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 Aerial friday5 hawaii yoga samadhi bikeventure school book concert lists science poweryoga hawaii bicycle food la dispute live show microbiology movie movies spring break spring break 2015 vinyl 2010 Book review cardistry cooking destiny phantogram reads rooster teeth silversun pickups touché amoré work EmM Stephen king beach books coffee metric movie review pyhiflygirls shopping virology b9 bacteria bus clothes comics deafheaven drug church interpol mlt roman literature socks vales 6131 YYY aerial problems amyz art balance and composure batman blood blood bank catullus ceremony cheese coffeesundays cover dengue earl grey exercise gymnastic linguist koji memories mlt adventures molecular motion city soundtrack needle work nightwing no sleep records octopus other realms pacific rim raveonettes recipe republik review saga scattegories sspu summer sunbather taking back sunday tattoo tiny cats tissu tycho wiwt yummiestars #hrf2015 2014 accident adult swim singles afi album review anakin anamanaguchi angels and airwaves asm beer best of blast bruises cadence caitlin doughty campy cards carnavas carpe noctem checklist manifesto chemistry chinese christmas concussion culture abuse dc comics deathwish descendents descender dress fight club fishing glow flow goodtime boys grouplove handstand heart shaped box hematology hip key how to with joel and adam hungry ear hurricane interstellar joe hill lab last look lily allen live lucky hell lululemon lyra makeup manoa megwin melissa modern baseball mr mercedes nails ncbi nerves never come undone orange bang poetry pool ps4 research rick and morty rohnert park rtpodcast sandwich sephora sequencing shameless shave ice sick sirens sleep society of s star wars static trap stereochemistry strava sviib sysmex the donnas the modern waikiki the winter passing third man records thrice tove lo training for aerial on the ground unpopular opinion waiola weezer xcm yeah yeah yeahs #hrf2016 #myhour #tdf2015 '68 007 100% 11-22-63 16s 1970s 1999 2011 2015 35mm 3hive 7seconds 90s :) @8_semesters @wild_twinkies 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 aculpture aesop rock agar aji fry ala moana alam alfa beach american hustle antics arcade fire art of play atlas genius atul gawande autoclave b sides backstage bai san bakasana baking bandcamp battlewagon bayonetta beach house beach slang beastie boys beats begiragons big bang theory big grams bike biochem biomusicology birds in a row bitter clarity uncommon grace black dress blaisdell blink 182 bond boscia brad meltzer brambles brazil brew bridge 9 bro force broke for free bronchial wash burrito buxom buy local calzone campy group campylobacter cancer candida candida albicans candy hearts carrot carrot cake cascade casket lottery catbird cayetana cdc cheap monday chet faker chinese new year chirashi chloe cho dang chores chozo cinerama circuits circulus cisco kid clarity clones of the queen cloning cls coexist coffee stout coheed and cambria coincidence coli com truise comicon common reactor conference constantine contortion cotq cremation crow csf cursive dads daiei dan and dave daniel dae kim dashboard david rees day by day records dead weather deadlines dealersgrip death deep blue defender class delerium demetri martin destiny 2 detective dee devart dhm diamond youth dick grayson dinosaur pile up disco discordia discount dance discovery distance/closure divination diy dl dna dntel does it work don quijote donnie darko doodles double ankle hang down the cliff dragonette dreamcar dreams dscvry dustin nguyen e. coli earth wind and fire ebola edge edward benz 27 times el pintor el-p electricity elle king emb emily haines enbalming enjoy your burrito epidemiology eqs esr evenings event evil needle exam exotica fall behind me family fashion fat bob fatale fck felix cartal fiction firefly fitz and the tantrums flash flash fact fletcher c johnson flight facilities flight of the conchords forever 21 forgettable formula x fountains of wayne frameworks franz ferdinand fresh cafe friday the 13th fuck yes fulton funeral game of thrones gate gatsby genetics ghost bc ghost feet ghost in the shell ghost key ghostly giant glass good charlotte gpc grant imahara grayson green river gtd guardians of the galaxy guest blogger gym class heroes habits haemophilus influenzae haim halloween handwriting harlem harmonica harrow heavy circles helena bertinelli hello hellogoodbye hematocrit heroquest high water marks history channel holiday mart homework houdini howl howl gaff gaff hummus hxc i love crazy tags iasip ice cream identity crisis in all dishonesty in and out of control in gratitude instagram instax interpro into the wayside intrepidity ipod iwant iyo udon jack johnson jack white japan jaspas jejuni jellys jimmy fallon jisuk jjak jld jock john wick joy departed joy division jpb julian casablancas justice league justice league dark kaimana kakaako kapiolani kat von d kegg keratin kiki kimchi kimchijeon kingsman kitsune kittygeddon klaxons koolau ballroom korean koundinyasana lab report latte art lazaretto le castle vania leahi left 4 dead 2 leotard leptospirosis les savy fav let me in lgc library light we made like like drive in lil gothams liquid nitrogen lismore lit little richard llea lol lolita loma prieta longboard lookbook 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 martin denny martin solveig marvel matty matheson mavica me earl and the dying girl me first and the gimme gimmes media memento memento bento meta metamorphoses mf doom michael crichton micr490L midweek milk tea mind of a chef miss manners mls adventures modest mouse mogwai mohawk monty python monuments men moon moose blood morbid mortdecai motown mr robot mr tea my chemical romance my sharona naked juice ncis ncisla nerd nerdist new eagle cafe new politics new scene nickleback nicole atkins nightmare before christmas nijiya nimitz no backup plans no doubt noisey nonfiction novo biocin ofelia only in youth origa osaka loop line ovid pai gow pam pancakes pandemic paramore paranormal activity part time job party past present felix patreon patrick kindlon pcr pe'ahi peanut butter and jelly peb pecan tree penny dreadful performance peter pan collar pfam photog physics pita pizza plants plastic metal platelets poker pole polefiction politics popo popoitis post office potpourri pow wow hawaii prey primer prince procrastination protein pu er pun punk pyogenes pyromaniac quiche r dorothy wayneright rage ramblings ramen random random weirdos rap record shop dude records red machine redbone reflective replacements revision rhcp ripd roller skates rooms of the house roxy rozwell kid rsd run for cover run the jewels sailor moon sakura mochi salem's lot salonpas samurai ice cake sapling scans scarjo scary scorpion sds page searching for a pulse sed rate seeking a friend for the end of the world self defense family self published separation sequins seven psychopaths shadowplay shaloha sheezus shimmering stars shoes shout out louds show sierra nevada sikdorak silly girl sinclair library skateboard skyfall slipshift small brown bike smart db smoke gets in your eyes snl somnio sorority noise soundcloud speech and debate sphere spyral stamps staph staphylococci star trek steam stellastarr* straddle ups string db suetonius suicide squad suis la lune sunday morning at a funeral sunny day real estate surya namaskar sushi sushi king swim swoon taco tuesday taiyaki tantalus target taste tea taunt tcb technical writing tempura tf2 tguk thanksgiving the angels the audition the bougies the calm blue sea the chase the get up kids the hives the killers the kingsmen the kinks the pipettes the quick the raconteurs the russian the saddest landscape 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 thursday ticket to ride tideland tigers jaw time travel tiny me title fight tittibhasana toby tokyo milk tom moffat topshelf records totoro tour de france transfection transformation tricks trojans tron tv two dots udon uniprot unknowns unrelatableexperience vacation valve vampire vegetables verde verse vertigo vibrio vice video dump volary walking backwards man walmart waning star want to read war wednesday addams welwing what we do in the shadows white house down white stripes whole foods wildhoney wildlife wired won tons workaholics wu xia xbox 360 xbox one xur yoga ball yogagypsy zatanna zero zippys zombie zones