P212121

I had the pleasure of taking my first crystallography course from Dr. Cora Lind. Cora was kind enough to ask me to speak at the American Crystallography Association meeting this year. In addition, she has always been patient and helpful with my crystallography questions.

Recently, Cora arranged for the video taping of her crystallography course.

I have not yet watched all the videos (in total they run nearly 23 hours!), but feel comfortable recommending them since I took the course. Also there are copies of the slides from each lecture to make it easy to follow along at home.

The relevance of the introductory lecture made me smile, ‘you may find publications with crystal data that may not make sense… you need to be able to judge that.’

I am really grateful for Cora putting this lecture series together.

If you find this video series helpful or think the crystallographic community would benefit from more lectures being posted, please drop a comment. Thanks.

The process in macromolecular crystallography for generating heavy atom derivatives can be tedious. Problems may arise from heavy atoms not being incorporated into your protein to difficulty in producing crystals for derivatization trials therefore making each attempt critical.

The Heavy-Atom Database System: HATODAS II has been created to address these problems. The database uses 93 known heavy atom binding motifs (derived from 3103 heavy atom binding sites) and can take into account the amino acid sequence as well as the crystallization condition (ref).

Here is an example of a prediction that HASTODAS generates for potential heavy-atom reagents:

The following is a list of the suggested motifs that are present in the submitted sequence:

If your protein does not contain a His, Cys or Met then you maybe forced to mutate a residue for derivatization, but which one do you choose? HASTODAS addresses this question by suggesting a point mutation(s) based on multiple sequence alignments of homologous proteins.

Points for creating a database with guts.

The context is always important, but this should give you a fighting chance at giving a meaningful response instead of a blank stare (if you want). Please note, this list is noninclusive.

1) ‘having trouble with the PCR’
meaning: rough start since it is nearly the first step a crystallographer does
response: oh, don’t worry it will work eventually

2) ‘having trouble with express’
meaning: the bacteria (or yeast etc..) are not producing their desired protein
response: bummer, what have you tried so far? (you can space out as they list things) then respond with ‘wow, can’t believe it hasn’t worked yet’

3) ‘I can’t get my protein out of the pellet’
meaning: once the cells have be lysed and centrifuged at a high speed their desired protein is no longer in solution
response: Have you tried lysing the pellet?

4) ‘I keep losing my protein’
meaning: this will happen during purification or using concentrators
response: Have you tried using a different concentrator? (virtually, no one does)

5) ‘precipitates out of solution’
meaning: the protein comes out of solution and can end up looking like dandruff
response: Have you tried finding another buffer for you protein?

6) ‘We only got (number greater than 4) angstrom diffraction’
meaning: the diffraction is not good
response: Do you have any more crystals?

7) ‘have a high mosaic spread’
meaning: the crystal is not well ordered
response: Do you have any more crystals?

‘not getting any hits when I set up expansions’
meaning: a hit is a solution (condition) that may yields a protein crystal
response: Strange.

9) ‘for some reason it won’t index’
meaning: just started data processing and trying to determine the space group
response: Are you sure the beam center is correct?

10) ‘the map doesn’t look right’
meaning: data processing is not going well
response: Hmm, are you sure the space group is right?

I have mentioned the Zhang server before and so thought I would share 2 ways this server can be helpful to crystallographers.

The Zhang Server (I-TASSER server) uses the amino acid sequence of a protein to predict its structure. The server is simple to use– enter in your desired amino acid sequence and email.

Note: You need to have an academic email address to use the server freely. (more information about the server).

Good:
1) Great resource: the server allows users to submit their sequences to be analyzed
2) Free to all academic users

Bad:
1) Turn around time: it took about two weeks before my predictions were complete (this will vary greatly depending on how many proteins are in the queue upon submission)

The two ways this server could be helpful:

First
I was able to find another protein that may have a similar fold to the one I am working on. Were it not for this server, this protein would have gone unnoticed. The Zhang server searches through the PDB looking for homology that you may miss.

Second:
In silico molecular replacement? Could the coordinates from the Zhang server then be used for molecular replacement? This idea is being explored by MR-CAFASP. I have not heard of many crystallographers testing whether in silico molecular replacement would work.

Has a novel protein been published using in silico predictions? In the future, I will play around with the coordinate file from the Zhang server to determine whether or not the results are robust enough to yield reasonable crystallographic statistics.