Dali Tutorial 2022: New features
Table of contents
What’s new?
Introduction
Case study: CASP target T1090
1. Finding structures for your sequence [*new features*]
2. Searching known structures in the Protein Data Bank
3. Pfam annotations [*new features*]
4. Searching AlphaFold Database (version 1) [*new features*]
5. Putting it all together: structural dendrogram and sequence signatures
6. Conclusion
Box 1: Tips for discovery

What’s new?
1) Support for AlphaFold database (version 1)
2) Possibility to generate stacked Pfam graphics, which is a useful tool to filter out redundant hits such asparalogs in AlphaFold database3) Web-server support to interactively visualize and link results to external sequence search, functionalannotation, and family classification resources.

Introduction
Dali (http://ekhidna2.biocenter.helsinki.fi/dali/) is a protein structure comparison server based on distancematrix comparison (https://onlinelibrary.wiley.com/doi/full/10.1002/pro.3749). In favourable cases,
structure comparison can reveal distant evolutionary relationships not seen by sequence comparison. The
web server supports searches in three databases [Protein Data Bank (PDB); PDB25, a representative subsetof PDB; AlphaFold (version 1)] and enables two customised types of structure comparisons: i) pairwise
structure comparison to one query structure and ii) all against all structure comparison.
The server takes the 3D coordinates of protein structures as input and returns a list of similar structures,
structural alignments and superimposed structures. The all against all comparison also returns a structural
dendrogram. The results are linked to sequence search and function prediction servers. Dali tutorial 2016
explains the web interface of the Dali server using live examples. It is still fully valid. This update, Dalitutorial 2022, explains more recently added features: enhanced visualization using stacked Pfam cartoons,and support for the AlphaFold Database. These features are illustrated with a case study (Table 1). Grayboxes mark exercises that you can try out interactively in the web server.
Table 1: Cross-mapped identifiers of proteins discussed in this tutorial. New findings highlighted in yellow.
PDB
4m8bR
n.a.
7k7vA

Dali
evi0A
ahoyA
gugsA

AlphaFold Database
YEAST:AF-P38867-F1
ARATH:AF-Q9FY74-F1
SCHPO:AF-Q9P7Y0-F1

Pfam
PF09729PF03859
PF08549

Pfam short name
Gt1/Pac2
CG-1
SWI-SNF_Ssr4_N

n.a.

abinA

ARATH:AF-Q9LUQ8-F1

n.a.

n.a.

1ut4B,
3swmA
4ywzA
4i90A
2rprA
2lexA
4lmgA
1odhA
n.a.

akgpA

ARATH:AF-Q9C932-F1

PF02365

NAM

Other
Pfam: CL0274
joins CL0274
CASP: T1090
joins CL0274
TAIR: At3g16750
joins CL0274
joins CL0274

gvtqA
esidA
e1hrA
ac70A
ewieA
gcg9A
h0rrA

STAA8:AF-Q2G2U4-F1
ECOLI :AF-P52129-F1
HUMAN: AF-Q4VC44-F1
ARATH: AF-Q9XI90-F1
YEAST: AF-Q08957-F1
MOUSE:AF-P70348-F1
SOYBN :AF-A0A0R0G5C4-F1

n.a.
PF03108
PF04500
PF3106PF08731PF03615PF03101

n.a.
DBD-Trp-Mut
FLYWCH
WRKY
AFT
GCM
FAR1

Pfam: CL0274
Pfam: CL0274
Pfam: CL0274
Pfam: CL0274
Pfam: CL0274
Pfam: CL0274

Description
WHITE-OPAQUE REGULATOR 1
Calmodulin-binding transcription activator 1
SWI/SNF and RSC complexes subunit ssr4 N-terminal
UNCHARACTERIZED PROTEIN
NAC DOMAIN-CONTAINING PROTEIN 19
SENSOR PROTEIN KINASE WALKmRNA endoribonuclease toxin LS
FLYWCH-type zinc finger-containing protein 1
Probable WRKY transcription factor 4
Iron-regulated transcriptional activator AFT2
Chorion-specific transcription factor GCMa
FAR1 domain-containing protein1Case study: CASP target T1090
CASP is a blind structure competition organized every two years (https://predictioncenter.org/casp14/). We
study CASP14 target T1090. The target is chromatin remodeling protein Ssr4 from Schizosaccharomyces
pombe. The protein was classified in CASP as a free modeling target, implying that template basedmodeling was not possible. The crystallographers wrote(https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7716260/): “PDBeFold (Krissinel & Henrick, 2004) wasused to compare the Ssr4 structure with all other structures in the PDB. … This suggests that this domain of
SSR4 adopts a fold that is not currently represented in the public PDB. For this reason it was submitted to
CASP14, with the results of the modelling to be presented in the future.” AlphaFold2 predicted thestructure with remarkably low RMSD and a clear gap to the next best predictor(https://onlinelibrary.wiley.com/doi/full/10.1002/prot.26172).

1. Finding structures for your protein [*new features*]First, we use the sequence to identify the corresponding PDB and AF-DB models. Paste the FASTA
formatted sequence (https://predictioncenter.org/casp14/target.cgi?id=130&view=all) to the SANSparallel
server at http://ekhidna2.biocenter.helsinki.fi/cgi-bin/daliviewer/call_sans.cgi. Select the searched
database using the radio buttons. We learn that the best PDB match is 7k7vA and the best AlphaFold
Database match is gugsA (Figure 1).

Figure 1. Interactive homology search using SANSparallel: a submission example (left) and outputs fromsearches in the PDB (middle) and AlphaFold Database (right).
Dali internally uses four-letter identifiers for the PDB entry plus chain identifier. PDB identifiers are
inherited from the Protein Data Bank and start with a number 1-9. Dali’s internal identifiers for AlphaFold
Database (AF-DB) models start with a letter a-h. The original AF-DB identifier can be recovered from thedescription field (cf. Figure 1).
Second, do a pairwise Dali alignment of gugsA and 7k7vA to verify the high accuracy of the model predicted
by AlphaFold2 with 0.2 A RMSD over 192 residues (Figure 2). Important to note that the PDB structure of
Ssr4 was released after the CASP14 competition.

Figure 2. Left: Summary statistics of pairwise alignment of AlphaFold2 model and experimentally
determined structure of CASP14 target T1090. Right: structural superimposition of PDB structure 7k7vA(yellow) and AF-DB model gugsA (green).

2

AlphaFold generates end-to-end models of the whole protein sequence. In the present case, only the Nterminal domain of gugsA is modelled as a compact domain. The C-terminal domain is not present in the
crystal structure. The crystal structure has an artificial N-terminal His tag, which is naturally not present in
the AF-DB model. Subsequent analysis will focus on the N-terminal domain of Ssr4, so we use 7k7vA as
query instead of the AF-DB model.

2. Searching known structures in the Protein Data Bank
Next, we did a PDB search for structural neighbors of 7k7vA. For initial screening, it is useful to filter
redundant hits by inspecting the top hits in the PDB25 subset (Figure 3-left). The stacked structuralalignment shows the extent of the common core (Figure 3-right). The top row shows the query sequence.
Structurally equivalent segments from the other structures are mapped onto it. We see that moststructures match the beta strands, but the alpha helix preceding the beta strands is present in only threestructural neighbors. We’ll select these three (1ut4B, 3m8bR, 4ywzA) for further comparison with 7k7vA.

Figure 3. Left: summary list of top hits (PDB25 subset) from PDB search. Right: stacked alignment of
selected hits (bottom, upper part shows amino acid sequences, lower part shows secondary structures).
Do a pairwise comparison of 7k7vA (first structure) against 1ut4B, 4m8bR and 4ywzA (second structures)using the pairwise comparison option of the Dali server. Pairwise comparisons are done interactively,
whereas there may be a long wait in the queue for PDB searches. You’ll get a summary list similar to Figure
3, only shorter. Select all structures and click the 3D Superimposition (PV) button. This opens a new the
Daliviewer window. Inspect the structural overlap between the structures. Hide all structures except thequery and color it by Structure Conservation. The common core (blue) forms a compact globule (Figure 4).

Figure 4. Structural conservation mapped onto 7k7vA. Highly conserved regions are blue.
Click on the PDB link in the summary list and check the HEADER record 1ut4B and 4m8bR. These entries
were released by PDB well before CASP14. (Hint: The coordinates are transformed to superimpose with thequery structure. Download them to generate publication-quality graphics in PyMol.)
1ut4B and 4m8bR were the top hits in the PDB search. The Z-scores are moderate (5.6-5.7) but thecommon core covers practically all secondary structure elements with an RMSD 3.1-3.3 A over 88-100 Catoms. We conclude that contrary to its classification as a free modeling target in CASP, T1090 did not
represent a novel fold but suitable templates were present in the PDB.
Continuing in the Daliviewer window, check the Load whole structures box at the bottom and check show
other chains of All. Now you see the DNA molecule complexed with 4mb8R; it is clearest in cartoon3

representation (Figure 5). The superimposition allows you to visualize a transplantation of the bound DNA
to the other structures. Toggle structures on/off and verify that 7k7vA and 1ut4B have open space where
the DNA binds, whereas 4ywzA has an extra helix that blocks the DNA binding site.

Figure 5. Superimposed structures side by side. From left to right: 7k7vA, 1ut4B, 4m8bR, 4ywzA.
4ywzA had a relatively low Z-score in the PDB search and has a different architecture. We’ll drop 4ywzAfrom further consideration and pursue the hypothesis that T1090 shares the DNA binding function with
4m8bR and 1ut4B.
The loop that connects the edge strands of the beta sheet is structurally conserved between the remaining
structures. It is the DNA recognition loop in 4m8bR, fitting into the major groove of dsDNA. We observethat this loop is disordered in 1ut4B, which causes chain breaks in the PDB structure. There are multiple
PDB structures for this NAC-domain containing protein 19 (https://www.uniprot.org/uniprot/Q9C932).
Some of these structures are complexed with DNA, e.g. PDB entry 3swm. However, they too havedisordered loops. Recalling that AlphaFold generates end-to-end models, we will see how these loops aremodelled by AlphaFold and whether they interfere with DNA binding. Dali’s internal identifier for the AFDB model is akgpA.
Make a new pairwise alignment of 7k7vA (first structure) against akgpA, 1ut4B, 3swmA, 3swmB, 3swmC,
3swmD and 4mb8R (second structures). Verify in the structural alignment view that akgpA models loops
missing from the 3swm chains. Inspect the contacts to DNA.

3. Pfam annotations [*new features*]
Next, we study what is known about the biological functions of our proteins of interest. Here, we exploit
information gathered in the Pfam database.
Return to the summary page, select all hits and click the Pfam button. The Pfam graphics show that the hits
belong to different protein families (Figure 6). Hover the cursor above a green cartouche for more
information on the domain family. Read the documentation of the families at the Pfam website, e.g.
https://pfam.xfam.org/family/PF08549. Substitute the Pfam identifier PFxxxxx for the other families.

Figure 6. Left: Stacked Pfam graphics of structural neighbors. Right: Meaning of visual cues. Placeholder
gaps are introduced in order to show structurally equivalent blocks aligned vertically throughout the stack.
4

The Pfam view gives a graphical overview of the structural alignment, adding Pfam annotations. The
members of a Pfam family share significant sequence similarity, modelled by Hidden Markov models(HMMs). Pfam annotations were taken from Pfam 35.0. Pfam annotations cover ∼75% of proteins and∼49% of residues (https://xfam.wordpress.com/2021/11/19/pfam-35-0-is-released/). Large families arewell covered by Pfam (https://academic.oup.com/nar/article/33/suppl_1/D188/2505389), smaller families
are collected in Pfam-B but not included in our Pfam annotation database. Pfam is derived from proteins in
the Uniprot database. Pfam annotations are available for PDB structures (in Pfam’s pdbmap table) and AFDB models (which are based on Uniprot sequences). User may upload their own structures to the Daliserver, but these will not have associated Pfam annotation. Since Pfam releases have an interval of a year,
annotations will also be missing for the most recent PDB structures.
It is common for distinct Pfam families to share structural similarity, either by common descent (homology)
or by physical convergence. In our example (Figure 4), we found that three domain families share acommon fold: SWI/SNF and RSC complexes subunit Ssr4 N-terminal (PF08549), no apical meristem (NAM)
protein (PF02365), and the Gt1/Pac2 family (PF09729). Intriguingly, they are all involved in gene regulation.
Pfam places the Gt1/Pac2 family in the WRKY-GCM1 clan (CL0274). Pfam clans unify remote homologs. No
clan is assigned to the other two families. WRKY and GCM1 are metal chelating DNA-binding domains (DBD)
which share a four-stranded fold (http://www.ncbi.nlm.nih.gov/pubmed/17130173). Our families share alarger core consisting of five strands and a helix (Figure 2).

4. Searching AlphaFold Database (version 1) [*new features*]
So far, we have done a PDB search and identified known structures with the same fold as T1090. Let’s moveon to check if AF-DB presents us with more potential members of the emerging superfamily of DNA-binding
domain (Box 1). PDB searches (PDB search and PDB25 search tabs) and AF-DB searches (AF-DB search tab)are kept separate in the Dali server. PDB contains experimental structures, whereas AF-DB contains(remarkably accurate) predictions.
AF-DB searches in Dali are currently limited to one model organism at a time. You choose from 21 modelorganisms, including human. AF-DB searches have long queueing times. Therefore, we show below the top
hits from precomputed searches of 7k7vA against HUMAN (Figure 7). Visual inspection shows thatmembers of the CG-1 family (PF03859), represented by e.g. e3ikA, resemble the DNA-binding domains. A
search against Arabidopsis again shows CG-1 as the top hit, an uncharacterized protein At3g16750 at rank
6, and the previously picked up NAM family as the next best hits (Figure 8). Also WRKY domains match thebeta sheet. A four-stranded beta sheet is a common structural motif, so it is noteworthy that WRKY
domains match better than other beta sheet proteins. A search against fission yeast recovers the query(Ssr4) and two paralogs of the Gt1/Pac2 family, already known members of our superfamily (Figure 9).
Other top hits did not pass the secondary structure filtering step.

# Job: HUMAN# Query: 7k7vA# No: ChainZ
rmsd lali nres
1: e3ik-A 9.6 3.4 115 1202
2: fckc-A 3.7 4.6
91 1070
3: e6yp-A 3.5 4.1
84626
4: e1v1-A 3.4 5.3
82 1724
5: fev8-A 3.1 4.4 124 1673
6: fdbh-A 3.1 3.4
86
186
7: e9iu-A 3.1 17.074608
8: e2gj-A 3.0 3.5
75
140
9: e62h-A 2.9 4.2
76 1332
10: fa7h-A 2.8 4.1
80 1400%id PDB Description
18
HUMAN:AF-O94983-F1 CALMODULIN-BINDING TRANSCRIPTION ACTIVATOR 2;
4
HUMAN:AF-O15063-F1 GRANULE ASSOCIATED RAC AND RHOG EFFECTOR PROTEIN
5
HUMAN:AF-Q9GZY0-F1 NUCLEAR RNA EXPORT FACTOR 2;
9
HUMAN:AF-Q9UKK3-F1 PROTEIN MONO-ADP-RIBOSYLTRANSFERASE PARP4;17
HUMAN:AF-Q9Y6Y1-F1 CALMODULIN-BINDING TRANSCRIPTION ACTIVATOR 1;
7
HUMAN:AF-Q9HD47-F1 RAN GUANINE NUCLEOTIDE RELEASE FACTOR;
7
HUMAN:AF-Q86X55-F1 HISTONE-ARGININE METHYLTRANSFERASE CARM1;7
HUMAN:AF-P35080-F1 PROFILIN-2;
7
HUMAN:AF-O95163-F1 ELONGATOR COMPLEX PROTEIN 1;
5
HUMAN:AF-Q5T011-F10 KICSTOR COMPLEX PROTEIN SZT2;

Figure 7. AF-DB search result against the human proteome. CG-1 family (PF03859) members are bold.

5

# Job: ARATH# Query: 7k7vA# No: Chain
Z
1: aulq-A 9.8
2: ahoy-A 9.7
3: akf2-A 9.4
4: aote-A 9.2
5: aanf-A 8.9
6: abin-A 6.5
7: apry-A 6.5
8: ah0e-A 6.3
9: aqv9-A 6.2
10: aki9-A 6.1// snip //75: aajg-A 4.8
76: ac99-A 4.8
77: arkn-A 4.8
78: asoz-A 4.8
79: addy-A 4.7
80: aqg2-A 4.6
81: am7i-A 4.6
82: amrf-A 4.6
83: ac8d-A 4.6
84: adw2-A 4.6

rmsd lali nres
2.8 106923
2.8 105 1007
2.7 104 1032
2.7 104 1050
3.0 106 1016
2.8 103194
5.5 112
322
3.4
99
276
3.3 103
320
3.2
97
175

%id PDB Description
22
ARATH:AF-O23463-F1 CALMODULIN-BINDING TRANSCRIPTION ACTIVATOR 5;
22
ARATH:AF-Q9FY74-F1 CALMODULIN-BINDING TRANSCRIPTION ACTIVATOR 1;
23
ARATH:AF-Q8GSA7-F1 CALMODULIN-BINDING TRANSCRIPTION ACTIVATOR 3;
24
ARATH:AF-Q6NPP4-F1 CALMODULIN-BINDING TRANSCRIPTION ACTIVATOR 2;
29
ARATH:AF-Q9FYG2-F1 CALMODULIN-BINDING TRANSCRIPTION ACTIVATOR 4;
17
ARATH:AF-Q9LUQ8-F1 UNCHARACTERIZED PROTEIN;
10
ARATH:AF-O80752-F1 APICAL MERISTEM FORMATION PROTEIN-RELATED;
9
ARATH:AF-F4IME8-F1 NAC DOMAIN CONTAINING PROTEIN 36;
13
ARATH:AF-O80756-F1 NAC DOMAIN CONTAINING PROTEIN 24;
13
ARATH:AF-Q1ECJ5-F1 AT1G60240;

4.2
3.3
4.6
4.5
3.4
3.0
3.6
4.7
3.9
4.9

11
14
10
8
13
14
11
10
6
6

107
99
79
89
98
94
97
81
84
87

469
528
274
195
212
451
303
337
374
557

ARATH:AF-Q9SCK6-F1 NAC DOMAIN-CONTAINING PROTEIN 62;
ARATH:AF-F4IED2-F1 NAC DOMAIN-CONTAINING PROTEIN 13;
ARATH:AF-Q9FHR7-F1 PROBABLE WRKY TRANSCRIPTION FACTOR 49;
ARATH:AF-Q8VWQ4-F1 PROBABLE WRKY TRANSCRIPTION FACTOR 56;
ARATH:AF-Q9FLM0-F1 AT5G41090;
ARATH:AF-Q9SQY0-F1 NAC DOMAIN CONTAINING PROTEIN 52;
ARATH:AF-Q9LSI4-F1 NAC (NO APICAL MERISTEM) DOMAIN TRANSCRIPTIONAL R
ARATH:AF-O22900-F1 WRKY TRANSCRIPTION FACTOR 23;
ARATH:AF-Q9C9F0-F1 PROBABLE WRKY TRANSCRIPTION FACTOR 9;
ARATH:AF-Q93WV0-F1 PROBABLE WRKY TRANSCRIPTION FACTOR 20;

Figure 8. AF-DB search result against the Arabidopsis proteome. CG-1 (bold), NAM and WRKY domain
families populate the list. abinA (rank 6) is not classified in Pfam.
# Job: SCHPO# Query: 7k7vA# No: Chain
Z
1: gugs-A 31.2
2: gshl-A 5.6
3: gs5d-A 5.4
4: guob-A 2.9
5: gtiw-A 2.9
6: gq17-A 2.8
7: gryg-A 2.5
8: gryy-A 2.5
9: gt0x-A 2.5
10: grce-A 2.4

rmsd lali nres
0.2 180
395
3.2
98
720
3.2 100
235
4.1
83
190
4.1
66
673
5.5
76 1611
4.1
77
434
4.2
81
832
4.0
69
141
4.5
75 1004%id PDB Description
99
SCHPO:AF-Q9P7Y0-F1 SWI/SNF AND RSC COMPLEXES SUBUNIT SSR4;12SCHPO:AF-O14367-F1 GLUCONATE TRANSPORT INDUCER 1;
9SCHPO:AF-Q10294-F1 CAMP-INDEPENDENT REGULATORY PROTEIN PAC2;5SCHPO:AF-O75002-F1 NUCLEAR IMPORT PROTEIN MOG1;5SCHPO:AF-O13650-F1 PUTATIVE TRANSCRIPTION FACTOR TAU SUBUNIT SFC9;4SCHPO:AF-O42854-F1 SH3 DOMAIN-CONTAINING PROTEIN C23A1.17;
9
SCHPO:AF-O94260-F1 PUTATIVE G3BP-LIKE PROTEIN;5SCHPO:AF-Q7Z9H9-F1 POLYPHOSPHOINOSITIDE PHOSPHATASE;
4SCHPO:AF-Q9USW0-F1 UNCHARACTERIZED PROTEIN C21B10.02;
5
SCHPO:AF-Q10408-F1 UNCHARACTERIZED PROTEIN C1F3.03;

Figure 9. AF-DB search result against the fission yeast proteome. Gt1/Pac2 family members are bold.

5. Putting it all together: structural dendrogram and sequence signatures
We performed an all-against-all comparison using representatives of old and new clan members (Figure10). For this analysis, we used cropped AF-DB models and a local DaliLite installation (see Download tab on
Dali server website). Cropped AF-DB models retain only residues that are confidently modelled (pLDDT>70).
Cropping removes non-compact loops in AF-DB models, like the C-terminal part of T1090 (see Figure 2),
which add spurious equivalences to Dali alignments that destroy the 3-D superimposition.
Copy-paste the resulting newick tree to iToL (https://itol.embl.de/):((((((((ahoyA_PF03959_CG1:38.5,abinA_n.a.:14.1):4.6,gugsA_PF08549_SWI/SNF:25.7):1.6,evi0A_PF09729_Gt1/Pac2:25.2):0.4,akgpA_PF02365_NAM:22.1):2.2,(gcg9A_PF036
15_GCM:23.5,ac70A_PF03106_WRKY:17.2):1.4):1.45714285714286,(((ewieA_PF08731_AFT:16.9,h0rrA_PF03101_FAR1:14.3):3.3,esidA_PF03108_DBDTrp-Mut:39.8):0.966666666666667,e1hrA_PF04500_FLYWCH:36.0666666666667):0.090476190476191):3.24285714285714,0.1:0):0.1);

Figure 10. Left: Structural dendrogram of extended WRKY-like clan using AF-DB models. Middle: internal
Dali-identifiers of family representatives. Right: Z-score matrix for cropped AF-DB models. Our study object
T1090/gugsA/SWI-SNF is bold. Z-scores between 2 and 5 are pink, Z-scores between 5 and 14 are yellow.
6

The crystal structure of SWI/SNF and the AF-DB models of CG-1 and unclassified abinA share strong
structural similarity and form a subgroup with the Gt1/Pac2 family. CG-1 is a specific DNA-binding protein(CG-1, a parsley light-induced DNA-binding protein - PubMed (nih.gov)). There are crystal structures of
NAM domains complexed with dsDNA (e.g. 3swm). SWI/SNF is involved in chromatin remodeling. However,electrostatic potential calculations do not show no clear face of potential that might signal specific binding
to DNA (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7716260/). abinA (At3g16750) is anuncharacterized protein. Sequence searches bring up few homologs which seem taxonomically restricted torosids. The protein has a negative net charge, making DNA binding unlikely. Nevertheless, there is striking
conservation of a key interaction stabilizing the DNA recognition loop in the Gt1/Pac2 family (Figure 11).
In structural alignment view, click the Show Stacked Sequence Logos button to compare sequenceconservation profiles between families. Convince yourself of the conservation of the DxxxW motif of therecognition loop (Figure 11).

Figure 11. Structural alignment of the recognition loop. The red arrow points to W72, which is essential for
DNA binding in the Gt1/Pac2 family (Crystal structure of the WOPR-DNA complex and implications for Wor1
function in white-opaque switching of Candida albicans | Cell Research (nature.com)). W72 forms ahydrogen bond to D68, stabilizing the loop. These residues are highly conserved in the other families.

6. Conclusion
Our analysis added three more Pfam families to the WRKY-GCM1 clan, namely CG-1, NAM, SWI/SNF, and asmall family represented by At3g16750 (abinA). Despite strong structural similarity to sequence-specific
DNA binding domains, electrostatic potential calculations (e.g. https://pdbj.org/eF-surf/top.do) do notsupport this function in the last two families. The starting point of this study, SWI/SNF, was accurately
modelled from sequence by AlphaFold. CG-1 and abinA were modelled by AlphaFold and still lackexperimental structures, making them interesting targets for structural genomics.

Box 1: Tips for discovery
1. Do a sequence search using SANSparallel. Terminate if found obvious homologs.
2. Do a PDB search using Dali server. Use PDB25 to remove redundancy.
3. Examine top-10 to top-20 hits. Define maximal common core guided by stacked structural aligment.
Terminate if no compact, globular core can be found.
4. Check Pfam annotations of retained hits. Terminate if homology (family or clan) is known already.
# At this point, we hypothesize a novel homologous relationship between protein/domain families.
5. Examine stacked Sequence Logos for sequence signals as possible evidence of homology.
6. Examine 3D superimpositions for peculiar structural features or conserved binding sites.
7. Do AF-DB searches using Dali server. Use Pfam annotations to select representative hits.
8. Add new Pfam families to the set of potential homologs, repeating steps 3-6.
# At this point, we should have evidence of conserved, unifying features of potential superfamily.
9. Synthesis: check congruence of structural trees with functional diversification.
10. Prioritize targets for experimental validation.
7