Structure-based Evidence for Function (TIGRfam, Pfam and PDB)

Structure-based Evidence for Function(TIGRfam, Pfam and PDB)

TIGRfams are protein familiescategorized by functional role

Concept: HMMs• HMM: A Hidden Markov Model is a probabilistic model developed from

observed sequences of proteins of a known function. The profile HMM is used to score the alignment of the amino acid sequence entered to other proteins base on amino acid identity and position

A concrete example of an HMM:

http://en.wikipedia.org/wiki/Hidden_Markov_model

Consider two friends, Alice and Bob, who live far apart from each other and who talk together daily over the telephone about what they did that day. Bob is only interested in three activities: walking in the park, shopping, and cleaning his apartment. The choice of what to do is determined exclusively by the weather on a given day. Alice has no definite information about the weather where Bob lives, but she knows general trends. Based on what Bob tells her he did each day, Alice tries to guess what the weather must have been like.

Alice believes that the weather operates as a discrete Markov chain (system in various states that can change randomly). There are two states, "Rainy" and "Sunny", but she cannot observe them directly, that is, they are hidden from her. On each day, there is a certain chance that Bob will perform one of the following activities, depending on the weather: "walk", "shop", or "clean". Since Bob tells Alice about his activities, those are the observations. The entire system is that of a hidden Markov model (HMM).

Alice knows the general weather trends in the area, and what Bob likes to do on average. In other words, the parameters of the HMM are known.

Follow this link from the lab notebook

TIGRfams:Haft et al. (2001)Nucleic Acids Research 29: 41-43.

Change Database to “TIGRFAMS” Change Scope to GLOBAL Change E-value cutoff to “0.01” Enter protein sequence in FASTA format in the box Click on “Start HMM search” Then wait…

Search TIGRFAM database

“Click”

Enter the TIGRfam number (format -- TIGRXXXXX) from 'Model' column into imgACT lab notebook in box for significant TIGRfam hit

Enter TIGRfam name from ‘Description’ column into notebook NOTE: If full name is cut off in ‘Description’ column, go to

http://cmr.jcvi.org/cgi-bin/CMR/shared/MakeFrontPages.cgi?page=text_search&crumbs=searches

Enter Score and E-value into Notebook as well

Score and E-Score and E-valuevalue

RESULTS:Only hits with

positive Score &E-value 10-3

should be recorded

“Click”



To obtain full TIGRfam name:

Then what?

Full name

Complete description

TIGRfam Results in imgACT Notebook

Terms to Know for Pfam

• Domain: A structural unit which can be found in multiple protein contexts.

e.g., zinc finger, leucine zipper

• Family: A collection of related proteins containingthe same domain.

e.g., immunoglobulins, CD4, MHC, TCR, etc.

• Clan: A collection of multiple protein families. The relationship may be defined by similarity of sequence,

structure, or profile-HMM.e.g., ATPase functioning in ETC vs. ATPase functioning in DNA replication.

Click on the link provided in your

notebook.

You know the Drill!

Enter your FASTA formatamino acid sequence

Change E-value to 0.001

“Click”

WAIT…this can sometimes take awhile

RESULTS!

Notice there may be two types of results based on your designated E-value:

Significant and insignificant matches. Only investigate significant matches.

NOTE: Insignificant matches may have valid E-value. . . but this Pfam result is considered insignificant because the length of the alignment is very short & Pfam has detected and flagged this.

If you do not have any significant matches, make a note of this in your notebook by creating a COMMENTS section, entering “No significant hits”.

Be sure your search criteria was accurate (e.g., E-value of 0.001)

Graphic view ofdomain organization

Investigate SIGNIFICANT matches

Click on [Show] to view the “pairwise alignment” for the Pfam match

Copy/paste this pair-wise alignment into designated box in your notebook.

Top row (#HMM): all capital letters indicateconserved residues in the HMM consensus sequence.Middle row (#MATCH): identical or functionallyconserved (similar) amino acids Bottom row (#SEQ): query sequence aligned toHMM representing the domain/family

How do I interpret the alignment?

Legend for #MATCH• Upper case = identical match (conserved and high frequency)

• Lower case = identical match (conserved but low frequency)

• + symbol = functionally similar (i.e. aspartic vs. glutamic acid)

• Space = no match

What is an HMM consensus sequence?

The HMM consensus sequence

Right “Click” Pfam link & open in new tab

On Pfam family summary page, click on

“Alignments”’


Full: Total number of sequences in database that have been categorized into this Pfam family

Seed: Number of sequences within multiple sequence alignment representing architectural variations within a single Pfam family

What does this mean?

Architecture Diversity

• Domain organization within context of full protein

Leave default settings and press the [View] button


Click on [Start Jalview] button to view the multiple sequence alignment


A new window will pop up as shown:

TOO MANY COLORS! How do we read this?!?


Another new window will pop up as shown:

Let’s make the view more manageable by simplifying the colors. . .


Select “Percentage Identity” from menu.

NOTE: Take the time to browse

other color schemes to learn more about your

protein.

Pay special attention to

BOTTOM graph: Consensus

sequence for protein family

This view reveals the amount of conservation in your amino acid sequence.Dark = highest frequency Light = lower frequency

Letters show which amino acids

occur most frequently at that

position.This consensus sequence is used to construct the HMM


Return to Summary page for Pfam family

What else do I need for my notebook?Pfam name and Pfam number

Pfam number

AbbreviatedPfam name

FullPfam name

Copy/paste full & abbreviated Pfam name as well as Pfam number into your lab notebook

Note: Pay Attention to possible 3D Image

• You may see a 3D image when you view your summary.

• If you see this image, then this is your first clue that you should expect to have significant hits in the PDB search (next section of this module).

• If you don’t see an image, then this suggests no structure has yet been solved for proteins containing the domain identified by Pfam.

What else do I need for my notebook?HMM Logo

On Summary page, click on “HMM logo”

SAVE this image in .png format and insert into your notebook.

What else do I need for my notebook?HMM Logo

How do we interpret the HMM Logo?HMM Logo:

-- Highly conserved amino acids are represented by wide letters-- Amino acids with a high frequency of occurrence in the alignment used to generate the HMM consensus sequence are represented by tall letters

Return to Summary page:

What else do I need for my notebook?Clan name and number

Click BROWSE to search for clan information

Use key words from Pfam family name for clan search

Investigate possible clans based on key word search from Pfam family description.

To learn more about the clan, click on hyperlink for more clan information.


AbbreviatedClan name Clan number

Tells you which Pfam families belong to this clan. If the Pfam family to which your protein belongs is not in this

list, then your protein is NOT a member of this clan.


FullClan name

NOTE: Not all Pfam families belong to a clan. If no clan is found, enter “None found” in your lab notebook.

What else do I need for my notebook?Key functional residues

You have THREE key tools to assist you in identifying theKEY FUNCTIONAL RESIDUES of your protein.

Tool #1: Pairwise Alignment

Tool #2: HMM Logo

Tool #3: Jalview consensus

Capital letter in #MATCH lineCapital letter in #MATCH line Tall, wide letter in HMM logoTall, wide letter in HMM logo

Tall bar in graphical depiction of consensus sequenceTall bar in graphical depiction of consensus sequence

How do we identify key functional residues?

Formula:Formula:AA(start+HMM#-1)AA(start+HMM#-1)

Example:

C(47+8-1)= C54

How do we report key functional residuesin the notebook?

HMM#HMM#

1. Use the HMM pair-wise alignment to identify possible key functional residues.

2. Use the HMM Logo and Jalview alignment tools to verify key functional residues.

3. Scan the entire amino acid sequence and record all key functional residues using proper notation.

SUMMARY: Identifying key functional residues

Recording results in your Lab Notebook

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REPEAT procedure for all significant Pfam hits

3 hits = 3 notebook entries

PDBProtein Data Bank

o Worldwide depository for three-dimensional structures of large biological molecules, including proteins and nucleic acids

o Contains information about structure such as. . .

Berman et al. (2003)Nature Structural Biology 10: 980.

• sequence details• atomic coordinates• crystallization conditions

• 3-D structure neighbors• derived geometric data• structure factors• 3-D images

Click on the link provided in your

notebook.

Select “Advanced Search”

Select “Sequence (Blast/Fasta)” option

Copy/paste your FASTA format protein sequence into query box

Click when readyto initiate search

Change E-valuecut off to 0.001

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Results of PDB Search

Search hits listed by ascending E-value

Alignmentand statistics

Assess quality of the alignment: Is the E-value less than 10-3?Is a significant proportion of the protein aligned?(Hint: compare alignment length to total length)

PDB NAME

PDB CODE Thumbnail of 3D structure.Click on it to get a high-resolution image for notebook.

Evaluating PDB Results

If so, good hit.

Citation

NOTE: Revise or add headings and boxes as needed


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Add to your notebook

You cannot simply copy/paste the entire alignment with correct formatting into your lab notebook…. DELETE THIS SECTION.

X

Documents

Structure-based Evidence for Function (TIGRfam, Pfam and PDB)