Reddit mentions of Crystallography Made Crystal Clear: A Guide for Users of Macromolecular Models (Complementary Science)

Here are some resources/tips that I've found helpful in my time working in a crystallography lab.

• Crystallography made Crystal Clear - good overview https://www.amazon.com/Crystallography-Made-Crystal-Clear-Macromolecular/dp/0125870736/ref=sr_1_1?ie=UTF8&qid=1536938710&sr=8-1&keywords=Crystallography+made+crystal+clear
  
• Tererese Bergfors website: http://xray.bmc.uu.se/terese/tutorials.html Good tutorials
  
• Look for Studier 2002- his formulations for Auto-induction media. Of interest is the formulation for PASM-5052 should you need to do experimental phasing via Selenomet labeling. As a side note, always look up the percent sequence identity of your protein to its homologs in the PDB. If its below 40%, definitely plan to do experimental phasing. Even at that, some labs have started hopping straight to expressing and crystallizing Selenomethionine labeled protein and collecting on the selenium edge from the get-go so that they hedge their bets on phasing. If doing experimental phasing, always collect until your crystal is dead dead dead.
  
• Most common issue with crystallization in my experience is a sample prep issue. This paper was very helpful to me when troubleshooting protein prep quality issues. http://dx.doi.org/10.1016/j.febslet.2013.10.044 One of my proteins was aggregating on the hours-days timescale. Don't neglect the utility of SEC, even if you see a single band on an SDS gel, you may have a mixture of oligomeric states which can only be detected by looking at the chromatogram from an SEC run.
  
• Look at the drops that do crystallize, then look at the neighbors in the same tray that don't have crystals. These observations can make it easier to figure out when you're close to the right crystallization conditions.
  
• High purity reagents- It's 2 O'clock in the morning, what is the purity of your Tween 20? 40%....40 fucking %. This is from Sigma aldrich, not some po dunk little chemical firm. Document your manufacturer if you need to use a detergent or reagent with purity this low. Regardless, document your fucking manufacturers of your reagents. One of the grad students in my lab was using TCEP and even though the purity was fairly high, a change of manufacturer stopped his protein from crystallizing. We also had a similar incident with a syringe filter manufacturer change.
  
• Submit samples for High-throughput screening at multiple concentrations, also recommended is to document the buffer your protein is in and try multiple different buffers so long as you get a clean chromatogram from SEC. Sometimes one buffer system works better than another for getting good quality crystals.
  
• Generally if possible, avoid glycerol. If your protein is having aggregation issues due to hydrophobic interactions, use a high-purity detergent like CHAPS, or n-Octyl-β-D-Glucopyranoside, or even sometimes an organic solvent like DMSO or isopropanol. All of the above mentioned compounds are the most cost-effective in my experience.
  
• Don't neglect the role of ligands. Sometimes you need to add a ligand or product to lock down the conformation.
  
• Don't neglect activity assays to make sure your sample preps are producing active protein.
  
• Don't count on an NMR structure to phase a crystal structure. I made this mistake, same protein, identical sequence, unable to phase with molecular replacement. I ultimately had to do a SAD experiment to phase.
  
• If your protein is badly behaved, you can try to rescue it with a maltose binding protein tag or a SUMO tag, etc. Sometimes they're helpful.
  
• DNA-binding proteins are often unstable in low salt when not bound to their target DNA sequence. Try binding the protein to its target DNA sequence then buffer exchanging into a low salt buffer.
  
• Seeding can sometimes be helpful.
  
• Make sure your sample purity is very high, the higher the better. Heterogeneity will fuck you on diffraction.
  
• Don't judge a book by its cover. Some crystals are ugly and diffract beautifully, others look beautiful and diffract terribly. I had these beautiful hexagonal crystals- no diffraction. In contrast, I had these plates that I expected nothing of that diffracted out to 1.7Angstroms.
  
• Common issue that can stop you from phasing is processing in the incorrect space group.
  
• Don't bother with HKL-2000 or HKL-3000. I strongly prefer XDS for data processing as it doesn't fail silently. There was a crystal dataset I was working on that had strong space group ambiguity and HKL-2k just wouldn't process it. XDS gave me enough feedback I was able to correctly diagnose and correct the issue. XDS is command line based but it gives you a lot more control and feedback.
  
• Get cozy with Phenix and Coot.
  
  I hope this helps. Let me know if you have any questions.

Crystallography Made Crystal Clear is a fantastic resource for learning the theory behind protein crystallography and structure solution. I used it to learn when I was starting out and I still consult it at times.

Crystallography Made Crystal Clear: A Guide for Users of Macromolecular Models (Complementary Science) https://www.amazon.com/dp/0125870736/ref=cm_sw_r_cp_api_Rp9MBb4PTEANT

Good luck!


Beyond this, I’ll add that there’s really no substitute for doing this hands on and learning from other students/postdocs in the lab. Crystallization feels like black magic a lot of the time, so the theory only gets you so far. Most of the learning really happens as you go along.