<p>If you read <a href="https://en.wikipedia.org/wiki/Position_weight_matrix#Creation" rel="nofollow noreferrer">wiki</a> carefully, you can see that PSSM is calculated in 3 steps. First frequency is calculated (how much times was the amino acid or nucleotide on that location in the motif), from that you can calculate probability (in the wiki example there were 10 sequences, so each freq. is divided by 10).</p>

<p>Then the log likelihood is calculated, which are the PSSM values. These are in your first matrix (rounded down). The second matrix shows how much the values are relative from your pseudocounts (I assume it was set to default = 0).</p>

<p>Lambda and kappa are estimated to calculate the normalized score (S') for HSP, if you never heard of this before I suggest you read the original psi-blast paper first which you can find <a href="https://www.ncbi.nlm.nih.gov/pubmed/9254694" rel="nofollow noreferrer">here</a>.  </p>

CC BY-SA 4.0
2018-06-12T20:23:16.693Z
2018-06-12T20:23:16.693Z
benn
<p>PSI-Blast on a sequence returns a PSSM matrix file containing two matrices, one to the left, one the the right, for all amino acids:</p>

<pre><code>A   R   N   D   C   Q   E   G   H   I   L   K   M   F   P   S   T   W   Y   V
</code></pre>

<p>What is the difference between these two matrices, how can I interpret them?</p>

<pre><code>Last position-specific scoring matrix computed, weighted observed percentages rounded down, information per position, and relative weight of gapless real matches to pseudocounts
            A   R   N   D   C   Q   E   G   H   I   L   K   M   F   P   S   T   W   Y   V   A   R   N   D   C   Q   E   G   H   I   L   K   M   F   P   S   T   W   Y   V
    1 R    -2   6  -1  -2  -4   1   0  -3   0  -3  -3   2  -2  -3  -2  -1  -1  -3  -2  -3    0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0  0.00     0.00
    2 G     0  -3   0  -2  -3  -2  -2   7  -2  -4  -4  -2  -3  -4  -2   0  -2  -3  -4  -4    0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0  0.00     0.00
    3 G     0  -3   0  -2  -3  -2  -2   7  -2  -4  -4  -2  -3  -4  -2   0  -2  -3  -4  -4    0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0  0.00     0.00
    4 G     0  -3   0  -2  -3  -2  -2   7  -2  -4  -4  -2  -3  -4  -2   0  -2  -3  -4  -4    0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0  0.00     0.00
    5 L    -2  -3  -4  -4  -1  -2  -3  -4  -3   2   4  -3   2   0  -3  -3  -1  -2  -1   1    0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0  0.00     0.00
    6 C     0  -4  -3  -4  10  -3  -4  -3  -3  -1  -1  -4  -2  -3  -3  -1  -1  -3  -3  -1    0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0  0.00     0.00
    7 Y    -2  -2  -2  -4  -3  -2  -2  -4   2  -2  -1  -2  -1   3  -3  -2  -2   3   8  -1    0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0  0.00     0.00
    8 C     0  -4  -3  -4  10  -3  -4  -3  -3  -1  -1  -4  -2  -3  -3  -1  -1  -3  -3  -1    0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0  0.00     0.00
    9 R    -2   6  -1  -2  -4   1   0  -3   0  -3  -3   2  -2  -3  -2  -1  -1  -3  -2  -3    0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0  0.00     0.00
   10 R    -2   6  -1  -2  -4   1   0  -3   0  -3  -3   2  -2  -3  -2  -1  -1  -3  -2  -3    0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0  0.00     0.00
   11 R    -2   6  -1  -2  -4   1   0  -3   0  -3  -3   2  -2  -3  -2  -1  -1  -3  -2  -3    0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0  0.00     0.00
   12 F    -3  -3  -3  -4  -3  -4  -4  -4  -1   0   0  -4   0   7  -4  -3  -2   1   3  -1    0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0  0.00     0.00
   13 C     0  -4  -3  -4  10  -3  -4  -3  -3  -1  -1  -4  -2  -3  -3  -1  -1  -3  -3  -1    0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0  0.00     0.00
   14 V     0  -3  -3  -4  -1  -3  -3  -4  -4   3   1  -3   1  -1  -3  -2   0  -3  -1   4    0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0  0.00     0.00
   15 C     0  -4  -3  -4  10  -3  -4  -3  -3  -1  -1  -4  -2  -3  -3  -1  -1  -3  -3  -1    0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0  0.00     0.00
   16 V     0  -3  -3  -4  -1  -3  -3  -4  -4   3   1  -3   1  -1  -3  -2   0  -3  -1   4    0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0  0.00     0.00
   17 G     0  -3   0  -2  -3  -2  -2   7  -2  -4  -4  -2  -3  -4  -2   0  -2  -3  -4  -4    0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0  0.00     0.00
   18 R    -2   6  -1  -2  -4   1   0  -3   0  -3  -3   2  -2  -3  -2  -1  -1  -3  -2  -3    0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0  0.00     0.00
</code></pre>

<p>Additionally, at the end of the file, there is additional information, what does it show exactly?</p>

<pre><code>                      K         Lambda
Standard Ungapped    0.1628     0.3454
Standard Gapped      0.0410     0.2670
PSI Ungapped         0.1628     0.3454
PSI Gapped           0.0410     0.2670
</code></pre>

CC BY-SA 4.0
2020-08-01T13:47:56.473Z
How do you actually obtain the PSSM matrix?  Can you only get it from command line?  The user interface on NCBI only allows download of a .asn file.
Brian Wiley
CC BY-SA 4.0
2020-08-01T15:43:38.007Z
@BrianWiley You can download the PSSM from the web search tools: make sure you select PSI Blast search and perform more than 1 iteration. The PSSM is only available after more than 1 iteration. Moreover, you can instead download and install NCBI BLAST+ suite from https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastDocs&DOC_TYPE=Download ...once installed, with a protein database also downloaded, also available from that page (usually people use NR, swissport or uniref), then you can search with the psiblast program.  Note: If you want to use uniref you will usually have to generate it.
Aalawlx
CC BY-SA 4.0
2020-08-02T02:39:58.647Z
Thanks.  How much space is required for the NR database?  I was able to get this format of the profile using psiblast with command line like you indicated.  Just made my own small database.
Brian Wiley
<blast><phylogenetics><pssm><psi-blast>
How to interpret a PSSM matrix from PSI-Blast - what do the numbers represent?
# How to interpret a PSSM matrix from PSI-Blast - what do the numbers represent?

(Asked by: username_0 on 2018-06-12)

PSI-Blast on a sequence returns a PSSM matrix file containing two matrices, one to the left, one the the right, for all amino acids:
A   R   N   D   C   Q   E   G   H   I   L   K   M   F   P   S   T   W   Y   V

What is the difference between these two matrices, how can I interpret them?
Last position-specific scoring matrix computed, weighted observed percentages rounded down, information per position, and relative weight of gapless real matches to pseudocounts
            A   R   N   D   C   Q   E   G   H   I   L   K   M   F   P   S   T   W   Y   V   A   R   N   D   C   Q   E   G   H   I   L   K   M   F   P   S   T   W   Y   V
    1 R    -2   6  -1  -2  -4   1   0  -3   0  -3  -3   2  -2  -3  -2  -1  -1  -3  -2  -3    0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0  0.00     0.00
    2 G     0  -3   0  -2  -3  -2  -2   7  -2  -4  -4  -2  -3  -4  -2   0  -2  -3  -4  -4    0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0  0.00     0.00
    3 G     0  -3   0  -2  -3  -2  -2   7  -2  -4  -4  -2  -3  -4  -2   0  -2  -3  -4  -4    0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0  0.00     0.00
    4 G     0  -3   0  -2  -3  -2  -2   7  -2  -4  -4  -2  -3  -4  -2   0  -2  -3  -4  -4    0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0  0.00     0.00
    5 L    -2  -3  -4  -4  -1  -2  -3  -4  -3   2   4  -3   2   0  -3  -3  -1  -2  -1   1    0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0  0.00     0.00
    6 C     0  -4  -3  -4  10  -3  -4  -3  -3  -1  -1  -4  -2  -3  -3  -1  -1  -3  -3  -1    0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0  0.00     0.00
    7 Y    -2  -2  -2  -4  -3  -2  -2  -4   2  -2  -1  -2  -1   3  -3  -2  -2   3   8  -1    0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0  0.00     0.00
    8 C     0  -4  -3  -4  10  -3  -4  -3  -3  -1  -1  -4  -2  -3  -3  -1  -1  -3  -3  -1    0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0  0.00     0.00
    9 R    -2   6  -1  -2  -4   1   0  -3   0  -3  -3   2  -2  -3  -2  -1  -1  -3  -2  -3    0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0  0.00     0.00
   10 R    -2   6  -1  -2  -4   1   0  -3   0  -3  -3   2  -2  -3  -2  -1  -1  -3  -2  -3    0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0  0.00     0.00
   11 R    -2   6  -1  -2  -4   1   0  -3   0  -3  -3   2  -2  -3  -2  -1  -1  -3  -2  -3    0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0  0.00     0.00
   12 F    -3  -3  -3  -4  -3  -4  -4  -4  -1   0   0  -4   0   7  -4  -3  -2   1   3  -1    0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0  0.00     0.00
   13 C     0  -4  -3  -4  10  -3  -4  -3  -3  -1  -1  -4  -2  -3  -3  -1  -1  -3  -3  -1    0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0  0.00     0.00
   14 V     0  -3  -3  -4  -1  -3  -3  -4  -4   3   1  -3   1  -1  -3  -2   0  -3  -1   4    0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0  0.00     0.00
   15 C     0  -4  -3  -4  10  -3  -4  -3  -3  -1  -1  -4  -2  -3  -3  -1  -1  -3  -3  -1    0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0  0.00     0.00
   16 V     0  -3  -3  -4  -1  -3  -3  -4  -4   3   1  -3   1  -1  -3  -2   0  -3  -1   4    0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0  0.00     0.00
   17 G     0  -3   0  -2  -3  -2  -2   7  -2  -4  -4  -2  -3  -4  -2   0  -2  -3  -4  -4    0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0  0.00     0.00
   18 R    -2   6  -1  -2  -4   1   0  -3   0  -3  -3   2  -2  -3  -2  -1  -1  -3  -2  -3    0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0  0.00     0.00

Additionally, at the end of the file, there is additional information, what does it show exactly?
                      K         Lambda
Standard Ungapped    0.1628     0.3454
Standard Gapped      0.0410     0.2670
PSI Ungapped         0.1628     0.3454
PSI Gapped           0.0410     0.2670

**Tags:** blast, phylogenetics, pssm, psi-blast
> **Comments:**
> **username_1**: How do you actually obtain the PSSM matrix?  Can you only get it from command line?  The user interface on NCBI only allows download of a .asn file.  
> **username_0**: @BrianWiley You can download the PSSM from the web search tools: make sure you select PSI Blast search and perform more than 1 iteration. The PSSM is only available after more than 1 iteration. Moreover, you can instead download and install NCBI BLAST+ suite from https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastDocs&DOC_TYPE=Download ...once installed, with a protein database also downloaded, also available from that page (usually people use NR, swissport or uniref), then you can search with the psiblast program.  Note: If you want to use uniref you will usually have to generate it.  
> **username_1**: Thanks.  How much space is required for the NR database?  I was able to get this format of the profile using psiblast with command line like you indicated.  Just made my own small database.  

---

## 1 answers

### Answer by username_2 on 2018-06-12. Score: 1 | Accepted answer

If you read [wiki](https://en.wikipedia.org/wiki/Position_weight_matrix#Creation) carefully, you can see that PSSM is calculated in 3 steps. First frequency is calculated (how much times was the amino acid or nucleotide on that location in the motif), from that you can calculate probability (in the wiki example there were 10 sequences, so each freq. is divided by 10).

Then the log likelihood is calculated, which are the PSSM values. These are in your first matrix (rounded down). The second matrix shows how much the values are relative from your pseudocounts (I assume it was set to default = 0).

Lambda and kappa are estimated to calculate the normalized score (S') for HSP, if you never heard of this before I suggest you read the original psi-blast paper first which you can find [here](https://www.ncbi.nlm.nih.gov/pubmed/9254694).


---

