Definition of a Protein Complex
A cellular component should include more than one gene product; complexes of one gene product with a cofactor, e.g. heme and chlorophyll, should not be included. Homomultimeric proteins, e.g. the homodimeric alcohol dehydrogenase, may be included as cellular component terms, as should heteromultimeric proteins, e.g. hemoglobin with alpha and beta chains. All complexes in the component ontology should be given parentage under the general term protein complex ; GO:0043234 . To distinguish cellular components from functions, use 'complex' in the term name of a component, and append enzyme names with the word 'activity'. For example, the molecular function term pyruvate dehydrogenase activity ; GO:0004738 describes the enzyme activity whereas the cellular component term pyruvate dehydrogenase complex ; GO:0045254 describes the multi-subunit structure in which the enzyme activity resides.
As a rule, GO terms to indicate association of a receptor with its ligand should not be created, as their complex may not always be stable, and there could be a potential explosion of terms. However, we should allow for exceptions. The IntAct database wouldn't curate receptor-ligand complexes if these consisted of a single chain of each, but it will curate complexes when the ligand is not monomeric and receptors oligomerize upon ligand binding. An example of this is GO:1990270 'platelet-derived growth factor receptor-ligand complex', where the ligands are always dimeric and the receptor dimerizes upon ligand binding. (Also, in the case of GO:1990270, the complex has been shown to exist in a variety of experiments, including crystals, pull downs, comigrations, competition assays and more.)
Notes from working group
- NOTE: This is a work in progress and includes continuing discussion with IntAct as well as the GOC.
- Last updated: 9/6/2015, Paola.
- Main working group: Birgit, David Osumi-Sutherland (DOS), Paola, Sandra.
Background and rationale
Recently, GO and IntAct have started to work together to improve the 'protein complex' branch in GO, making it less flat and more informative, and to provide species-agnostic GO terms that IntAct can reference to for their species-specific curation projects, namely the Complex Portal. At the time of writing (Spring 2015) the focus of the Complex Portal lies on human, mouse, yeast and E.coli, but it can take direct curation requests as well (firstname.lastname@example.org). Other MODs are encouraged to collaborate directly.
Here, we collect current guidelines on protein complex terms, to aid GO curators in discerning whether a protein complex belongs in GO or not, and if yes, in including all necessary information when requesting a new protein complex.
Protein complexes in GO
Rule 1: Is the complex stable?
In GO, 'protein complex' is defined as "A stable macromolecular complex composed (only) of two or more polypeptide subunits along with any covalently attached molecules (such as lipid anchors or oligosaccharide) or non-protein prosthetic groups (such as nucleotides or metal ions). Prosthetic group in this context refers to a tightly bound cofactor. The component polypeptide subunits may be identical."
When in doubt, how can a curator figure out if a complex is really stable?
We can refer to the IntAct Complex Portal Rules . These are reported below for reference, and in a definition comment to 'protein complex' to serve as annotation guidance:
What can be described as a complex?
A stable set of (two or more) interacting macromolecules such as proteins which can be co-purified by an acceptable method and have been shown to exist as an isolated, functional unit in vivo. Any interacting non-protein molecules (e.g. small molecules, nucleic acids) will also be included.
What should not be captured:
- Enzyme/substrate, receptor/ligand or any similar transient interactions unless these are a critical part of the complex assembly (e.g. PDGF receptors only become 'dimeric' when linked by the dimeric ligand forming a tetramer).
- Proteins associated in a pulldown/coimmunoprecipitation assay with no functional link or any evidence that this is a defined biological entity rather than a loose affinity complex.
- Any literature complex where the only evidence is based on genetic interaction data.
- Partial complexes.
- If the complex is not stable, it's just protein binding. Interactions can then be captured by a protein-protein interaction DB such as IntAct.
- Beware of partial complexes shown experimentally, especially when crystallised. Some subunits (e.g. transmembrane subunits) cannot be expressed as recombinant proteins and are 'left out' of detailed studies. More reading is often necessary to find out what the full complex is thought to be.
Tricky cases we DO (or could) capture in the IntAct Complex Portal:
- Substrates or ligands if the enzyme or receptor complex only forms in their presence (see PDGF receptors above, e.g. EBI-9082861). These terms would also qualify for GO.
- Homologous proteins, with the same functionality, which would be inferred by sequence similarity to form a complex but for which no physical link has been demonstrated, e.g. proteins A and B have been shown to physically interact and form a functional complex, protein C is a homologue of protein B by sequence similarity and is know to have the same function as B but protein A-C interaction has not been demonstrated experimentally. E.g. SUMO - E1 ligase complexes where there is interaction evidence for binding with SUMO1 (EBI-9345927) but not with SUMO2 (EBI-9349603).
- The Complex Portal could also hold transient complexes, e.g. signaling complexes. We have not created any of these to date but they are possible, and controlled vocabulary terms exist to distinguish the two classes. BUT - they would probably fall outside the scope of GO if GO limit themselves to stable complexes.
- We can also curate complexes that lack full experimental evidence but are commonly regarded as existing, e.g. complexes submitted by ChEMBL for which we only have pharmacological evidence. These complexes are tagged with ECO:0000306 - inferred from background scientific knowledge by manual assertion. E.g GABA (EBI-9008426) receptors and many other transmembrane receptors.
Expanded definition comment for GO:0043234 ‘protein complex’:
"A protein complex in this context is meant as a stable set of interacting proteins which can be co-purified by an acceptable method, and where the complex has been shown to exist as an isolated, functional unit in vivo. Acceptable experimental methods include stringent protein purification followed by detection of protein interaction. The following methods should be considered non-acceptable: simple immunoprecipitation, pull-down experiments from cell extracts without further purification, colocalization and 2-hybrid screening. Interactions that should not be captured as protein complexes include: 1) enzyme/substrate, receptor/ligand or any similar transient interactions, unless these are a critical part of the complex assembly or are required e.g. for the receptor to be functional; 2) proteins associated in a pull-down/co-immunoprecipitation assay with no functional link or any evidence that this is a defined biological entity rather than a loose-affinity complex; 3) any complex where the only evidence is based on genetic interaction data; 4) partial complexes, where some subunits (e.g. transmembrane ones) cannot be expressed as recombinant proteins and are excluded from experiments (in this case, independent evidence is necessary to find out the composition of the full complex, if known). Interactions that may be captured as protein complexes include: 1) enzyme/substrate or receptor/ligand if the complex can only assemble and become functional in the presence of both classes of subunits; 2) complexes where one of the members has not been shown to be physically linked to the other(s), but is a homologue of, and has the same functionality as, a protein that has been experimentally demonstrated to form a complex with the other member(s); 3) complexes whose existence is accepted based on localization and pharmacological studies, but for which experimental evidence is not yet available for the complex as a whole."
Rule 2: Is the complex species-agnostic?
- GO should host species-agnostic complexes, ideally conserved across taxa. Where this isn't known, we should still make the definition generic, and add 'For example, in human this complex contains...' as a definition gloss or definition comment.
- Species-specific complexes don't belong in GO, but IntAct/Complex Portal and/or PRO can take them. (We acknowledge that GO contains many historic terms that contravene this rule. For the time being, the agreement is that we will not review them globally, though we may fix them if and when we come across them.)
- We may, however, need taxon restrictions on a case-by-case basis such as complexes that only exist in prokaryots or eukaryotes. Curators are encouraged to provide these information, if applicable, when they request a new term (or come across an existing one).
Rule 3: Does the complex have a molecular function?
- If yes, add capable_of link(s) to molecular function terms. These links are used by the reasoner to place the complex into the correct branch under 'protein complex'.
Rule 4: Is the complex known to be involved in one or more biological processes?
- If yes, add capable_of_part_of links to biological process(es).
- Note: we decided not to use BP as a qualifier for making grouping terms for complexes as these would become too unspecific, e.g. 'regulatory complex' could include most complexes!
Rule 5: Does the complex contain conserved subunits?
- Many complexes are defined by their subunit composition rather than their activity. Such complexes will still be accepted into the GO. The curator should endeavour to place the new complex as child of a functional parent or a parent term also defined by its composition, in some cases this may be difficult and those complex terms will be direct children of 'protein complex'.
- Complex definitions should include their function or the process they are involved in as well as the defining subunits (using the style "In human, it is composed of..."). However, we need to be careful not to explode the subunit composition in the definition if they turn out to be rather different in the different branches of the tree of life. In such a situation, the curator and editor should consider broadening the term name and adding the various subunit-defined names as narrow synonyms. This should be handled on a case-by-case basis.
- Complexes defined by their subunits but functionally identical to a more generic parent term should not be created as separate GO terms but added to the parent term as narrow synonyms. The specific complex belongs in the Complex Portal.
[DOS to look into some automatic reasoning across subunits but we think it may become tricky. To be discussed among editors.]
Rule 6: Where is the complex located?
- Indicate cellular location as specifically as possible, unless parent already has one.
- The CC location is meant for the complex as a whole. We discussed this in the context of transmembrane complexes where one or more members of the complex are located on one side of the membrane only or have no membrane attachment at all. As gene products have the part_of relationship with the complexes this is fine (and the only way of reflecting the CC for the complex as a whole).
- If we have complexes defined by their location (see below under 'Futures Plans'), does the reasoner take the part_of relationship to place them automatically into the right complex-by-location branch? [DOS?]
- Complexes simply defined by the cell type they are expressed in are not permitted.
Rule 7: Adding appropriate is_a relationships
- We are trying to avoid placing complexes as direct is_a children of 'protein complex', by adding some granularity to this ontology branch.
- An is_a parent of a complex can be a
- complex defined by its activity, via the complex-by-activity TG template
- complex defined by its location, such as 'plasma membrane complex'. [Update: DOS added some useful protein complex grouping terms based on location, such as 'membrane protein complex'.]
- complex defined by its subunit composition. This may be related to protein families but it may be difficult to make it a rule/template (see above).
- We decided NOT to define complexes by their process or MF binding as they would become too generic.
- Note: Complexes can have multiple parents!
- Note: if capable_of MF links are added, and/or if location information is provided as part_of CC, the automatic assert-inference script will take care of placing most newly created protein complex terms more granularly in the ontology.
Rule 8: Adding appropriate part_of relationships
- All complexes should have a part_of link to a cellular component term, even if it's very generic, such as 'cell'.
- CC does not have to be added manually if it's the same as the parent term as it will be inferred.
- If the CC is more specific than the parent, the part_of relationship must be added manually.
- Complexes can be subcomplexes of larger entities and can therefore be part_of another protein complex. If the larger complex necessarily needs the smaller one as its component in order to be functional, a has_part link should also be added (larger complex has_part smaller complex).
- Complexes cannot have several part_of relationships to different CCs, as part_of must ALWAYS be true. If a complex can be part of several larger complexes or be found in several locations, such as cytoplasm and nucleus where it may have different functions, separate terms may have to be considered. [This point is still open to discussion, see https://sourceforge.net/p/geneontology/ontology-requests/10745/, now with DOS. To be discussed on Editor's call.]
How to request protein complexes in GO based on the above (TG template, TG freeform)
- If the complex is generic and its function exists as a GO term, use the TG complex-by-activity template (and add relevant synonyms as discussed above).
- If the function does not yet exist in GO but is clearly defined, create the new MF term first (via SF or TG FF (freeform) depending on the curator's experience), then create the new complex term via the TG complex-by-activity template.
- If the complex-by-activity template is not applicable, create the complex term either via SF or TG FF depending on the curator's experience.
- If a complex is known to be involved in a broader biological process (but not to have a specific molecular function), request the new term using TG FF (by filling in the capable_of_part_of field), or using SF depending on curator's experience. TG FF allows both capable_of and capable_of_part_of links in case a function is known and a process too, but the function is not part of that process.
- IntAct is happy to curate requested complexes into the Complex Portal at the same time as adding to the GO structure. Curators are encouraged to curate complexes directly into the Complex Portal after being trained by IntAct. SGD and UCL are doing this already. Contact email@example.com for either use case.
Complex Definition field
The way definitions of older protein complex terms are structured is not always consistent. Currently, we recommend the following:
- Start the definition referring to the function (if applicable), such as 'A protein complex capable of X function...'.
- Processes can also be mentioned in the def.
- Subunits should only be listed in the definition if the complex is defined by its composition rather than function (see Rule 5 above). If the complex is defined by its function, subunit composition should go into a definition comment or be added as narrow synonyms.
- Complexes should NOT be defined by their stoichiometry, though this may be mentioned in the def as a 'soft' comment (definition gloss), or in a definition comment. The rationale behind this recommendation is that, as knowledge advances and more examples are found, stoichiometry defs would have to be updated, causing a lot of work. It is perfectly fine though to mention something like 'usually consists of a catalytic and a regulatory subunit and possibly further accessory subunits...'.
- We are aware that many older terms' definitions do not conform to these guidelines. At the moment, however, we do not plan on fixing all of them. We may make corrections on a case-by-case basis and/or when working on a given branch.
as discussed in a meeting with Birgit Meldal, Sandra Orchard, David Osumi-Sunderland and Paola Roncaglia on 28/4/2015
We discussed how we can make 'quick gains' in making the ontology more granular beyond the fixes Birgit does on a case by case basis. This is to target historic terms that have only 'protein complex' as a parent because they have no annotation extensions. The aim is to have most complexes grouped either by their function, location or subunit composition.
- Do a pass through term names and definitions to find major groups of complexes that can be grouped by function, e.g. 'catalytic complex' (the term exists but many historic terms have not automatically been classified as such as they have no capable_of extensions). Other keywords: kinase, activity, viral, receptor, respiratory chain... [BM, SO & DOS]
- Add parent terms based on location, such as 'membrane complex' and children or 'mitochondrial complex'. Can the reasoner place complexes automatically into this branch based on their part_of relationship (see above Rule 6)? Should we have a TG template for this? [BM, DOS]
- Combine activity and location, such as 'membrane receptor complex'.
- We discussed grouping by protein families but this may be tricky. Decide on a case by case basis. A working example are the BCL protein family complexes which cannot be grouped by function as they may be pro- and/or antiapoptotic.
Emily started documentation here, in case it's helpful, but this wasn't worked on since 2011: http://wiki.geneontology.org/index.php/Protein_Complex_ids_as_GO_annotation_objects
Birgit's comments [from email to Paola]:
Inheritance of annotations: I agree with the wiki, you cannot inherit MF from a complex to a subunit and even a CC is problematic, see the transmembrane example above. This needs more thinking about. I don't know what you are doing right now...
Orthologies: We infer within taxon groups, e.g. human to mouse to rat or any other mammal etc, depending on where the exp evidence comes from. We systematically infer human-mouse. We have a few pombe complexes inferred from yeast (Sc!) but we don't do it systematically.
Paralogues: We make inferences between related complexes in the same species when the gene products are very similar, e.g. hemoglobin chains for adult and developmental complexes.
'Large' complexes: We have tackled the 'mediator' and we can now link to RNACentral for RNAs so time permitting we'll tackle the 'biggies' soon!
Pro: We have a list of Pro complexes that we consult for refs.
IntAct Complex Portal, http://www.ebi.ac.uk/intact/complex/