Summary: Luciferase helical bundle domain
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Luciferase Edit Wikipedia article
|Structure of Photinus pyralis firefly luciferase.|
|PDB||1LCI More structures|
|Bacterial Luciferase monooxygenase family|
|Dinoflagellate Luciferase catalytic domain|
crystal structure of a luciferase domain from the dinoflagellate Lingulodinium polyedrum
|Dinoflagellate Luciferase/LBP N-terminal domain|
|Dinoflagellate Luciferase helical bundle domain|
crystal structure of a Dinoflagellate luciferase domain from the dinoflagellate Lingulodinium polyedrum
Luciferase is a generic term for the class of oxidative enzymes used in bioluminescence and is distinct from a photoprotein. The name is derived from Lucifer, the root of which means 'light-bearer' (lucem ferre). One example is the firefly luciferase (EC 188.8.131.52) from the firefly Photinus pyralis. "Firefly luciferase" as a laboratory reagent often refers to P. pyralis luciferase although recombinant luciferases from several other species of fireflies are also commercially available.
A variety of organisms regulate their light production using different luciferases in a variety of light-emitting reactions. The most famous are the fireflies, although the enzyme exists in organisms as different as the Jack-O-Lantern mushroom (Omphalotus olearius) and many marine creatures.
 Firefly and click beetle
The luciferases of fireflies - of which there are over 2000 species - and of the Elateroidea (fireflies, click beetles and relatives) in general - are diverse enough to be useful in molecular phylogeny. In fireflies, the oxygen required is supplied through a tube in the abdomen called the abdominal trachea. One well-studied luciferase is that of the Photinini firefly Photinus pyralis, which has an optimum pH of 7.8.
 Sea pansy
Also well studied is the luciferase from Renilla reniformis. In this organism, the luciferase (Renilla-luciferin 2-monooxygenase) is closely associated with a luciferin-binding protein as well as a green fluorescent protein (GFP). Calcium triggers release of the luciferin (coelenterazine) from the luciferin binding protein. The substrate is then available for oxidation by the luciferase, where it is degraded to coelenteramide with a resultant release of energy. In the absence of GFP, this energy would be released as a photon of blue light (peak emission wavelength 482 nm). However, due to the closely associated GFP, the energy released by the luciferase is instead coupled through resonance energy transfer to the fluorophore of the GFP, and is subsequently released as a photon of green light (peak emission wavelength 510 nm). The catalyzed reaction is:
Bacterial bioluminescence is seen in Photobacterium species, Vibrio fischeri, haweyi, and harveyi. Light emission in some utilize 'antenna' such as 'lumazine protein' to accept the energy from the primary excited state on the luciferase, resulting in an excited lulnazine chromophore which emits light that is of a shorter wavelength (more blue), while in others use a yellow fluorescent protein (YFP) with FMN as the chromophore and emits light that is red-shifted relative to that from luciferase.
Dinoflagellate luciferase is a multi-domain protein, consisting of an N-terminal domain, and three catalytic domains, each of which preceded by a helical bundle domain. The structure of the dinoflagellate luciferase catalytic domain has been solved. The core part of the domain is a 10 stranded beta barrel that is structurally similar to lipocalins and FABP. The N-terminal domain is conserved between dinoflagellate luciferase and luciferin binding proteins (LBPs). It has been suggested that this region may mediate an interaction between LBP and luciferase or their association with the vacuolar membrane. The helical bundle domain has a three helix bundle structure that holds four important histidines that are thought to play a role in the pH regulation of the enzyme.
Newer luciferases have recently been identified that, unlike other luciferases above, are naturally secreted molecules. One such example is the Metridia luciferase (MetLuc) that is derived from the marine copepod Metridia longa. The Metridia longa secreted luciferase gene encodes a 24 kDa protein containing an N-terminal secretory signal peptide of 17 amino acid residues. The sensitivity and high signal intensity of this luciferase molecule proves advantageous in many reporter studies. Some of the benefits of using a secreted reporter molecule like MetLuc is its no-lysis protocol that allows one to be able to conduct live cell assays and multiple assays on the same cell.
 Mechanism of reaction
The chemical reaction catalyzed by firefly luciferase takes place in two steps:
Luciferyl adenylate can additionally participate in a side reaction with O2 to form hydrogen peroxide and dehydroluciferyl-AMP. About 20% of the luciferyl adenylate intermediate is oxidized in this pathway.
The reaction catalyzed by bacterial luciferase is also an oxidative process:
- FMNH2 + O2 + RCHO â FMN + RCOOH + H2O + light
In the reaction, a reduced flavin mononucleotide oxidizes a long-chain aliphatic aldehyde to an aliphatic carboxylic acid. The reaction forms an excited hydroxyflavin intermediate, which is dehydrated to the product FMN to emit blue-green light.
Nearly all of the energy input into the reaction is transformed into light. The reaction is 80% to 90% efficient. As a comparison, the incandescent light bulb only converts about 10% of its energy into light. and a 150 lumen per Watt (lm/W) LED converts 20% of input energy to visible light.
Firefly luciferase generates light from luciferin in a multistep process. First, D-luciferin is adenylated by MgATP to form luciferyl adenylate and pyrophosphate. After activation by ATP, luciferyl adenylate is oxidized by molecular oxygen to form a dioxetanone ring. A decarboxylation reaction forms an excited state of oxyluciferin, which tautomerizes between the keto-enol form. The reaction finally emits light as oxyluciferin returns to the ground state.
Luciferase can function in two different pathways: a bioluminescence pathway and a CoA-ligase pathway. In both pathways, luciferase initially catalyzes an adenylation reaction with MgATP. However, in the CoA-ligase pathway, CoA can displace AMP to form luciferyl CoA.
Fatty acyl-CoA synthetase similarly activates fatty acids with ATP, followed by displacement of AMP with CoA. Because of their similar activities, luciferase is able to replace fatty acyl-CoA synthetase and convert long-chain fatty acids into fatty-acyl CoA for beta oxidation.
The protein structure of firefly luciferase consists of two compact domains: the N-terminal domain and the C-terminal domain. The N-terminal domain is composed of two Î²-sheets in an Î±Î²Î±Î²Î± structure and a Î² barrel. The two Î²-sheets stack on top of each other, with the Î²-barrel covering the end of the sheets.
The C-terminal domain is connected to the N-terminal domain by a flexible hinge, which can separate the two domains. The amino acid sequences on the surface of the two domains facing each other are conserved in bacterial and firefly luciferase, thereby strongly suggesting that the active site is located in the cleft between the domains.
During a reaction, luciferase has a conformational change and goes into a âclosedâ form with the two domains coming together to enclose the substrate. This ensures that water is excluded from the reaction and does not hydrolyze ATP or the electronically excited product.
 Spectral differences in bioluminescence
Firefly luciferase bioluminescence color can vary between yellow-green (Î»max = 550 nm) to red (Î»max = 620). There are currently several different mechanisms describing how the structure of luciferase affects the emission spectrum of the photon and effectively the color of light emitted.
One mechanism proposes that the color of the emitted light depends on whether the product is in the keto or enol form. The mechanism suggests that red light is emitted from the keto form of oxyluciferin, while green light is emitted from the enol form of oxyluciferin. However, 5,5-dimethyloxyluciferin emits green light even though it is constricted to the keto form because it cannot tautomerize.
Another mechanism proposes that twisting the angle between benzothiazole and thiazole rings in oxyluciferin determines the color of bioluminescence. This explanation proposes that a planar form with an angle of 0Â° between the two rings corresponds to a higher energy state and emits a higher-energy green light, whereas an angle of 90Â° puts the structure in a lower energy state and emits a lower-energy red light.
The most recent explanation for the bioluminescence color examines the microenvironment of the excited oxyluciferin. Studies suggest that the interactions between the excited state product and nearby residues can force the oxyluciferin into an even higher energy form, which results in the emission of green light. For example, Arg 218 has electrostatic interactions with other nearby residues, restricting oxyluciferin from tautomerizing to the enol form. Similarly, other results have indicated that the microenvironment of luciferase can force oxyluciferin into a more rigid, high-energy structure, forcing it to emit a high-energy green light.
D-luciferin is the substrate for firefly luciferaseâs bioluminescence reaction, while L-luciferin is the substrate for luciferyl-CoA synthetase activity. Both reactions are inhibited by the substrateâs enantiomer: L-luciferin and D-luciferin inhibit the bioluminescence pathway and the CoA-ligase pathway, respectively. This shows that luciferase can differentiate between the isomers of the luciferin structure.
L-luciferin is able to emit a weak light even though it is a competitive inhibitor of D-luciferin and the bioluminescence pathway. Light is emitted because the CoA synthesis pathway can be converted to the bioluminescence reaction by hydrolyzing the final product via an esterase back to D-luciferin.
Luciferase activity is additionally inhibited by oxyluciferin  and allosterically activated by ATP. When ATP binds to the enzymeâs two allosteric sites, luciferaseâs affinity to bind ATP in its active site increases.
Luciferase can be produced in the lab through genetic engineering for a number of purposes. Luciferase genes can be synthesized and inserted into organisms or transfected into cells. Mice, silkworms, and potatoes are just a few of the organisms that have already been engineered to produce the protein.
In the luciferase reaction, light is emitted when luciferase acts on the appropriate luciferin substrate. Photon emission can be detected by light sensitive apparatus such as a luminometer or modified optical microscopes. This allows observation of biological processes. Since light excitation is not needed for luciferase bioluminescence, there is minimal autofluorescence and therefore virtually background-free fluorescence.  Therefore, as little as 0.02pg can still be accurately measured using a standard scintillation counter. 
In biological research, luciferase is commonly used as a reporter to assess the transcriptional activity in cells that are transfected with a genetic construct containing the luciferase gene under the control of a promoter of interest. Additionally proluminescent molecules that are converted to luciferin upon activity of a particular enzyme can be used to detect enzyme activity in coupled or two-step luciferase assays. Such substrates have been used to detect caspase activity and cytochrome P450 activity, among others.
Luciferase can also be used to detect the level of cellular ATP in cell viability assays or for kinase activity assays. Luciferase can act as an ATP sensor protein through biotinylation. Biotinylation will immobilize luciferase on the cell-surface by binding to a streptavidin-biotin complex. This allows luciferase to detect the efflux of ATP from the cell and will effectively display the real-time release of ATP through bioluminescence. Luciferase can additionally be made more sensitive for ATP detection by increasing the luminescence intensity through genetic modification.
Whole animal imaging (referred to as in vivo or, occasionally, ex vivo imaging) is a powerful technique for studying cell populations in live animals, such as mice. Different types of cells (e.g. bone marrow stem cells, T-cells) can be engineered to express a luciferase allowing their non-invasive visualization inside a live animal using a sensitive charge-couple device camera (CCD camera).This technique has been used to follow tumorigenesis and response of tumors to treatment in animal models. However, environmental factors and therapeutic interferences may cause some discrepancies between tumor burden and bioluminescence intensity in relation to changes in proliferative activity. The intensity of the signal measured by in vivo imaging may depend on various factors, such as D-luciferin absorption through the peritoneum, blood flow, cell membrane permeability, availability of co-factors, intracellular pH and transparency of overlying tissue, in addition to the amount of luciferase.
Luciferase can be used in blood banks to determine if red blood cells are starting to break down. Forensic investigators can use a dilute solution containing the enzyme to uncover traces of blood remaining on surfaces at a crime scene. Luciferase is a heat sensitive protein that is used in studies on protein denaturation, testing the protective capacities of heat shock proteins. The opportunities for using luciferase continue to expand.
 See also
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- Baldwin TO (March 1996). "Firefly luciferase: the structure is known, but the mystery remains". Structure 4 (3): 223â8. doi:10.1016/S0969-2126(96)00026-3. PMID 8805542.
- Steghens JP, Min KL, Bernengo JC (November 1998). "Firefly luciferase has two nucleotide binding sites: effect of nucleoside monophosphate and CoA on the light-emission spectra". Biochem. J. 336 (1): 109â13. PMC 1219848. PMID 9806891.
- Shimomura O (1985). "Bioluminescence in the sea: photoprotein systems". Symp. Soc. Exp. Biol. 39: 351â72. PMID 2871634.
- Baldwin TO, Christopher JA, Raushel FM, Sinclair JF, Ziegler MM, Fisher AJ, Rayment I (1995 Dec). "Structure of bacterial luciferase". Curr Opin Struct Biol. 5 (6): 798â809. PMID 8749369.
- Schultz LW, Liu L, Cegielski M, Hastings JW (February 2005). "Crystal structure of a pH-regulated luciferase catalyzing the bioluminescent oxidation of an open tetrapyrrole". Proc. Natl. Acad. Sci. U.S.A. 102 (5): 1378â83. doi:10.1073/pnas.0409335102. PMC 547824. PMID 15665092.
- Okamoto OK, Liu L, Robertson DL, Hastings JW (December 2001). "Members of a dinoflagellate luciferase gene family differ in synonymous substitution rates". Biochemistry 40 (51): 15862â8. doi:10.1021/bi011651q. PMID 11747464.
- Baldwin TO; Lee, Jongsung; Jung, Eunsun; Kim, Sang-Cheol; Kang, Jung-Il; Lee, Jienny; Kim, Yong-Woo; Sung, Young Kwan et al. (June 2009). "A cell-based system for screening hair growth-promoting agents.". Archives of Dermatological Research 301 (3): 381â385. doi:10.1007/s00403-009-0931-0. PMID 19277688.
- Fraga H, Fernandes D, Novotny J, Fontes R, Esteves da Silva JC (June 2006). "Firefly luciferase produces hydrogen peroxide as a coproduct in dehydroluciferyl adenylate formation". Chembiochem 7 (6): 929â35. doi:10.1002/cbic.200500443. PMID 16642538.
- Fisher AJ, Thompson TB, Thoden JB, Baldwin TO, Rayment I (September 1996). "The 1.5-A resolution crystal structure of bacterial luciferase in low salt conditions". J. Biol. Chem. 271 (36): 21956â68. PMID 8703001.
- Elizabeth Wilson (Jan 18, 1999). "What's That Stuff?". Chemical and Engineering News 77 (3): 65.
- Vanessa Knivett (2009). "Lighting the way". EE times.
- General Electric TP-110, page 23, table.
- Nakamura M, Maki S, Amano Y, Ohkita Y, Niwa K, Hirano T, Ohmiya Y, Niwa H (June 2005). "Firefly luciferase exhibits bimodal action depending on the luciferin chirality". Biochem. Biophys. Res. Commun. 331 (2): 471â5. doi:10.1016/j.bbrc.2005.03.202. PMID 15850783.
- Oba Y, Ojika M, Inouye S (April 2003). "Firefly luciferase is a bifunctional enzyme: ATP-dependent monooxygenase and a long chain fatty acyl-CoA synthetase". FEBS Lett. 540 (1-3): 251â4. doi:10.1016/S0014-5793(03)00272-2. PMID 12681517.
- Conti E, Franks NP, Brick P (March 1996). "Crystal structure of firefly luciferase throws light on a superfamily of adenylate-forming enzymes". Structure 4 (3): 287â98. doi:10.1016/S0969-2126(96)00033-0. PMID 8805533.
- Ugarova NN (July 1989). "Luciferase of Luciola mingrelica fireflies. Kinetics and regulation mechanism". J. Biolumin. Chemilumin. 4 (1): 406â18. doi:10.1002/bio.1170040155. PMID 2801227.
- White EH, Rapaport E, Hopkins TA, Seliger HH (April 1969). "Chemi- and bioluminescence of firefly luciferin". J. Am. Chem. Soc. 91 (8): 2178â80. PMID 5784183.
- Fraga H (February 2008). "Firefly luminescence: a historical perspective and recent developments". Photochem. Photobiol. Sci. 7 (2): 146â58. doi:10.1039/b719181b. PMID 18264582.
- Branchini BR, Southworth TL, Murtiashaw MH, et al. (June 2004). "An alternative mechanism of bioluminescence color determination in firefly luciferase". Biochemistry 43 (23): 7255â62. doi:10.1021/bi036175d. PMID 15182171.
- McCapra F,Gilfoyle DJ, Young DW, et al. (1994). Bioluminescence and Chemiluminescence: Fundamentals and Applied.
- Nakatani N, Hasegawa JY, Nakatsuji H (July 2007). "Red light in chemiluminescence and yellow-green light in bioluminescence: color-tuning mechanism of firefly, Photinus pyralis, studied by the symmetry-adapted cluster-configuration interaction method". J. Am. Chem. Soc. 129 (28): 8756â65. doi:10.1021/ja0611691. PMID 17585760.
- Nakamura M, Niwa K, Maki S, et al. (December 2006). "Construction of a new firefly bioluminescence system using L-luciferin as substrate". Anal. Biochem. 47 (1): 1197â1200. doi:10.1016/j.tetlet.2005.12.033.
- Lembert N (July 1996). "Firefly luciferase can use L-luciferin to produce light". Biochem. J. 317 (1): 273â7. PMC 1217473. PMID 8694774.
- Nakamura M, Maki S, Amano Y, et al. (June 2005). "Firefly luciferase exhibits bimodal action depending on the luciferin chirality". Biochem. Biophys. Res. Commun. 331 (2): 471â5. doi:10.1016/j.bbrc.2005.03.202. PMID 15850783.
- Ribeiro C, Esteves da Silva JC (September 2008). "Kinetics of inhibition of firefly luciferase by oxyluciferin and dehydroluciferyl-adenylate". Photochem. Photobiol. Sci. 7 (9): 1085â90. doi:10.1039/b809935a. PMID 18754056.
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This article incorporates text from the public domain Pfam and InterPro IPR018804 This article incorporates text from the public domain Pfam and InterPro IPR007959 This article incorporates text from the public domain Pfam and InterPro IPR018475
This tab holds the annotation information that is stored in the Pfam database. As we move to using Wikipedia as our main source of annotation, the contents of this tab will be gradually replaced by the Wikipedia tab.
Luciferase helical bundle domain Provide feedback
This domain is found associated with the the catalytic domain of dinoflagellate luciferase . Luciferase is involved in catalysing the light emitting reaction in bioluminescence. The structure of this domain has been solved . This domain has a three helix bundle structure that holds four important histidines that are thought to play a role in the pH regulation of the enzyme.
Schultz LW, Liu L, Cegielski M, Hastings JW; , Proc Natl Acad Sci U S A. 2005;102:1378-1383.: Crystal structure of a pH-regulated luciferase catalyzing the bioluminescent oxidation of an open tetrapyrrole. PUBMED:15665092 EPMC:15665092
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This tab holds annotation information from the InterPro database.
InterPro entry IPR018475
This domain is found associated with the the catalytic domain of dinoflagellate luciferase. Luciferase is involved in catalysing the light emitting reaction in bioluminescence. This domain has a three helix bundle structure that holds four important histidines that are thought to play a role in the pH regulation of the enzyme [PUBMED:15665092].
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|Seed source:||Bateman A|
|Number in seed:||7|
|Number in full:||51|
|Average length of the domain:||65.80 aa|
|Average identity of full alignment:||70 %|
|Average coverage of the sequence by the domain:||21.82 %|
|HMM build commands:||
build method: hmmbuild -o /dev/null HMM SEED
search method: hmmsearch -Z 23193494 -E 1000 --cpu 4 HMM pfamseq
|Family (HMM) version:||4|
|Download:||download the raw HMM for this family|
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- 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 Luciferase_3H domain has been found. There are 1 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.
Loading structure mapping...