVIVASVPHFIKILIDYFSKSEKISNAFYIRTIATKNINNIYSNFNKNVFTYGVEGQFLLLDTYNNYGVLPGAKIYKIMNNILNPALAKDILTSITFISEQNNLNRLKYSVQYDLLTSNKLPILREKIRDLIYKKILQNGQAIKIDYVKGILRYDSKLKEGLEEITITP-------------NYFIERTVKGVDAKEFTEELNGVSFKNVKYTGITNSIINDNLNKEKLLELLYKLVKPLNKQKL---------RQRKD------LTEEEIVDIQFREGLENGEYDGQF-----WRN-IQGLILPHHPKKDEFIEEYLKQEEVRINQINEQLQQEWETWKQVYDK

7jh6A

0.18

0.04

1.23

0.54

CNFpred

----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------EIFDKGDDDSLEQVLEEIEELIQKHRQLASELPKLELQAIKQYR------EALEYVKLPVLAKILEEEKHIEWLKEAAKQGDQWVQLFQRFRE

6reyc

0.06

0.04

1.81

0.67

DEthreader

YELVSIQAR-LI-LLKKKELLSRADL-ELPWRPLY---------------LVSCRPYFPAD--------------------------P-SLPPEHATDNIGYMMGGVVRRLHRERYKKPSWLT----------PVPD-------SH-K-TD-DVTLALMRPVIVTQLTATLS--DFSKCMIFQLVELGS--IGGNPPLKGEPVTNLV-ELQFSHKHFDSWKS--------QV---AQQTFFALGNFCLHDTIGLRDGILKQLKRT-P-LGRSREDM--------E---Q-IFDHFSIFDDAFLIGLIWCITSCVGGLAQLLLLKLTQVYKNVR--------TQGIKLLKTILKWLASAGSTAVTQL-L-------------LLYPHQVPLVLQVLKQTAWARYLEDEQLVREMAATTLSG-

5wznA

0.06

2.60

0.97

MapAlign

FSFAGAATVTHERIETGTGVGVRSVFAGFAGADYAFETYIWIESGDVLCEWVPLRECRIDRVLWPAPLSFDRADAHDVTLITHEQGVMIPNSWPTEVTDAVSFGGRFETAGGYMPWFAQLRSDGHAETPWNAGYDIDHPAGGPYTHVGMWFEPSLGRMDYRRVVRYRLLDHADHTAICKTYRAYVNERGRLRTLAEKAARNPSVRDLLGRSWVHVGIKTNVQPDSSFYDPAQPGKNDSLVTFAQRERQMRTLHEMGAGRLYLHLDGWAQPGYDNGHPDYLPACREAGGWKGMKSLIDACHAPDYVRRNFSEIATHGIVLDCAYLDVFTCNEGDECSHPEHRMTRRECYERRAPVPLYNLVYHDCVIQPWMMRVAGGDDYMPYLNDIERCAVVAGLHRRVLVQRSVFADGTAVTCDFHAQTY

5n8oA

0.18

5.70

0.79

MUSTER

DRRSQMNLKQDERL--SGELITGRRQLLEGMAFTPGSVDQGEKLSNVQSGQRTGTGSALMAMKDAGVNRWQGGEQRPATIISEPDRNVRYARLAGDFAASVKAGEESVAQVSGVREQAILTQAIRSELKTQGVLGHPEVTMTALSPVWLDSRSRYLRDMYRPGMVMEQ----WNPETRSHDRYVIDRVTAQSHSLTLRDAQGETQVVRISSLDSSWSRPEKMPVADGERTGKIPGLRVSGGDRLQVASVSEDVVPGRAEPASLPVSDSPFTTPGHSVSDSSVTQMAMNATLNGLARSGRDVRLYSSLDETRTAEKLARHPSFTVVSEQIKARAGETL------LETAISLQKAGLHT-PAQQAIHLALPVLESKNLAFSM-----VDLLTEAKSTELGGEINAQIKRGDLLYVDVAKGYGT

5ng8Av

0.24

0.03

0.89

0.44

HHsearch

---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------IEII---RSKEFSLKPMDSEEMNLLGHDFFVFTDRETDGTSIVRKDGKLIQT------------------------------

6wknA

0.07

2.86

0.48

CEthreader

LTGLLEYLRWNSKYLTDSAYTEGSTGKTCLMKAVLNLQDGVNACIMPLLQIDKDSGNPKLLVNAQCTDEFYQGHSALHIAIEKRSLQCVKLLVENGADVHLRACGRFFQKHQGTCFYFGELPLSLAACTKQWDVVTYLLENPHQPASLEATDSLGNTVLHALVMIADNSPENSALVIHMYDGLLQMGARLCPTVQLEEISNHQGLTPLKLAAKEGKIEIFRHILQREFSGPYQPLSRKFTEWCYGPVRVSLYDLSSVDSWEKNSVLEIIAFHCKSPNRHRMVVLEPLNKLLQEKWDRLVSRFFFNFACYLVYMFIFTVVAYHQPFGESMLLLGHILILLGGIYLLLGQLWYFWRRRFMDSYFEILFLLQALLTVLSQVLRFMETEWYLPLLVLSLVLGWLNLLYYTRGFQHTGIYSVMIQK

(a)	ID1 is the number of template residues identical to query divided by number of aligned residues.
(b)	ID2 is the number of template residues identical to query divided by query sequence length.
(c)	Cov is equal the number of aligned template residues divided by query sequence length.
(d)	Norm. Zscore is the normalized Z-score of the threading alignments. A Normalized Z-score >1 means a good alignment and is highlighted in bold.
(e)	Download alignment lists the threading program used to identify the template, and provide the 3D structure of aligned regions of threading templates (threading[1-10].pdb.gz).
(f)	Template residues identical to query sequence are highlighted in color.

Top 1 final models from D-I-TASSER

Click to view	Rank^a	Download	Estimated TM-score^b
	1	model1.pdb.gz	0.3836

(a)

D-I-TASSER simulations generate a large ensemble of structural conformations, i.e. decoys. These decoys are clustered by SPICKER based on pairwise structure similarity to report up to five final models from the five largest clusters. Models are ranked in descending order of cluster size. If the simulations converge well, it is possible to have less than 5 models generated, which is usually an indication of good model quality.

(b)

The model confidence is quantitatified by estimated TM-score (eTM-score), calculated based on significance of threading template alignments, contact map satisfaction rate, mean absolute error between distance of model and distance of AttentionPotential, and convergence of D-I-TASSER simulations. eTM-score is typically in the range of [0, 1], with higher eTM-score signifies higher model confidence.

Proteins with similar structure

Top 10 structural analogs in PDB (as identified by TM-align)

Rank	PDB Hit	TM-score	RMSD^a	IDEN^a	Cov.	Download Alignment
1	6reyc	0.41	7.79	0.041	0.739	model1_6reyc.pdb.gz
2	4o9xA	0.40	6.72	0.072	0.637	model1_4o9xA.pdb.gz
3	7dxjA	0.37	7.17	0.019	0.618	model1_7dxjA.pdb.gz
4	4iglA2	0.37	6.90	0.031	0.603	model1_4iglA2.pdb.gz
5	3ecqB	0.36	7.28	0.046	0.618	model1_3ecqB.pdb.gz
6	6gauA	0.36	7.49	0.057	0.625	model1_6gauA.pdb.gz
7	5flmA	0.36	6.89	0.034	0.577	model1_5flmA.pdb.gz
8	6l7iF	0.36	7.26	0.046	0.611	model1_6l7iF.pdb.gz
9	3gtgA	0.35	7.04	0.047	0.577	model1_3gtgA.pdb.gz
10	7jptA	0.35	7.36	0.034	0.603	model1_7jptA.pdb.gz

(a)	Query structure is shown in cartoon, while the structural analog is displayed using backbone trace.
(b)	Ranking of proteins is based on TM-score of the structural alignment between the query structure and known structures in the PDB library.
(c)	RMSD^a is the RMSD between residues that are structurally aligned by TM-align.
(d)	IDEN^a is the percentage sequence identity in the structurally aligned region.
(e)	Cov. represents the coverage of the alignment by TM-align and is equal to the number of structurally aligned residues divided by length of the query protein.

Predicted Gene Ontology (GO) Terms

Molecular Function (MF)

GO term	Cscore^GO	Name
GO:0044822	1.00	poly(A) RNA binding

Download full result of the above consensus prediction.

Click the graph to show a high resolution version.

(a)

Cscore^GO is the confidence score of predicted GO terms. Cscore^GO values range in between [0-1]; where a higher value indicates a better confidence in predicting the function using the template.

(b)

The graph shows the predicted terms within the Gene Ontology hierachy for Molecular Function. Confidently predicted terms are color coded by Cscore^GO:

[0.4,0.5)

[0.5,0.6)

[0.6,0.7)

[0.7,0.8)

[0.8,0.9)

[0.9,1.0]

Biological Process (BP)

GO term	Cscore^GO	Name
GO:0008380	1.00	RNA splicing
GO:0006351	0.90	transcription, DNA-templated

Download full result of the above consensus prediction.

Click the graph to show a high resolution version.

(a)

Cscore^GO is the confidence score of predicted GO terms. Cscore^GO values range in between [0-1]; where a higher value indicates a better confidence in predicting the function using the template.

(b)

The graph shows the predicted terms within the Gene Ontology hierachy for Biological Process. Confidently predicted terms are color coded by Cscore^GO:

[0.4,0.5)

[0.5,0.6)

[0.6,0.7)

[0.7,0.8)

[0.8,0.9)

[0.9,1.0]

Cellular Component (CC)

GO term	Cscore^GO	Name
GO:0005634	1.00	nucleus
GO:0005813	0.90	centrosome

Download full result of the above consensus prediction.

Click the graph to show a high resolution version.

(a)

Cscore^GO is the confidence score of predicted GO terms. Cscore^GO values range in between [0-1]; where a higher value indicates a better confidence in predicting the function using the template.

(b)

The graph shows the predicted terms within the Gene Ontology hierachy for Cellular Component. Confidently predicted terms are color coded by Cscore^GO:

[0.4,0.5)

[0.5,0.6)

[0.6,0.7)

[0.7,0.8)

[0.8,0.9)

[0.9,1.0]

Predicted Enzyme Commission (EC) Numbers

Top 5 enzyme homologs in PDB

Rank	Cscore^EC	PDB Hit	TM-score	RMSD^a	IDEN^a	Cov.	EC Number	Predicted Active Site Residues
1	0.060	1vlbA	0.280	7.69	0.036	0.501	1.2.99.7	NA
2	0.060	1zxvA	0.289	7.30	0.050	0.494	3.4.24.83	NA
3	0.060	1rw9A	0.281	6.61	0.052	0.442	4.2.2.5	NA
4	0.060	3iydD	0.294	7.01	0.043	0.489	2.7.7.6	NA
5	0.060	2ckjA	0.289	7.77	0.045	0.527	1.17.1.4 1.17.3.2	NA

	Click on the radio buttons to visualize predicted active site residues.
(a)	Cscore^EC is the confidence score for the Enzyme Commission (EC) number prediction. Cscore^EC values range in between [0-1]; where a higher score indicates a more reliable EC number prediction.
(b)	TM-score is a measure of global structural similarity between query and template protein.
(c)	RMSD^a is the RMSD between residues that are structurally aligned by TM-align.
(d)	IDEN^a is the percentage sequence identity in the structurally aligned region.
(e)	Cov. represents the coverage of global structural alignment and is equal to the number of structurally aligned residues divided by length of the query protein.

Predicted Ligand Binding Sites

Template proteins with similar binding site:

Click to view	Rank	Cscore^LB	PDB Hit	TM-score	RMSD^a	IDEN^a	Cov.	BS-score	Lig. Name	Download Complex	Predicted binding site residues
	1	0.01	2ja54	0.118	4.10	0.022	0.145	0.25	III	complex1.pdb.gz	80,84,87,88,90,91,94,95,96,97,98,100,101,102,103,104
	2	0.01	2hgu3	0.135	5.68	0.032	0.195	0.35	III	complex2.pdb.gz	74,75,77,78,79

	Click on the radio buttons to visualize predicted binding site and residues.
(a)	Cscore^LB is the confidence score of predicted binding site. Cscore^LB values range in between [0-1]; where a higher score indicates a more reliable ligand-binding site prediction.
(b)	BS-score is a measure of local similarity (sequence & structure) between template binding site and predicted binding site in the query structure. Based on large scale benchmarking analysis, we have observed that a BS-score >1 reflects a significant local match between the predicted and template binding site.
(c)	TM-score is a measure of global structural similarity between query and template protein.
(d)	RMSD^a the RMSD between residues that are structurally aligned by TM-align.
(e)	IDEN^a is the percentage sequence identity in the structurally aligned region.
(f)	Cov. represents the coverage of global structural alignment and is equal to the number of structurally aligned residues divided by length of the query protein.

[Click result.zip to download all results on this page]

References:
1.	Wei Zheng, Chengxin Zhang, Yang Li, Robin Pearce, Eric W. Bell, Yang Zhang. Folding non-homology proteins by coupling deep-learning contact maps with I-TASSER assembly simulations. In preparation, 2020.