Summary: Alkaline phosphatase
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Alkaline phosphatase Edit Wikipedia article
|PDB structures||RCSB PDB PDBe PDBsum|
|Gene Ontology||AmiGO / EGO|
Structure of alkaline phosphatase.
Alkaline phosphatase (ALP, ALKP, ALPase, Alk Phos) (EC 188.8.131.52) or basic phosphatase is a homodimeric protein enzyme of 86 Kilo-Daltons, containing two zinc atoms crucial to its catalytic function per monomer, and is optimally active at alkaline pH environments. As its name indicates, ALP functions best under alkaline pH environments and has the physiological role of dephosphorylating compounds. The enzyme is found across a plethora of organisms, prokaryotes and eukaryotes alike, with the same general function but in different structural forms suitable to the environment they function in. In humans for example, it is found in many forms depending on its origin within the body - it plays an integral role in metabolism within the liver and development within the skeleton. Due to its widespread prevalence in these areas, its concentration in the bloodstream is used by diagnosticians as a biomarker in helping determine diagnoses such as hepatitis or osteomalacia. The level of alkaline phosphatase in the blood is checked through the ALP test, which is often part of routine blood tests. The levels of this enzyme in the blood depend on factors such as age, gender, blood type and whether an individual is pregnant or not. Additionally, abnormal levels of Alkaline phosphatase in the blood could indicate issues relating to the liver, gall bladder or bones. Kidney tumors, infections as well as malnutrition has also shown abnormal level of alkaline phosphatase in blood.
In Gram-negative bacteria, such as Escherichia coli (E.coli), alkaline phosphatase is located in the periplasmic space, external to the inner cell membrane and within the peptidoglycan portion of the cell wall. With the periplasmic gap being more prone to environmental variation than the inner cell, alkaline phosphatase is suitably resistant to inactivation, denaturation, or degradation. This characteristic of the enzyme is uncommon to many other proteins.
The precise structure and function of the isozyme in E.coli is solely geared to supply a source of inorganic phosphate when the environment lacks this metabolite.
While the outer membrane of E.coli contains porins that are permeable to phosphorylated compounds the inner membrane does not. Then, an issue arises in how to transport such compounds across the inner membrane and into the cytosol. Surely, with the strong anionic charge of phosphate groups along with the remainder of the compound they are very much immiscible in the nonpolar region of the bilayer. The solution arises in cleaving the phosphate group away from the compound via ALP. In effect, along with the concomitant compound the phosphate was bound to this enzyme yields pure inorganic phosphate which can be ultimately targeted by the pst transport system for translocation into the cytosol. As such, the main purpose of dephosphorylation by alkaline phosphatase is to increase the rate of diffusion of the molecules into the cells and inhibit them from diffusing out.
Alkaline phosphatase is a zinc-containing dimeric enzyme with the MW: 86,000 Da, each subunit containing 429 amino acids with four cysteine residues linking the two subunits. Alkaline phosphatase contains four Zn ions and two Mg ions, with Zn occupying active sites A and B, and Mg occupying site C, so the fully active native alkaline phosphatase is referred to as (ZnAZnBMgC)2 enzyme. The mechanism of action of alkaline phosphatase involves the geometric coordination of the substrate between the Zn ions in the active sites, whereas the Mg site doesn’t appear to be close enough to directly partake in the hydrolysis mechanism, however, it may contribute to the shape of the electrostatic potential around the active center. Alkaline Phosphatase has a Km of 8.4 x 10^-4.
Alkaline phosphatase in E.coli is uncommonly soluble and active within elevated temperature conditions such as 80 degrees Celsius. Due to the kinetic energy induced by this temperature the weak hydrogen bonds and hydrophobic interactions of common proteins become degraded and therefore coalesce and precipitate. However, upon dimerization of ALP the bonds maintaining its secondary and tertiary structures are effectively buried such that they are not affected as much at this temperature. Furthermore, even at more elevated temperatures such as 90 degrees Celsius ALP has the uncommon characteristic of reverse denaturation. Due to this, while ALP ultimately denatures at about 90 degrees it has the added ability to accurately reform its bonds and return to its original structure and function once cooled back down.
Alkaline phosphatase in E. coli is located in the periplasmic space and can thus be released using techniques that weaken the cell wall and release the protein.Due to the location of the enzyme, and the protein layout of the enzyme the enzyme is in solution with a small amount of proteins than there are in another portion of the cell.  The proteins' heat stability can also be taken advantage of when isolating this enzyme(through heat denaturation).In addition, alkaline phosphatase can be assayed using p-Nitrophenyl phosphate. A reaction where alkaline phosphatase desphosphorylates the non-specific substrate, p-Nitrophenyl phosphate in order to produce p-Nitrophenol(PNP) and inorganic phosphate. PNP's yellow color, and it's λmax at 410 allows spectophotemetry to determine important information about enzymatic activity. Some complexities of bacterial regulation and metabolism suggest that other, more subtle, purposes for the enzyme may also play a role for the cell. In the laboratory, however, mutant Escherichia coli lacking alkaline phosphatase survive quite well, as do mutants unable to shut off alkaline phosphatase production.
The optimal pH for the activity of the E. coli enzyme is 8.0 while the bovine enzyme optimum pH is slightly higher at 8.5. Alkaline Phosphatase accounts for 6% of all proteins in depressed cells.
Use in research
By changing the amino acids of the wild-type alkaline phosphatase enzyme produced by Escherichia coli, a mutant alkaline phosphatase is created which not only has a 36-fold increase in enzyme activity, but also retains thermal stability. Typical uses in the lab for alkaline phosphatases include removing phosphate monoesters to prevent self-ligation, which is undesirable during plasmid DNA cloning.
Common alkaline phosphatases used in research include:
- Shrimp alkaline phosphatase (SAP), from a species of Arctic shrimp (Pandalus borealis). This phosphatase is easily inactivated by heat, a useful feature in some applications.
- Calf-intestinal alkaline phosphatase (CIP)
- Placental alkaline phosphatase (PLAP) and its C terminally truncated version that lacks the last 24 amino acids (constituting the domain that targets for GPI membrane anchoring) - the secreted alkaline phosphatase (SEAP). It presents certain characteristics like heat stability, substrate specificity, and resistance to chemical inactivation.
- Human-intestinal alkaline phosphatase. The human body has multiple types of alkaline phosphatase present, which are determined by a minimum of three gene loci. Each one of these three loci controls a different kind of alkaline phosphatase isozyme. However, the development of this enzyme can be strictly regulated by other factors such as thermostability, electrophoresis, inhibition, or immunology.
Human-intestinal ALPase shows around 80% homology with bovine intestinal ALPase, which holds true their shared evolutionary origins. That same bovine enzyme has more than 70% homology with human placental enzyme. However, the human intestinal enzyme and the placental enzyme only share 20% homology despite their structural similarities.
Alkaline phosphatase has become a useful tool in molecular biology laboratories, since DNA normally possesses phosphate groups on the 5' end. Removing these phosphates prevents the DNA from ligating (the 5' end attaching to the 3' end), thereby keeping DNA molecules linear until the next step of the process for which they are being prepared; also, removal of the phosphate groups allows radiolabeling (replacement by radioactive phosphate groups) in order to measure the presence of the labeled DNA through further steps in the process or experiment. For these purposes, the alkaline phosphatase from shrimp is the most useful, as it is the easiest to inactivate once it has done its job.
Another important use of alkaline phosphatase is as a label for enzyme immunoassays.
Because undifferentiated pluripotent stem cells have elevated levels of alkaline phosphatase on their cell membrane, therefore alkaline phosphatase staining is used to detect these cells and to test pluripotency (i.e., embryonic stem cells or embryonal carcinoma cells).
Current researchers are looking into the increase of tumor necrosis factor-α and its direct effect on the expression of alkaline phosphatase in vascular smooth muscle cells as well as how alkaline phosphatase (AP) affects the inflammatory responses and may play a direct role in preventing organ damage.
- Alkaline phosphatase (AP) affects the inflammatory responses in patients with Chronic kidney disease and is directly associated with Erythropoiesis stimulating agent resistant anemia.
- Intestinal alkaline phosphatase (IAP) and the mechanism it uses to regulate pH and ATP hydrolysis in rat duodenum.
- Testing the effectiveness of the inhibitor and its impact on IAP in acute intestinal inflammation as well as explore the molecular mechanisms of IAP in "ameliorating intestinal permeability."
Alkaline phosphatase is commonly used in the dairy industry as an indicator of successful pasteurization. This is because the most heat stable bacterium found in milk, Mycobacterium paratuberculosis, is destroyed by temperatures lower than those required to denature ALP. Therefore, ALP presence is ideal for indicating successful pasteurization.
Pasteurization verification is typically performed by measuring the fluorescence of a solution which becomes fluorescent when exposed to active ALP. Fluorimetry assays are required by milk producers in the UK to prove alkaline phosphatase has been denatured, as p-Nitrophenylphosphate tests are not considered accurate enough to meet health standards.
Alternatively the colour change of a para-Nitrophenylphosphate substrate in a buffered solution (Aschaffenburg Mullen Test) can be used. Raw milk would typically produce a yellow colouration within a couple of minutes, whereas properly pasteurised milk should show no change. There are exceptions to this, as in the case of heat-stable alkaline phophatases produced by some bacteria, but these bacteria should not be present in milk.
All mammalian alkaline phosphatase isoenzymes except placental (PALP and SEAP) are inhibited by homoarginine, and, in similar manner, all except the intestinal and placental ones are blocked by levamisole. Heating for ~2 hours at 65 °C inactivates most isoenzymes except placental isoforms (PALP and SEAP). Phosphate is another inhibitor which competitively inhibits alkaline phosphatase.
Another known example of an alkaline phosphatase inhibitor is [(4-Nitrophenyl)methyl]phosphonic acid.
In humans, alkaline phosphatase is present in all tissues throughout the entire body, but is particularly concentrated in the liver, bile duct, kidney, bone, intestinal mucosa and placenta. In the serum, two types of alkaline phosphatase isozymes predominate: skeletal and liver. During childhood the majority of alkaline phosphatase are of skeletal origin. Humans and most other mammals contain the following alkaline phosphatase isozymes:
- ALPI – intestinal (molecular weight of 150 kDa)
- ALPL – tissue-nonspecific (liver/bone/kidney)
- ALPP – placental (Regan isozyme)
Alkaline Phosphatase in cancer cells
Studies show that the alkaline phosphatase protein found in cancer cells has similar characteristics to that found in non-malignant body tissues and that the protein originates from the same gene in both the malignant and the non-malignant cells. One study tested the structural comparison between the alkaline phosphatase proteins found in liver giant-cell carcinoma and non-malignant placental cells. In this study, an alkaline phosphatase that was immunochemically similar to placental alkaline phosphatase was purified from metastases of giant-cell carcinoma of the lung and its physical and chemical properties were determined. Thereafter, these were compared with purified placental alkaline phosphatase. The results showed great similarity in both based on evaluations of NH2-terminal sequence, peptide map, subunit molecular weight, and isoelectronic point. Overall, this study strongly supports the supposition that the alkaline phosphatase protein in both tumor and non-malignant placental cells are derived from the same gene.
In a different study in which scientists examined alkaline phosphatase protein presence in a human colon cancer cell line, also known as HT-29, results showed that the enzyme activity was similar to that of the non-malignant intestinal type. However, this study revealed that without the influence of sodium butyrate, alkaline phosphatase activity is fairly low in cancer cells. A study based on sodium butyrate effects on cancer cells conveys that it has an effect on androgen receptor co-regulator expression, transcription activity, and also on histone acetylation in cancer cells. This explains why the addition of sodium butyrate show increased activity of alkaline phosphatase in the cancer cells of the human colon. In addition, this further supports the theory that alkaline phosphatase enzyme activity is actually present in cancer cells.
In another study, choriocarcinoma cells were grown in the presence of 5-bromo-2’-deoxyuridine (BrdUrd) and results conveyed a 30- to 40- fold increase in alkaline phosphatase activity. This procedure of enhancing the activity of the enzyme is known as enzyme induction. The evidence shows that there is in fact activity of alkaline phosphatase in tumor cells, but it is minimal and needs to be enhanced. Results from this study further indicate that activities of this enzyme vary among the different choriocarcinoma cell lines and that the activity of the alkaline phosphatase protein in these cells is lower than in the non-malignant placenta cells. but levels are significantly higher in children and pregnant women. Blood tests should always be interpreted using the reference range from the laboratory that performed the test. High ALP levels can occur if the bile ducts are obstructed. Also, ALP increases if there is active bone formation occurring, as ALP is a byproduct of osteoblast activity (such as the case in Paget's disease of bone). Levels are also elevated in people with untreated coeliac disease. Lowered levels of ALP are less common than elevated levels. The source of elevated ALP levels can be deduced by obtaining serum levels of gamma glutamyltransferase (GGT). Concomitant increases of ALP with GGT should raise the suspicion of hepatobiliary disease.
Some diseases do not affect the levels of alkaline phosphatase, for example, hepatitis C. A high level of this enzyme does not reflect any damage in the liver, even though high alkaline phosphatase levels may result from a blockage of flow in the biliary tract or an increase in the pressure of the liver.
If it is unclear why alkaline phosphatase is elevated, isoenzyme studies using electrophoresis can confirm the source of the ALP. Skelphosphatase (which is localized in osteoblasts and extracellular layers of newly synthesized matrix) is released into circulation by a yet unclear mechanism. Placental alkaline phosphatase is elevated in seminomas and active forms of rickets, as well as in the following diseases and conditions:
- Biliary obstruction
- Bone conditions
- Osteoblastic bone tumors
- Liver disease or hepatitis
- Paget's disease
- Myocardial infarction
The following conditions or diseases may lead to reduced levels of alkaline phosphatase:
- Hypophosphatasia, an autosomal recessive disease
- Postmenopausal women receiving estrogen therapy because of aging
- Men with recent heart surgery, malnutrition, magnesium deficiency, hypothyroidism, or severe anemia
- Children with achondroplasia and cretinism
- Children after a severe episode of enteritis
- Pernicious anemia
- Aplastic anemia
- Chronic myelogenous leukemia
- Wilson's disease
In addition, the following drugs have been demonstrated to reduce alkaline phosphatase:
- Oral contraceptives
Measuring alkaline phosphatase (along with prostate specific antigen) during, and after six months of hormone treated metastatic prostate cancer was shown to predict the survival of patients.
Leukocyte alkaline phosphatase
Leukocyte alkaline phosphatase (LAP) is found within mature white blood cells. White blood cell levels of LAP can help in the diagnosis of certain conditions.
- Higher levels are seen in the physiological response, the leukemoid reaction, and in pathologies that include mature white blood cells, such as polycythemia vera (PV), essential thrombocytosis (ET), and in primary myelofibrosis (PM).
- Lower levels are found in pathologies that involve undeveloped leukocytes, such as chronic myelogenous leukemia (CML), paroxysmal nocturnal hemoglobinuria (PNH) and acute myelogenous leukaemia (AML).
Structure and Properties
Essentially, an alkaline phosphatase is homodimeric enzyme, meaning it is formed with two molecules. Three metal ions are contained in the catalytic sites, which are two Zn atoms and one Mg atom, both types are crucial enzymatic activity to occur. The enzymes will catalyze the hydrolysis of monoesters in phosphoric acid which can additionally catalyze a transphosphorylation reaction with large concentrations of phosphate acceptors. While the main features of the catalytic mechanism and activity are conserved when comparing mammalian and bacterial alkaline phosphate, however mammalian alkaline phosphatase has higher specific activity and Km values thus a lower affinity; have a more alkaline pH optimum; showcase a lower heat stability; are typically membrane bound and are inhibited by l-amino acids and peptides via a means of uncompetitive mechanism. These properties noticeably contrast among different mammalian alkaline phosphatase isozymes and therefore showcase a difference in in vivo functions. Alkaline phosphatase have homology in a large number of other enzymes and compose part of a superfamily of enzymes with several overlapping catalytic aspects substrate traits. This explains why most salient structural features of mammalian alkaline are the way they are and reference their substrate specificity and homology to other members of the nucleoside pyrophosphatase/phosphodiesterase family of isozyme.
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- Alkaline phosphatase at Lab Tests Online
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Internal database links
|SCOOP:||DUF229 Metalloenzyme Phosphodiest Sulfatase|
|Similarity to PfamA using HHSearch:||Sulfatase Metalloenzyme|
External database links
This tab holds annotation information from the InterPro database.
InterPro entry IPR001952
This entry represents alkaline phosphatases (EC) (ALP), which act as non-specific phosphomonoesterases to hydrolyse phosphate esters, optimally at high pH. The reaction mechanism involves the attack of a serine alkoxide on a phosphorus of the substrate to form a transient covalent enzyme-phosphate complex, followed by the hydrolysis of the serine phosphate. Alkaline phosphatases are found in all kingdoms of life, with the exception of some plants. Alkaline phosphatases are metalloenzymes that exist as a dimer, each monomer binding metal ions. The metal ions they carry can differ, although zinc and magnesium are the most common. For example, Escherichia coli alkaline phosphatase (encoded by phoA) requires the presence of two zinc ions bound at the M1 and M2 metal sites, and one magnesium ion bound at the M3 site [PUBMED:15938627]. However, alkaline phosphatases from Thermotoga maritima and Bacillus subtilis require cobalt for maximal activity [PUBMED:11910033].
In mammals, there are four alkaline phosphatase isozymes: placental, placental-like (germ cell), intestinal and tissue-nonspecific (liver/bone/kidney). All four isozymes are anchored to the outer surface of the plasma membrane by a covalently attached glycosylphosphatidylinositol (GPI) anchor [PUBMED:17520090]. Human alkaline phosphatases have four metal binding sites: two for zinc, one for magnesium, and one for calcium ion. Placental alkaline phosphatase (ALPP or PLAP) is highly polymorphic, with at least three common alleles [PUBMED:11124260]. Its activity is down-regulated by a number of effectors such as l-phenylalanine, 5'-AMP, and by p-nitrophenyl-phosphonate (PNPPate) [PUBMED:15946677]. The placental-like isozyme (ALPPL or PLAP-like) is elevated in germ cell tumours. The intestinal isozyme (ALPI or IAP) has the ability to detoxify lipopolysaccharide and prevent bacterial invasion across the gut mucosal barrier [PUBMED:18292227]. The tissue-nonspecific isozyme (ALPL) is, and may play a role in skeletal mineralisation. Defects in ALPL are a cause of hypophosphatasia, including infantile-type (OMIM:241500), childhood-type (OMIM:241510) and adult-type (OMIM:146300). Hhypophosphatasia is an inherited metabolic bone disease characterised by defective skeletal mineralisation [PUBMED:17719863].
This entry also contains the related enzyme streptomycin-6-phosphate phosphatase (EC) (encoded by strK) from Streptomyces species. This enzyme is involved in the synthesis of the antibiotic streptomycin, specifically cleaving both streptomycin-6-phosphate and, more slowly, streptomycin-3-phosphate [PUBMED:1654502].
The mapping between Pfam and Gene Ontology is provided by InterPro. If you use this data please cite InterPro.
|Molecular function||phosphatase activity (GO:0016791)|
|Biological process||metabolic process (GO:0008152)|
Below is a listing of the unique domain organisations or architectures in which this domain is found. More...
The graphic that is shown by default represents the longest sequence with a given architecture. Each row contains the following information:
- the number of sequences which exhibit this architecture
a textual description of the architecture, e.g. Gla, EGF x 2, Trypsin.
This example describes an architecture with one
Gladomain, followed by two consecutive
EGFdomains, and finally a single
- a link to the page in the Pfam site showing information about the sequence that the graphic describes
- the UniProt description of the protein sequence
- the number of residues in the sequence
- the Pfam graphic itself.
Note that you can see the family page for a particular domain by clicking on the graphic. You can also choose to see all sequences which have a given architecture by clicking on the Show link in each row.
Finally, because some families can be found in a very large number of architectures, we load only the first fifty architectures by default. If you want to see more architectures, click the button at the bottom of the page to load the next set.
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The members of this clan all share a common structure of their catalytic domains, which contain conserved metal binding residues .
The clan contains the following 10 members:Alk_phosphatase DUF1501 DUF229 DUF4976 Metalloenzyme PglZ Phosphodiest Phosphoesterase Sulfatase Sulfatase_C
We store a range of different sequence alignments for families. As well as the seed alignment from which the family is built, we provide the full alignment, generated by searching the sequence database (reference proteomes) using the family HMM. We also generate alignments using four representative proteomes (RP) sets, the UniProtKB sequence database, the NCBI sequence database, and our metagenomics sequence database. More...
There are various ways to view or download the sequence alignments that we store. We provide several sequence viewers and a plain-text Stockholm-format file for download.
We make a range of alignments for each Pfam-A family:
- the curated alignment from which the HMM for the family is built
- the alignment generated by searching the sequence database using the HMM
- Representative Proteomes (RPs) at 15%, 35%, 55% and 75% co-membership thresholds
- alignment generated by searching the UniProtKB sequence database using the family HMM
- alignment generated by searching the NCBI sequence database using the family HMM
- alignment generated by searching the metagenomics sequence database using the family HMM
You can see the alignments as HTML or in three different sequence viewers:
- a Java applet developed at the University of Dundee. You will need Java installed before running jalview
- an HTML page showing the whole alignment.Please note: full Pfam alignments can be very large. These HTML views are extremely large and often cause problems for browsers. Please use either jalview or the Pfam viewer if you have trouble viewing the HTML version
- an HTML-based representation of the alignment, coloured according to the posterior-probability (PP) values from the HMM. As for the standard HTML view, heatmap alignments can also be very large and slow to render.
You can download (or view in your browser) a text representation of a Pfam alignment in various formats:
You can also change the order in which sequences are listed in the alignment, change how insertions are represented, alter the characters that are used to represent gaps in sequences and, finally, choose whether to download the alignment or to view it in your browser directly.
You may find that large alignments cause problems for the viewers and the reformatting tool, so we also provide all alignments in Stockholm format. You can download either the plain text alignment, or a gzipped version of it.
We make a range of alignments for each Pfam-A family. You can see a description of each above. You can view these alignments in various ways but please note that some types of alignment are never generated while others may not be available for all families, most commonly because the alignments are too large to handle.
1Cannot generate PP/Heatmap alignments for seeds; no PP data available
Key: available, not generated, — not available.
Format an alignment
We make all of our alignments available in Stockholm format. You can download them here as raw, plain text files or as gzip-compressed files.
You can also download a FASTA format file containing the full-length sequences for all sequences in the full alignment.
HMM logos is one way of visualising profile HMMs. Logos provide a quick overview of the properties of an HMM in a graphical form. You can see a more detailed description of HMM logos and find out how you can interpret them here. More...
If you find these logos useful in your own work, please consider citing the following article:
This page displays the phylogenetic tree for this family's seed alignment. We use FastTree to calculate neighbour join trees with a local bootstrap based on 100 resamples (shown next to the tree nodes). FastTree calculates approximately-maximum-likelihood phylogenetic trees from our seed alignment.
Note: You can also download the data file for the tree.
Curation and family details
This section shows the detailed information about the Pfam family. You can see the definitions of many of the terms in this section in the glossary and a fuller explanation of the scoring system that we use in the scores section of the help pages.
|Number in seed:||10|
|Number in full:||3399|
|Average length of the domain:||365.50 aa|
|Average identity of full alignment:||26 %|
|Average coverage of the sequence by the domain:||77.85 %|
|HMM build commands:||
build method: hmmbuild -o /dev/null HMM SEED
search method: hmmsearch -Z 26740544 -E 1000 --cpu 4 HMM pfamseq
|Family (HMM) version:||19|
|Download:||download the raw HMM for this family|
Weight segments by...
Change the size of the sunburst
selected sequences to HMM
a FASTA-format file
- 0 sequences
- 0 species
This visualisation provides a simple graphical representation of the distribution of this family across species. You can find the original interactive tree in the More....
This chart is a modified "sunburst" visualisation of the species tree for this family. It shows each node in the tree as a separate arc, arranged radially with the superkingdoms at the centre and the species arrayed around the outermost ring.
How the sunburst is generated
The tree is built by considering the taxonomic lineage of each sequence that has a match to this family. For each node in the resulting tree, we draw an arc in the sunburst. The radius of the arc, its distance from the root node at the centre of the sunburst, shows the taxonomic level ("superkingdom", "kingdom", etc). The length of the arc represents either the number of sequences represented at a given level, or the number of species that are found beneath the node in the tree. The weighting scheme can be changed using the sunburst controls.
In order to reduce the complexity of the representation, we reduce the number of taxonomic levels that we show. We consider only the following eight major taxonomic levels:
Colouring and labels
Segments of the tree are coloured approximately according to their superkingdom. For example, archeal branches are coloured with shades of orange, eukaryotes in shades of purple, etc. The colour assignments are shown under the sunburst controls. Where space allows, the name of the taxonomic level will be written on the arc itself.
As you move your mouse across the sunburst, the current node will be highlighted. In the top section of the controls panel we show a summary of the lineage of the currently highlighed node. If you pause over an arc, a tooltip will be shown, giving the name of the taxonomic level in the title and a summary of the number of sequences and species below that node in the tree.
Anomalies in the taxonomy tree
There are some situations that the sunburst tree cannot easily handle and for which we have work-arounds in place.
Missing taxonomic levels
Some species in the taxonomic tree may not have one or more of the main eight levels that we display. For example, Bos taurus is not assigned an order in the NCBI taxonomic tree. In such cases we mark the omitted level with, for example, "No order", in both the tooltip and the lineage summary.
Unmapped species names
The tree is built by looking at each sequence in the full alignment for the family. We take the name of the species given by UniProt and try to map that to the full taxonomic tree from NCBI. In some cases, the name chosen by UniProt does not map to any node in the NCBI tree, perhaps because the chosen name is listed as a synonym or a misspelling in the NCBI taxonomy.
So that these nodes are not simply omitted from the sunburst tree, we group them together in a separate branch (or segment of the sunburst tree). Since we cannot determine the lineage for these unmapped species, we show all levels between the superkingdom and the species as "uncategorised".
Since we reduce the species tree to only the eight main taxonomic levels, sequences that are mapped to the sub-species level in the tree would not normally be shown. Rather than leave out these species, we map them instead to their parent species. So, for example, for sequences belonging to one of the Vibrio cholerae sub-species in the NCBI taxonomy, we show them instead as belonging to the species Vibrio cholerae.
Too many species/sequences
For large species trees, you may see blank regions in the outer layers of the sunburst. These occur when there are large numbers of arcs to be drawn in a small space. If an arc is less than approximately one pixel wide, it will not be drawn and the space will be left blank. You may still be able to get some information about the species in that region by moving your mouse across the area, but since each arc will be very small, it will be difficult to accurately locate a particular species.
The tree shows the occurrence of this domain across different species. More...
We show the species tree in one of two ways. For smaller trees we try to show an interactive representation, which allows you to select specific nodes in the tree and view them as an alignment or as a set of Pfam domain graphics.
Unfortunately we have found that there are problems viewing the interactive tree when the it becomes larger than a certain limit. Furthermore, we have found that Internet Explorer can become unresponsive when viewing some trees, regardless of their size. We therefore show a text representation of the species tree when the size is above a certain limit or if you are using Internet Explorer to view the site.
If you are using IE you can still load the interactive tree by clicking the "Generate interactive tree" button, but please be aware of the potential problems that the interactive species tree can cause.
For all of the domain matches in a full alignment, we count the number that are found on all sequences in the alignment. This total is shown in the purple box.
We also count the number of unique sequences on which each domain is found, which is shown in green. Note that a domain may appear multiple times on the same sequence, leading to the difference between these two numbers.
Finally, we group sequences from the same organism according to the NCBI code that is assigned by UniProt, allowing us to count the number of distinct sequences on which the domain is found. This value is shown in the pink boxes.
We use the NCBI species tree to group organisms according to their taxonomy and this forms the structure of the displayed tree. Note that in some cases the trees are too large (have too many nodes) to allow us to build an interactive tree, but in most cases you can still view the tree in a plain text, non-interactive representation. Those species which are represented in the seed alignment for this domain are highlighted.
You can use the tree controls to manipulate how the interactive tree is displayed:
- show/hide the summary boxes
- highlight species that are represented in the seed alignment
- expand/collapse the tree or expand it to a given depth
- select a sub-tree or a set of species within the tree and view them graphically or as an alignment
- save a plain text representation of the tree
Please note: for large trees this can take some time. While the tree is loading, you can safely switch away from this tab but if you browse away from the family page entirely, the tree will not be loaded.
There is 1 interaction for this family. More...
We determine these interactions using iPfam, which considers the interactions between residues in three-dimensional protein structures and maps those interactions back to Pfam families. You can find more information about the iPfam algorithm in the journal article that accompanies the website.
For those sequences which have a structure in the Protein DataBank, we use the mapping between UniProt, PDB and Pfam coordinate systems from the PDBe group, to allow us to map Pfam domains onto UniProt sequences and three-dimensional protein structures. The table below shows the structures on which the Alk_phosphatase domain has been found. There are 232 instances of this domain found in the PDB. Note that there may be multiple copies of the domain in a single PDB structure, since many structures contain multiple copies of the same protein seqence.
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