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Frequently Asked Questions in MACiE


The Answers

Navigating around MACiE

MACiE can be navigated from the left hand pannel of the web pages. The link at the very top of this pannel (MACiE Home) will always take the user back to the home page. The four links shown below this (in red) detail the number of entries, EC numbers, PDB codes and CATH codes in MACiE, and clicking on one of these will take the user to a list of the MACiE entries ordered by the given identifier (e.g. clicking on the EC Numbers link will show all MACiE entries sorted by EC number). The "Database Analysis and Statistics" link will take the user to the analysis pages and the "Search MACiE" link will take the user to the query home page, and will also expand out to show all the queries available as a new menu. This top section is always available.

The next menu section links the user to more general information regarding the MACiE database, such as this FAQ and other documentaion. Whilst this section is always shown, when the user is in the entry view, it will appear below the entry specific menu.

The entry view menu shows the overall reaction overview (Overview), this menu item expands to show links to the structureal overview, similar reactions (from the manual annotation) atthe overall reaction level and similar reactions (again, from the manual annotation) at the composite reaction level. New, when there is an animation available, this link is shown, and finally, a list of all the reaction steps, which take the user to a specific step view.

This side bar also links the user directly to our sister databases Metal-MACiE, CoFactor as well as the group pages of the main people involved in the MACiE project.

We have included tool-tips on the homepage to aid you in using the searches available there. Tool-tips are also interspersed around MACiE for definitions of terms, most notable for amino acid function.



What is in a MACiE Entry

Each entry in MACiE consists of an overall reaction, individual reaction stages and many of the entries up to M0202 also contain an animated reaction. You will also find a 3D analysis page for each entry and similarity searches (based on either the overall bond changes or composite bond changes (mechanism) of the entry.



The Overall Reaction

Each overall reaction in MACiE contains a pictorial representation of the overall transformation occurring, the names and various identifiers of the chemical compounds involved, a list of the amino acid residues involved, the species name in which the reaction occurs, and the reference we have used to create the entry. Wherever appropriate, a reference contains a PubMed Number which links to the relevant PubMed entry. Each entry also contains links to the following databases:

  • PDB-SUM (from the links section)
  • Enzyme Structures Database (from the links section)
  • Catalytic Site Atlas (from the links section)
  • IntEnz (from the menu section)
  • EzCatDB, where there is a corresponding entry in MACiE (from the links section)
  • SFLD, where there is a corresponding entry in MACiE (from the links section)
  • BRENDA (from the links section)
  • KEGG (from the links section)
  • The Enzyme Commission entry (from the links section)
  • PDBe entry (from the menu section and the links section)
  • RCSB PDB entry (from the links section)
  • CATH Code entry (from the menu section)
  • KEGG identifier (from the reactants and products list)
  • A MACiE identifier, this is only assigned where there is no other code assignable, e.g. the molecule does not have a KEGG code (from the reactants and products list)
  • A PDB identifier, this is assigned protein overall reactants and products (from the reactants and products list)
  • The ChEBI Identifier (from the reactants and products list)
  • Uniprot entries (from the general information section and the menu section)
  • MetaCyc (from the links section)
  • Metal-MACiE (when there is a metal cofactor involved in the reaction)
  • CoFactor (when there is an organic cofactor involved in the reaction)

The overall reaction is the page at which a MACiE entry is accessed (although some searches will allow a user to go directly to the returned stage). On the left hand side of the screen is a menu which contains basic information on the entry (enzyme name, PDB code, EC number and CATH code) as well as a list of links to all the steps involved, similar reaction lists, the animation and the structural summary. The user will also find the references used to create the entry here and details of other "similar" entries in MACiE, either at the EC number level of catalytic CATHC code level, as well as the homologues present. This section also detailes the steps involved as a breif free text field, and the overview of the catalytic residues and cofactors involved.



The Animations

The animations are automatically generated from the raw CML as scalable vector graphics (SVG). Unfortunately, due to circumstances beyond our control, these currently only work well on Internet Explorer and Avant with the Adobe SVG plug-in, which is available from adobe.com. Whilst Firefox and some other browser do support SVGs, the do not seem to currently support the animation elements. This seems to be a case of Adobe removing support for browsers other than Internet Explorer from their software. We will continue to try to resolve this issue.

We are currently aware of problems with the following browsers:

  • Firefox
  • Opera
  • Netscape
  • Konqueror
  • Safari
  • Camino


The Reaction Stages

Each reaction stage in MACiE, which is also termed a reaction step, contains a pictorial representation of the chemical changes taking place, a textual description of the mechanism and any mechanism components taking place in the step, comments on the step are also included. A full, and detailed, listing of all catalytic residues is also present, including the functions those residues are performing. Each residue function is linked to a tool-tip which gives the MACiE dictionary definition of that particular function. The reaction stage also contains a full and detailed listing of the cofactors (both metal and organic) which are involved in the step and their functions. Finally, the reaction stage contains a listing of the bonds involved and the reactive centres.



Mechanism

In MACiE we limit the definition of mechanism to that of the Ingold definitions, plus those that we have added to explicitly deal with mechanisms which we felt did not comfortably fall into the Ingold classification. For more information on Ingold mechanisms and the mechanisms defined in MACiE, please see here.



Mechanism Components

A mechanism component is that which is part of the mechanism, but not a mechanism in itself. For example, a bond cleavage is an integral part of an elimination mechanism, but it is not a mechanism in itself. Mechanism components also include alternative descriptions of the chemical change occurring, e.g. hydrolysis, dephosphorylation, etc. For more information on the mechanism components, please see here.



Alternative Mechanisms

There is often more than one possible mechanism that fits all the available experimental data. In MACiE, we have attempted, where possible, to annotate cases of these alternative mechanisms. Unfortunately, in the current release these are only available as stage or overall reaction comments, although it is our intention to further develop MACiE such that we can fully represent these alternatives..



Downloading MACiE

Unfortunately, it is currently not possible to download a copy of the MACiE database from the website. However, if you wish to obtain a copy of MACiE, please email me. We simply ask that you tell us what you're planing on doing with MACiE (because we're interested in what research MACiE is being used for) and ask that you respect the copyright and do not further distribute MACiE. If you add any new data into MACiE, we would also ask that you allow us to incorporate that new data into the next release of MACiE. If you publish, please cite:
G. L. Holliday, D. E. Almonacid, G. J. Bartlett, N. M. O'Boyle, J. W. Torrance, P. Murray-Rust, J. B. O. Mitchell and J. M. Thornton. MACiE (Mechanism, Annotation and Classification in Enzymes): novel tools for searching catalytic mechanisms. (2007) Nucleic Acids Research, 35, D515-D520.



How do I report a bug in MACiE

Please email me with any bugs that you think need attention, with as much detail as possible, including the overall reaction identifier (and step identifier, were applicable), the nature of the bug and what needs to be done to fix it. Please also include any references that might be necessary for us to refer back to in confirming and fixing the error.



How do I request a new Entry or Feature be added to MACiE

Please email me with any new entries that you would like to see included, with as much detail as possible. We do have a long back-log of potential new entries, but user requests will be added to the top of the queue. Please also email me if there is a specific feature or query that you would like to see added to MACiE. We cannot guarentee that new features will be added.



Homology and Heuristics in MACiE

Homology in MACiE is determined by the Catalytic Site Atlas homologies. These are found using a PSI-BLAST search (using an e value cut-off of 0.0005 and five iterations) against all sequences currently in the Protein Databank, plus all sequences in a non-redundant subset of UniProt. In the CSA, and thus MACiE, homologous entries are only included if the residues which align with the catalytic residues in the parent literature entry are identical in residue type. In other words, there must be no mutations at the catalytic residue positions. There are, however, a few exceptions to this rule:

  1. In order to allow for the many active site mutants in the PDB, one (and only one) catalytic residue per site can be different in type from the equivalent in the parent literature entry. This is only permissible if all residue spacing is identical to that in the parent literature entry, and there are at least two catalytic residues.
  2. Sites with only one catalytic residue are permitted to be mutant provided that the residue number is identical to that in the parent entry.
  3. Fuzzy matching of residues is permitted within the following groups: [V,L,I],[F,W,Y],[S,T],[D,E],[K,R],[D,N],[E,Q],[N,Q]. This fuzzy matching cannot be used in combination with rules (1) or (2) above.

It is always possible that the catalytic residue assignments in a homologous entry are incorrect. This is a particular danger when the catalytic function of the homologous entry differs from that of the original literature entry. For this reason, homologous entries which have a different EC code from the original literature entry are clearly marked.

STRICT HOMOLOGY TO DO

In MACiE the search for PDB codes implements the PDB homology, the search is detailed in the figure following.

PDB Search Heuristics

In MACiE, homology has only been included for the PDB searches. However, due to the fact that we only have a very few EC numbers listed the EC search will step up through the entered EC code to find EC codes similar to the one that was looked for. This assumes that (except for a few cases) similar EC codes will have similar functions. The search works in the following manner (and is shown in the figure below) and can be entered at any point on the flow diagram:

  1. EC 1.1.1.1 does not exist in the current database, the search will search for EC 1.1.1.1 in the obsolete EC codes.
  2. EC 1.1.1.1 does not exist in the current, or obsolete codes, the search will discard the final digit and search for any enzymes with the code EC 1.1.1.-
  3. EC 1.11.99.- does not exist in the current database, the search will discard the final digit and search for any enzymes with the code EC 1.11.-.-
  4. EC 1.99.-.- does not exist in the current database, the search will discard the final digit and search for any enzymes with the code EC 1.-.-.-

EC Search Heuristics

Obviously, the more generic the search becomes, the less likely the mechanisms are to be the same. The same heuristics are also offered for the obsolete EC codes.

If the search starts discarding digits, then the user is offered an advanced search option (see following figure), which allows for a structural homology search based on the EC number. However, it should be noted that this is not automatically run, and is no guarantee of a similar mechanism.

EC Advanced Search Heuristics

Finally, the enzyme name is searchable, and this also employs heuristics to widen the coverage of names in MACiE, the search is detailed in the figure following.

Name Search Heuristics



Why are some CATH codes shown in bold?

In MACiE there are two types of CATH domain:

  1. Catalytic CATH domains. These are those domains in the protein that furnish at least one catalytic amino acid residue. Whenever these domains appear in a results table, they are highlighted by bold text, to distinguish them from the "other" domains which are not defined as catalytic.
  2. "Other" CATH domains. These include all the CATH domains in the protein that are not annotated as catalytic, at present this includes the cofactor (metal and organic) and substrate binding domains as well as those domains which are not annotated with any role in the reaction.


How is reaction similarity calculated?

Each reaction in MACiE, which includes all reaction steps as well as the overall reactions, has reactive centre information in the form of bonds formed, cleaved and changed in order. At the very least this information is garnered from manual annotation of the reaction, but where possible we also perform automatic atom-atom mapping and generate reactive centre information from this information. The information in the reactive centre is then coded up into a fingerprint, which is stored in the database. This fingerprint contains the specific count of the bonds changed in the reaction, which includes:

  • Bonds formed
  • Bonds cleaved
  • Bonds changed in order (each different order change is considered separately, i.e. a bond order change of 2 to 1 is different to a bond order change of 1 to 2)

There are three different "types" of fingerprint in MACiE, which can be calculated as either direction dependent or direction independent:

  1. Overall reaction based off the manual annotation of the bond changes for the overall substrates to products only
  2. Overall reaction based off the manual annotation of teh bond changes for the composite bond changes of all the required reaction steps
  3. Reaction steps based off the manual annotation of the bond changes

These data can then be compared using a Tanimoto score for continuous variables:

Tanimoto Similarity Equation

where x jA is the value of the j th attribute in object A and x jB is the value of the j th attribute in object B. The value of the Tanimoto similarity score may be between 0 and 1, where 0 indicates no bits in common and 1 indicates that the two fingerprints are identical.

References:

  • P. Willett, John M. Barnard and Geoffry M. Downs; Chemical Similarity Searching. J. Chem. Inf. Comput. Sci., 38, 983-996, 1998.


What data collections are available in MACiE?

Originally, MACiE was an extension of the CatRes dataset, which was designed to be non-homologous at the “H” level of the CATH domain classification. Whilst some homologoues sneaked in due to the fact that CATH defines single domains rather than entire proteins, the dataset was more-or-less non-homologous. Therefore the first version of MACiE was also largely non-homologous, and where homologues existed, the mechanisms were considerably different.

For the extension of the dataset to Version 2.0 of MACiE, we attempted to populate the database with examples of each EC sub-subclass where there was an available mechanism and crystal structure.

However, for population of the MACiE dataset after version 2.0, we have followed less stringent selection criteria, with the selection of new entries mostly being governed by interest within our own research groups, as well as external user suggestions. Thus, some significant homology has crept in, most notably in the case of the terpenoid synthase family.

Therefore, we have decided to create a number of datasets based on the MACiE database, which cater for a number of different criteria:

Currently, these datasets are not available for individual download, however they can be viewed in overvew in the statistics and analysis section of this website.



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