BIN-SX-Domains

Structure domains

 

Keywords:  Structural domains, domain databases - CATH, SCOP, cDART

 

 

Caution!

This unit is under development. There is some contents here but it is incomplete and/or may change significantly: links may lead to nowhere, the contents is likely going to be rearranged, and objectives, deliverables etc. may be incomplete or missing. Do not work with this material until it is updated to "live" status.

 

Abstract

...

 

This unit ...

Prerequisites

You need to complete the following units before beginning this one:

• BIN-SX-Chimera (UCSF Chimera: Structure Visualization and Analysis)

 

Objectives

This unit will ...

• ... introduce concepts of structural domains, the hierarchical nature of protein structure; that domains are folding units, units of inheritance and functional modules; that domains are ubiquitous in proteins; that domain assignment allows to to organize structures into domain databases;
• ... teach how to access domain databases and get the data for structural domain annotations;

 

Outcomes

After working through this unit you ...

• ... are familar with domain annotations obtained via the PDB or CDD, and derived from CATH or Pfam and know how to annotate proteins based on those.

 

Deliverables

• Time management: Before you begin, estimate how long it will take you to complete this unit. Then, record in your course journal: the number of hours you estimated, the number of hours you worked on the unit, and the amount of time that passed between start and completion of this unit.
• Journal: Document your progress in your Course Journal. Some tasks may ask you to include specific items in your journal. Don't overlook these.
• Insights: If you find something particularly noteworthy about this unit, make a note in your insights! page.

 

Evaluation

Evaluation: NA

This unit is not evaluated for course marks.

 

Contents

Task:

• Read the introductory notes on protein domains defined by 3D structure analysis.

 

CATH

 

APSES domains in Chimera

 

What precisely constitutes an APSES domain is a matter of definition, as we will explore in the following task.

Task:

• Open Chimera and load the 1MB8 structure.
• Display the protein in ribbon style, e.g. with the Interactive 1 preset.
• Access the Interpro information page for Mbp1 at the EBI: http://www.ebi.ac.uk/interpro/protein/P39678
• In the section Domains and repeats, mouse over the red annotations and note down the residue ranges for the annotated domains covering the N-terminus. You should find:
  • IPR003163 (InterPro) Transcription regulator HTH, APSES-type DNA-binding domain (IPR003163) annotated on the Mbp1 sequence;
  • IPR018004 (InterPro; same as SMART SM01252) The KilA, N-terminal/APSES-type HTH, DNA-binding definition annotated on the Mbp1 sequence;
  • PF04383 (Pfam): the KilA-N domain definition, which is also the one that is annotated to the Mbp1 protein sequence by CDD.

• Follow the links to the respective Interpro and Pfam domain definition pages and read about the domain. Each domain definition describes essentailly the same biomolecule, but the have distinct and partially overlapping sequence rangex.

• Navigate to the NCBI page for the Mbp1 protein and click on Identify conserved domains.
• Note the range of the Pfam KilA-N annotation in the linked page: hint - it is different from the annotation you find at Interpro.

Back at Chimera...

• Select the entire protein chain and colour it white (residues 4 to 102, practically identical to the IPR003163 APSES domain definition.)

Next, use the "Sequence Window" to select specific residue ranges:

• Choose Tools → Sequence → Sequence to open the sequence window, select the sequence corresponding to IPR018004 (Kil-A N) annotation and colour this fragment yellow. You can get the sequence numbers of a residue in the sequence window when you hover the pointer over it - but do confirm that the sequence numbering that Chimera displays matches the numbering of the Interpro domain definition.

• Then select the residue range of Pfam 04383, the KilA-N domain as defined by CDD and colour that fragment orange.

• Finally, choose the residues for PF04383, the KilA-N domain as defined by InterPro and color them red.

• Study this in a side-by-side stereo view and get a sense for how the extra sequence beyond the Kil-A N domain(s) is part of the structure, and how the integrity of the folded structure would be affected if these fragments were missing.

• Display Hydrogen bonds, to get a sense of interactions between residues from the differently colored parts. First show the protein as a stick model, with sticks that are thicker than the default to give a better sense of sidechain packing:
  

(i) Select → Select all

(ii) Actions → Ribbon → hide

(iii) Select → Structure → protein

(iv) Actions → Atoms/Bonds → show

(vi) click on the looking glass icon at the bottom right of the graphics window to bring up the inspector window and choose Inspect ... Bond. Change the radius to 0.4.

• Then calculate and display the hydrogen bonds:
  

(vii) Tools → Surface/Binding Analysis → FindHbond

(viii) Set the Line width to 3.0, leave all other parameters with their default values an click Apply

Clear the selection.

Study this view, especially regarding side chain H-bonds. Are there many? Do side chains interact more with other sidechains, or with the backbone?

• Let's now simplify the scene a bit and focus on backbone/backbone H-bonds:
  

(ix) Select → Structure → Backbone → full

(x) Actions → Atoms/Bonds → show only

Clear the selection.

In this way you can appreciate how H-bonds build secondary structure - α-helices and β-sheets - and how these interact with each other ... in part across the KilA N boundary.

• Finally, we add H-bonding side-chains back in. We display the sidechains of D, E, H, K, N, Q, R, S, T, W, Y, but reduce the bond radius for clarity:

(xi) Select → Clear selection

(xi) Select → Selection mode → append

(xi) Select → Residue → ARG ... then one after another also

(xi) Select → Structure → Backbone → full

(xi) Select → Structure → Backbone → full

(xi) Select → Structure → Backbone → full

• Orient the protein well, save the resulting image as a jpeg and upload it to your Journal on the Wiki.

There is a rather important lesson in this: domain definitions may be fluid, and their boundaries may be computationally derived from sequence comparisons across many families, and do not necessarily correspond to individual structures. In our example, you saw that the more restrictive KilA-N domain definition Make sure you understand this well.

Given this, it seems appropriate to search the sequence database with the sequence of an Mbp1 structure–this being a structured, stable, subdomain of the whole that presumably contains the protein's most unique and specific function. Let us retrieve this sequence. All PDB structures have their sequences stored in the NCBI protein database. They can be accessed simply via the PDB-ID, which serves as an identifier both for the NCBI and the PDB databases. However there is a small catch (isn't there always?). PDB files can contain more than one protein, e.g. if the crystal structure contains a complex. Each of the individual proteins gets a so-called chain ID–a one letter identifier– to identify them uniquely. To find their unique sequence in the database, you need to know the PDB ID as well as the chain ID. If the file contains only a single protein (as in our case), the chain ID is always A. make sure you understand the concept of protein chains, and chain IDs.

 

Further reading, links and resources

Dawson et al. (2017) CATH: an expanded resource to predict protein function through structure and sequence. Nucleic Acids Res 45:D289-D295. (pmid: 27899584)

[ PubMed ] [ DOI ] The latest version of the CATH-Gene3D protein structure classification database has recently been released (version 4.1, http://www.cathdb.info). The resource comprises over 300 000 domain structures and over 53 million protein domains classified into 2737 homologous superfamilies, doubling the number of predicted protein domains in the previous version. The daily-updated CATH-B, which contains our very latest domain assignment data, provides putative classifications for over 100 000 additional protein domains. This article describes developments to the CATH-Gene3D resource over the last two years since the publication in 2015, including: significant increases to our structural and sequence coverage; expansion of the functional families in CATH; building a support vector machine (SVM) to automatically assign domains to superfamilies; improved search facilities to return alignments of query sequences against multiple sequence alignments; the redesign of the web pages and download site.

Das & Orengo (2016) Protein function annotation using protein domain family resources. Methods 93:24-34. (pmid: 26434392)

[ PubMed ] [ DOI ] As a result of the genome sequencing and structural genomics initiatives, we have a wealth of protein sequence and structural data. However, only about 1% of these proteins have experimental functional annotations. As a result, computational approaches that can predict protein functions are essential in bridging this widening annotation gap. This article reviews the current approaches of protein function prediction using structure and sequence based classification of protein domain family resources with a special focus on functional families in the CATH-Gene3D resource.

Sillitoe et al. (2015) CATH: comprehensive structural and functional annotations for genome sequences. Nucleic Acids Res 43:D376-81. (pmid: 25348408)

[ PubMed ] [ DOI ] The latest version of the CATH-Gene3D protein structure classification database (4.0, http://www.cathdb.info) provides annotations for over 235,000 protein domain structures and includes 25 million domain predictions. This article provides an update on the major developments in the 2 years since the last publication in this journal including: significant improvements to the predictive power of our functional families (FunFams); the release of our 'current' putative domain assignments (CATH-B); a new, strictly non-redundant data set of CATH domains suitable for homology benchmarking experiments (CATH-40) and a number of improvements to the web pages.

 

Notes

 

Self-evaluation

 

 

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If in doubt, ask! If anything about this learning unit is not clear to you, do not proceed blindly but ask for clarification. Post your question on the course mailing list: others are likely to have similar problems. Or send an email to your instructor.

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About ...
 
Author:

Boris Steipe <boris.steipe@utoronto.ca>

Created:

2017-08-05

Modified:

2017-08-05

Version:

0.1

Version history:

• 0.1 First stub

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