A MORE CHALLENGING SEQUENCING EXERCISE

some more sequencing work, this one a little heavier than earlier 16s work.
(the figure numbers are off because this is the part 2 of a long paper...)

Protein Sequence: pro.50_orf_10
 (488 letters)
MHISSLPGKFGIGTFGKSAFEFVDFLAETKQTYWQILPLTTTSFGDSPYQSFSAIAGNTHFIDFELLVDDELLEAADLCDITFGTNPEAVDYAQLFQVRRPLLEKAVRAFVAEQENVCKLEAFETASSWLTDFAEFMALKEYFNNKALQDWDDETVIKRQEDSLNNYRELLAKKITYHKVCQYFFYQQWSALKTYANHKGIEIIGDMPIYVSADSVEVWTMPELFKVDSDKKPLFIAGVPADGFSEDGQLWGNPTYNWSAHEKSNFAWWIYRIQESFKLYDQLRIDHFKGFSDFWEIPAGDKTARNGHWASAPGIALFSAVREALGELPIIAENLGYIDEKAEQLLASTGFPGMKILEFGLFDITSQSIDLPHYYDRNCVAYTGTHDNEVVNGWYDNLSEEQVHFVNNYLHKHADESITKAMLRTIFASVCDTAILCIQDLLDKDGKSRMNMPNTIGGNWQWRMLDGELNQDHKDYLIYLTDLYGRAR

NCBI BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) was used to search pro.50_orf_10 



Figure 27: NCBI BLAST results for pro.50_orf_10
The protein sequence was searched in NCBI BLAST and was identified as PRK14508, part of the Glyco_hydro_77 superfamily. 


Figure 28: NCBI BLAST color key for alignment scores for pro.50_orf_10
The color key for alignment scores is a graphical representation of E values for the matching sequences.  The shortened line is a partial match to a sequence, while the full length lines have the best E values; are more complete matches. 

Figure 29: NCBI BLAST partial match for pro.50_orf_10
The image above is an example of a partial match.  The E value is 0 and it is a 99% identity (the mismatched amino acid can be seen in the first line, there is a threonine instead of an isoleucine) however the sequence begins matching from the 167th amino acid and matches until the end (letter 488).  A better match would be a more complete sequence match as follows:


Figure 30: NCBI BLAST top match for pro.50_orf_10
The top match for pro.50_orf_10 is 4-alpha-glucanotransferase from organism Streptococcus pyogenes M1 GAS.  There is 100% identity and no gaps.  The E value is 0 and the match is from the first amino acid through the last (letter 488).    




Figure 31: NCBI BLAST Gene Details of 4-alpha-glucanotransferase
Going to the gene details of the top match, a list of related proteins is shown.  The protein from our match is fourth from the top of the list, from S. pyogenes M1 GAS.  the gene is called “malM” and is known as SPy_1292 in this particular organism. 



Figure 32: NCBI BLAST Gene Details of malM SPy_1292

Figure 33: NCBI BLAST Pathway information for malM SPy_1292
Selecting malM Spy_1292 more details are obtained on 4-alpha-glucanotransferase.  The gene is protein coding, is involved in starch and sucrose metabolism, and may also be called amylomaltase.  Two of the pathways listed are specific to S. pyogenes M1 GAS and a preview image is provided:



Figure 34: malM SPy_1292 pathway involvement in S. pyogenes M1 GAS

Figure 35: malM SPy_1292 starch and sucrose metabolism pathway involvement in S. pyogenes M1 GAS

KEGG database ( http://www.kegg.jp ) was searched for Spy_1292 to confirm NCBI BLAST pathway results. 


Figure 36: KEGG database results for SPy_1292
Both results from KEGG database search are for the correct organism, S. pyogenes M1 GAS but the first match is for the correct protein.  Selecting this provides detailed information:

Figure 37: KEGG database results for Spy_1292: malM; 4-alpha-glucanotransferase; K00705 4-alpha-glucanotransferase
KEGG database search confirms NCBI BLAST results.  Gene malM is also known as 4-alpha-glucotransferase from Streptococcus pyogenes serotype M1.  It is involved in two pathways for this organism, carbohydrate metabolism and starch and sucrose metabolism.  It is in the superfamily glyco_hydro_77 as identified by the conserved motif.


Figure 38: Metabolic pathways involved in S. pyogenes M1 GAS

The overview metabolic pathway for S. pyogenes M1 GAS shows applicable pathways in colors (all other are grayed out) and the specific gene searched is indicated in red.  KEGG shows malM as part of the sucrose and starch metabolism pathway. 
The sucrose and starch pathway for S. pyogenes M1 GAS is shown in detail:

Figure 39: KEGG database starch and sucrose metabolism pathway for S. pyogenes M1 GAS
malM is indicated in red (2.4.1.25) and is involved in metabolizing Maltose to alpha-D-Glucose.


To confirm NCBI BLAST results pro.50_orf_10 was searched in interpro database (https://www.ebi.ac.uk/interpro/ )

Figure 40: Interpro results for pro.50_orf_10 search
Interpro confirmed NCBI BLAST results and provided more information about the superfamily in which malM belongs to and its biological function (4-alpha-glucotransferase activity in carbohydrate metabolic process). Glycol_hydro_77 superfamily is Glycoside hydrolase family 77. 


Figure 41: Details of glyco_hydro_77 from Interpro search of pro.50_orf_10
Details of glycol_hydro_77 confirm KEGG results; the family is involved in metabolism of alpha-D-Glucan.


Pfam database (http://pfam.xfam.org) is searched for pro.50_orf_10

Figure 42: Pfam results for pro.50_orf_10
Pfam confirms Interpro results, showing 4-alpha-glucanotransferase in the glycol_hydro_77 family.  The detailed information also confirms the description by Interpro.


Figure 43: Detailed results of Pfam search for pro.50_orf_10
2557 different organisms use the Glyco_hydro_77 family. 

Figure 44: Sunburst diagram of Glyco_hydro_77 family distribution among domains
A sunburst diagram shows the distribution is highest among bacteria although other domains contain this family.  Streptococcus pneumoniae has a higher frequency of the family (170 species) among Streptococcus species; S. pyogenes distribution is shown in the alt-text box (15 species).
Pro.50_orf_10 was searched in SMART database but did not have any results.  Instead, pro.50_orf_10 was searched in Uniprot BLAST (http://www.uniprot.org ) to obtain an accession number. 

Figure 45: Uniprot BLAST result for pro.50_orf_10
Uniprot BLAST search resulted in accession number Q99ZC2.
The accession number obtained was searched in SMART database but had no results, possibly because the protein is an enzyme and is only a predicted structure (SMART specializes in evolutionary conserved, regulatory domains: http://smart.embl-heidelberg.de/help/FAQ.shtml )
Instead, STRING database was accessed directly (http://string-db.org/newstring_cgi/show_input_page.pl?UserId=fhcW4mKEyaBx&sessionId=6yzve2qn3GK1 ) to search for malM in S. pyogenes M1 GAS.

Figure 46: STRING database results for malM in S. pyogenes M1 GAS
malM is confirmed as 4-alpha-glucotransferase in STRING database and has functional partners glycogen phophorylase (glgP) and cyclomaltodextrinase (amyB). 


SUMMARY

Two unknown amino acid sequences were researched across five databases... The second sequence, pro.50_orf_10, comes from Streptococcus pyogenes M1 GAS and is in the family Glycoside Hydrolase Family 77.  The gene is malM also known as 4-alpha-glcotransferase and is involved in the metabolism of starch and sucrose. 


and now we party because it's over!



No comments:

Post a Comment

INSTANT TIME MACHINE!

CERCA TROVA:

TAG MANIA

music 30 days of music Aerial friday5 20Q hawaii samadhi yoga vinyl bikeventure la dispute school touché amoré book concert live show silversun pickups bicycle lists metric science cooking destiny poweryoga hawaii rooster teeth EmM food microbiology movie movies phantogram shopping spring break spring break 2015 2010 Book review Stephen king cardistry koji reads tissu work beach books ceremony coffee cover deafheaven movie review pyhiflygirls recol sick tycho virology aerial problems art b9 bacteria bus clothes comics drug church earl grey emily haines interpol lyra mlt no sleep records other realms pacific rim raveonettes republik roman literature sephora socks sspu summer sviib taking back sunday tattoo vales wiwt 6131 YYY amyz arcade fire balance and composure batman blood blood bank brambles carnavas casket lottery catullus cheese chinese christmas coffeesundays contortion culture abuse death cab for cutie dengue destiny 2 exercise grouplove gymnastic linguist handstand jawbreaker lily allen makeup me first and the gimme gimmes memories minecraft mlt adventures modest mouse molecular motion city soundtrack needle work nightwing octopus recipe records review saga scattegories small brown bike sunbather thursday tigers jaw tiny cats weezer xbox one yummiestars #hrf2015 2014 2018 7 inch accident adult swim singles afi album review anakin anamanaguchi angels and airwaves asm atul gawande beach house beer best of bike biomusicology birds in a row blast bruises cadence caitlin doughty campy candy hearts cards carpe noctem cayetana charcoal checklist manifesto chemistry chicago chinese new year clones of the queen concussion dads dc comics deathwish delerium descendents descender dragonette dress earth wind and fire evenings felix cartal fight club fishing fletcher c johnson ghost feet glow flow goodtime boys heart shaped box hematology hip key how to with joel and adam hungry ear hurricane interstellar jack white jjak joe hill john wick katy perry kintsugi lab last look live lucky hell lululemon manoa martin solveig megwin melissa mls adventures modern baseball mr mercedes nails ncbi nerves never come undone nicole atkins no doubt ofelia orange bang pixies poetry pool portal prawn ps4 research rick and morty rohnert park rsd rsd2018 rtpodcast sandwich school of seven bells sequencing shameless shave ice shimmering stars sirens six feet under sleep society of s sorority noise star wars static trap steam stereochemistry strava strfkr sysmex the donnas the killers the modern waikiki the saddest landscape the winter passing there is no learning curve third man records thrice tokyo milk topshelf records tour de france tove lo training for aerial on the ground tron unpopular opinion waiola xcm xur yeah yeah yeahs #hrf2016 #hrf2017 #myhour #tdf2015 #teampretzeltank '68 007 100% 11-22-63 16s 1970s 1999 2011 2015 2017 35mm 3hive 7seconds 90s :) @8_semesters @wild_twinkies Booker T. and the M.G.'s CD Carrie Fishbones GITS Godzilla Groundhog Day HST L-cuts L4D2 MCR Pink vans RIP SEM Streptococcus The Great Wall UH a favor house atlantic a girl walks home alone at night abraham lincoln vampire hunter achievement hunter aculpture aesop rock agar aji fry akari ala moana alam alfa beach allister alt/air amanda tastes ambient american hustle amoeba music antics arsenic art of play atlas genius autoclave b sides backstage bai san bakasana baking ballet bandcamp barbra streisand bargain basement battle lines battlewagon bayonetta beach slang beastie boys beats begiragons big bang theory big grams biochem bitter clarity uncommon grace black dress blaisdell blink 182 blondie bob bond boscia brad meltzer brand new brave new world brazil brew bridge 9 bro force broke for free bronchial wash burrito buxom buy local calligraphy calzone campy group campylobacter cancer candida candida albicans carrot carrot cake carsick cars cartwheel cascade casket girls catbird cave in cdc chairlift charlie day cheap monday chet faker chirashi chloe cho dang choir of the mind chores chozo christmas movies cinelli cinerama circuits circulus cisco kid clarity cloning cls coexist coffee stout coheed and cambria coincidence coli com truise comicon common reactor conference constantine cotq country comfort credence clearwater revival cremation crow csf cults cursive daiei dan and dave dan deacon daniel dae kim dashboard david rees day by day records dead weather deadlines dealersgrip death deep blue deer tick defender class delicious demetri martin deodato depression cherry detective dee devart devrim kay dhm diamond youth dick grayson dim mak dinosaur pile up disco discordia discount dance discovery distance/closure divination diy dl dna dntel does it work dog don quijote donnie darko doodles double ankle hang doubles down the cliff dreamcar dreams dscvry duolingo dupe dustin nguyen e. coli ebola edge edward benz 27 times el pintor el-p electric president electricity elle king emb embroidery enbalming enjoy your burrito epidemiology eqs eraas erik kwakkel esr event evil needle exam exotica eye shadow facebook fall behind me family fashion fat bob fatale father/daughter records fck feather fiction firefly fireworks fitz and the tantrums flash flash fact flight facilities flight of the conchords flower drum song forever 21 forgettable forgetters formula x fountains of wayne frameworks franz ferdinand fresh cafe friday the 13th fuck yes fuji fulton funeral game of thrones gate gatsby genetics ghost bc ghost in the shell ghost key ghostly giant glass glass animals good charlotte google gpc grant imahara grayson green river gtd guardians of the galaxy guest blogger gym class heroes h&m habits haemophilus influenzae haim hair half gramme holiday halloween handwriting haribo harlem harmonica harold harrow hawaii problems heavy circles helena bertinelli hell bunny hello hellogoodbye hematocrit heroquest high water marks history history channel hl2 hohokum holiday mart homework houdini howl howl gaff gaff hummus hxc hyper light drifter i love crazy tags iasip ice cream idea's music and books identity crisis in all dishonesty in and out of control in gratitude inside insomnia insound instagram instax instrumental interpro into the wayside intrepidity ipod iwant iyo udon jack johnson japan jaspas jejuni jellys jeniferever jimmy fallon jisuk jld jock journey joy departed joy division jpb julian casablancas julien baker justice league justice league dark kaimana kakaako kalapana kapiolani kat von d keep talking and nobody explodes kegg kent keratin kickstarter kiki kimchi kimchijeon kingsman kitsune kittygeddon klaxons knives don't have your back koolau ballroom korean koundinyasana lab problems lab report labyrinth ear lali puna last gang records latte art layers of fear lazaretto le castle vania leahi left 4 dead 2 leggings leotard leptospirosis les savy fav let me in lgc library light we made like like drive in lil gothams lipstick lipstories liquid nitrogen lismore lit little richard llea lol lolita loma prieta longboard lookbook looming love luster luck lust lust lust lymbyc systym magic magic island magnolia cafe mah jong maiden heist mail day mais2 major league makapuu maldi-tof mandarin martin denny marvel matty matheson mavica me earl and the dying girl media medieval memento memento bento mental block gaming meta metamorphoses mf doom mgmt michael crichton micr490L midweek mika milk tea mind of a chef miss manners modcloth mogwai mohawk monty python monuments men moon moose blood morbid mortdecai motown mr robot mr tea musubi my chemical romance my sharona naked juice narcissus ncis ncisla neo classical nerd nerdist new eagle cafe new politics new scene new year nickleback nicole atikins nightmare before christmas nijiya nimitz no backup plans no better noisey nonfiction novo biocin now now oats obituary oblivion old navy ombre omori boy on the spot on! air! library! only in youth opera origa osaka loop line ost outdoor voices outkast overcooked ovid pacific rim 2 pai gow pam pancakes pandemic paramore paranormal activity part time job party past is prologue past present felix pat mcgrath labs patreon patrick kindlon pcr pe'ahi peanut butter and jelly peb pecan tree penny dreadful peony perennial flux performance perfume peter pan collar pfam photog physics pikul pita pizza plants plastic metal platelets playdead pointe poker pole polefiction politics popo popoitis portugal the man post office potpourri pow wow hawaii prey primer prince prismatic procrastination protein pu er pun punk pusheen pyogenes pyromaniac quiche r dorothy wayneright rage ramblings ramen ramones random random weirdos rap record shop dude red hot chili peppers red machine redbone reflective replacements revision rhcp ringtone ripd roller skates rooms of the house roxy rozwell kid rumu run for cover run the jewels sailor moon sakura mochi salem's lot salonpas samurai ice cake san francisco sapling satoshi kon saves the day scans scar tissue scarjo scary science fiction scorpion scrabble scrubs sds page sean kingston searching for a pulse sed rate seeking a friend for the end of the world self defense family self published separation sequins serein settled seven psychopaths sewing sex pistols sgl shadowplay shaloha sheezus shipping shoes shout out louds show shrimp sia sierra nevada sikdorak silly girl sinclair library skateboard skyfall sleeve scribbles slipshift smart db smashing pumpkins smoke gets in your eyes snl somnio soundcloud speech and debate sphere spill spyral stamps staph staphylococci star trek starcrawler stellastarr* still corners straddle ups string db suckerpunch sudafed suetonius suicide squad suis la lune sunday morning at a funeral sunny day real estate superhot surya namaskar sushi sushi king swedish swim swoon taco tuesday taiyaki tantalus target taste tea taunt tcb technical writing tempura tf2 tguk thank jose thanksgiving the angels the artist the audition the bougies the byrds the calm blue sea the chase the get up kids the hives the kingsmen the kinks the las the pipettes the quick the raconteurs the russian the supremes the things we think we're missing the voices the weight of the world the wolfpack the xx there's nothing thieves like us threadless three cheers for sweet revenge ticket to ride tideland time travel tiny echo tiny me title fight tittibhasana toby todor kobakov tokyo godfathers tom moffat totoro touche amore tower records transfection transformation tricks trojans tumblr turnover turnstile tv twenty-two twitter two dots udon undercut uniprot unknowns unrelatableexperience vacation valve vampire vegetables verde verse vertigo vibrio vice video dump violet viva fest volary wakey wakey walking backwards man walmart waning star want to read war washed out wednesday addams welwing wharf cat what is free to a good home what we do in the shadows white house down white stripes whole foods wildhoney wildlife wired won tons word freak workaholics wu xia xbox 360 yoga ball yogagypsy yuck zatanna zero zippys zombie zones zotero