1. Target mass/size

X-Ray Crystallography serves a wide range of protein sizes, even being amenable to protein complexes 150 kDa or above. While larger proteins certainly crystallize, the smaller, more globular proteins generally are more crystallizable due to their ability to pack in nice lattices (i.e., crystal packing).

2. Purity

Purity is the most critical variable for crystallization. Having aggregates or multiple species in solution (polydispersity) can lead to every crystallographer’s nightmare… no crystals. Samples should be at 95% purity or above.

3. Solubility

Much like purity, the sample needs to be soluble upon crystallization setup. If the sample is aggregated before it touches the crystallization solution, the likelihood of it forming diffraction-quality crystals is close to zero.

4. Unstructured Regions

To increase a sample’s ability to crystallize, it is many times necessary to trun-cate unstructured or flexible regions from the full-length molecule. These un-structured regions or flexible parts perturb the molecule’s ability to pack in a crystal lattice.

For this reason, scrutinizing homology models in the PDB or predicted AlphaFold models is a primary tactic to increase success rates in crystallography.

Sample Requirements

1. Preferred protein concentration and volume

The standard “go-to” concentration for protein & nucleotide crystallography is 10 mg/mL; however, there are plenty of cases where the optimal concentration is less than or greater than 10 mg/ml. It is common for proteins to require 20, 50, or even 100 mg/mL concentrations before saturation levels are conducive for crystallization to occur.

Before setting up high throughput commercial screens, Helix uses a PCT (Pre-Crystallization Test) and a commercial PEG screen to analyze the concentration and determine whether it suffices for full commercial screening.

The volume of protein required depends on how many crystallization screens are being set up. For ten commercial screens + crystal optimization, 750 μl of the sample at crystallization concentration is required.

2. Sample buffer

The sample buffer should be as simple as possible to keep the sample happy and in solution (i.e., stable and soluble). pH, ionic strength, excipients, and additives should be refined to promote sample stability, solubility, and homogeneity.

It is important to include ionic strength, often in the form of NaCl, to keep the sample soluble and table. We recommend keeping the salt in the sample buffer 150mM or below to avoid salt crystal formation during commercial screening.

Additives or excipients, such as ligands, metals, detergents, sugars, etc., should be considered, especially if the inclusion of these in the sample buffer aids in sample stability, solubility, or homogeneity. These additives can also be added during the crystallization setup.

Reducing agents (i.e., BME, DTT, TCEP) may be included, but preferably at low concentrations that do not negatively impact the sample’s ability to crystallize.

Glycerol or other cryo-preservatives should be avoided in the sample buffer if possible or dialyzed out before crystallization. These additives, especially 5% or greater, can inhibit the sample from crystallizing.

Protein Crystallography Milestone-based Project Workflow

Milestone 1: High-throughput Commercial Crystallization

Milestone 2: Crystal Optimization

Milestone 3: Synchrotron X-Ray Data Collection

Milestone 4: Atomic Structure Solution & Refinement

• AlphaFold – Construct design and homology model generation

• XDS/DIALS – Synchrotron X-Ray data processing

• AlphaHelix – Automated Structure Solution Pipeline

• CCP4 – Data Analysis, Molecular Replacement, Autobuilding, Refinement

• Phenix – Data Analysis, Molecular Replacement, Autobuilding, Ligand Fitting, Refinement, Structure Analysis

• ARI Gryphon LCP – Robotic setup of crystallization plates

• ARI CrysCam – Automated imaging of crystal plates

• Leica M205C – Crystallography Microscope

• Synchrotron Facilities – NSLS2, APS, CLS, PetraIII, ESRF, SOLEIL, ALBA