The Protein Crystallization Starter Kit is designed to introduce students to the field of protein crystallization. It contains all material you need to start to grow great looking Lysozyme crystals – a real highlight in Biology or Chemistry courses!

Two different experiments can be carried out using the Protein Crystallization Starter Kit:

• Growing Lysozyme crystals using the hanging drop method, demonstrating the dependence of crystal formation and crystal growth on salt concentration and buffer pH
• Growing Lysozyme crystals within minutes using the batch crystallization method

The kit contains two crystallization plates, cover slides and a microscope slide, a syringe pre-filled with sealing grease, all necessary buffer and salt stock solutions and a small tube containing pre-filtered Lysozyme solution.

If you are a school or any other educational organization, please contact us for educational discounts!

Application

The JBS Crystallization Freshman Kit – Junior is addressed to newcomers in the field of protein crystallography. It is designed for screening of initial crystallization conditions of proteins, peptides, nucleic acids and macromolecular complexes in order to grow single crystals suitable for X-ray diffraction analysis.

Kit Contents

The JBS Crystallization Freshman Kit – Junior contains the required material to crystallize your protein under investigation using the “Hanging Drop Method”:

• 4 Crystalgen SuperClear™ Plates, pregreased (CPL-132)
• 100 Thick Circular Cover Slides, siliconized (CSL-107)
• 96 unique screening conditions, 1.7 ml each: JBScreen JCSG++ HTS (CS-206L)
• a detailed User Guide

How does one manage to grow crystals from such complicated molecules like proteins?

If you are new in the field of macromolecular crystallography we recommend to read the background information before starting the experiment. The Scoring Sheet will provide help to analyse the results of your crystallization experiment.

Our Crystallization Model Proteins can be utilized in crystallization experiments as well as crystallization training and demos.

We offer 2 proteins which can be easily crystallized within days as lyophilized powder or in a stabilization buffer:

• Lysozyme (Chicken egg white)
• Proteinase K (Tritirachium album)

The Crystal Handling Kit will help you to acquire skills in protein crystallization, crystal mounting and data collection.

Each kit contains:

• 3 proteins, i.e. Lysozyme, Xylanase and Proteinase K
• optimized solubilization and crystallization buffers for each protein
• MicroMounts™ and Goniometer Bases, as well as
• a user manual with instructions for protein crystallization using the hanging-drop vapor diffusion method and crystal mounting using MiTeGen’s MicroMounts™

A major challenge in drug discovery is the identification of chemical moieties that specifically interact with a particular protein target. Traditionally, this was addressed by High Throughput Screening (HTS) however, recently “Fragment Screening” has become increasingly popular. In a Fragment Screen a set of small molecules (“fragments”), typically with MW < 300 Da and with low affinities, are evaluated for specific interaction with the target. Crystallography/X-ray diffraction shows not only whether a fragment binds to the protein but also where and how the binding occurs and is therefore the favored screening method. Hit-fragments are subsequently chemically modified in several optimization/screening cycles until a high affinity lead structure is obtained. Since such a fragmented approach allows screening of broader chemical space compared to large, distinct libraries, the hit rates of Fragment Screens are believed to be 10-1000x higher than those in traditional HTS^[5].
The Frag Xtal Screen offers an easy entry to fragment-based lead discovery (FBLD) by crystallographic screening:

• 96 fragments
• High fragment solubility allows high soaking concentrations (> 90 mM; may depend on soaking conditions)
• In-house tests with high crystallographic hit rates
• Validated X-Ray hits for diverse target classes in the PDB
• Diverse and representative fragment library for large chemical space
• Straight-forward follow-up compounds available

References / Recommended Literature

[1] Wollenhaupt et al. (2021) Workflow and Tools for Crystallographic Fragment Screening at the Helmholtz-Zentrum Berlin. J. Vis. Exp. 169:e62208.
[2] Huschmann et al. (2016) Structures of endothiapepsin-fragment complexes from crystallographic fragment screening using a novel, diverse and affordable 96-compound fragment library. Acta Cryst F 72:346.
[3] Schiebel et al. (2016) Six Biophysical Screening Methods Miss a Large Proportion of Crystallographic Discovered Fragment Hits: A Case Study. ACS Chem. Biol. 11:1693.
[4] Schiebel et al. (2015) One Question, Multiple Answers: Biochemical and Biophysical Screening Methods Retrieve Deviating Fragment Hit Lists. ChemMedChem 10:1511.
[5] Hajduk and Greer (2007) A decade of fragment-based drug design: strategic advances and lessons learned. Nature Reviews Drug Discovery 6:211.
[6] Rees et al. (2004) Fragment-based lead discovery. Nature Reviews Drug Discovery 3:660.

Upgraded with the Anderson−Evans polyoxotungstate [TeW₆O₂₄]⁶⁻ (TEW) as universal additive, the XP Screens promote protein crystallization even for most challenging targets and improve diffraction quality of protein crystals^[1]. Its potential has been shown in the new protein structures of aurone synthase from Coreopsis grandiflora^[2-4] (PDB code: 4Z12, 4Z13) and mushroom tyrosinase PPO4 from Agaricus bisporus^[6,7] (PDB code: 4OUA). The model protein lysozyme crystallized into a new crystal form^[5] (PDB code: 4PHI).
The XP Screens are TEW-optimized JBScreen Basics: 96 of the most prominent crystallization conditions complemented with TEW as “glue” for protein molecules.

• is highly soluble in aqueous solutions and stable over a wide pH range
• has a high negative charge that links positively charged protein surface regions, the electrostatic spacer effect prevents steric interference between protein molecules
• provides a valuable anomalous signal for phasing
• can act as linker in various orientations (pictured below) and even structurally adapt to fit into the protein molecule^[3]
• is able to induce heterogeneous crystallization, e.g. two different protein forms in one single crystal^[6]

Protein-protein bridging by TEW in different orientations
Image from [1], used by courtesy of Prof. Annette Rompel, University of Vienna, Austria

• Sobala et al. (2020) Structure of human endo-α-1,2-mannosidase (MANEA), an antiviral host-glycosylation target. PNAS 117 (47):29595.
• Ames et al. (2020) Identifying a Molecular Mechanism That Imparts Species-Specific Toxicity to YoeB Toxins. Front Microbiol 11:959.

References / Recommended Literature

[1] Bijelic et al. (2017) Ten Good Reasons for the Use of the Tellurium-Centered Anderson-Evans Polyoxotungstate in Protein Crystallography. Acc. Chem. Res. 50:1441.
[2] Molitor et al. (2016) Aurone synthase is a catechol oxidase with hydroxylase activity and provides insights into the mechanism of plant polyphenol oxidases. Proc. Natl. Acad. Sci. 113:E1806.
[3] Molitor et al. (2016) In situ formation of the first proteinogenically functionalized [TeW₆O₂₄O₂(Glu)]^7- structure reveals unprecedented chemical and geometrical features of the Anderson-type cluster. Chem. Commun. 52:12286.
[4] Molitor et al. (2015) Crystallization and preliminary crystallographic analysis of latent, active and recombinantly expressed aurone synthase, a polyphenol oxidase, from Coreopsis grandiflora. Acta Cryst. F 71:746.
[5] Bijelic et al. (2015) Hen Egg-White Lysozyme Crystallisation: Protein Stacking and Structure Stability Enhanced by a Tellurium(VI)-Centred Polyoxotungstate. ChemBioChem 16:233.
[6] Mauracher et al. (2014) Latent and active abPPO4 mushroom tyrosinase cocrystallized with hexatungstotellurate(VI) in a single crystal. Acta Cryst. D 70:2301.
[7] Mauracher et al. (2014) Crystallization and preliminary X-ray crystallographic analysis of latent isoform PPO4 mushroom (Agaricus bisporus) tyrosinase. Acta Cryst. F 70:263.

The Thermofluor Screens JBScreen FUNDAMENT and JBScreen SPECIFIC allow identification of protein-stabilizing buffer conditions which is pivotal for protein purification, characterization and crystallization.
Undesired overlay of screening of interdependent variables is prevented by strictly categorizing stability screening of proteins into

1. FUNDAMENTAL factors that influence the whole protein molecule (pH and ionic strength)

2. SPECIFIC factors that affect energetically important hot spots on the protein (substrates and their analogs, cations, anions, …)

The protein’s melting temperature (Tm) is used as reporter for protein stability and determined by a thermal shift assay (96-well plate format) monitoring the unfolding of a protein in a temperature-dependent manner. The higher the Tm, the higher is the thermostability of the protein in that specific environment.

JBScreen Thermofluor FUNDAMENT and SPECIFIC are provided in a deep-well block at 0.5 ml each, which allows a flexible application of this alternative approach.

Example: Stabilizing effects on α-Chymotrypsinogen A are directly correlated with crystallizability

α-Chymotrypsinogen A was applied in JBScreen Thermofluor FUNDAMENT and SPECIFIC.
Stabilizing conditions (high ionic strength, neutral pH, CaCl₂ & FeCl₃) were combined without further optimization. A crystallization screen was set up:

• without any specific ion
• with 20 mM CaCl₂
• with 10 mM FeCl₃

Crystal growth was promoted by the stabilizing specific ions CaCl₂ & FeCl₃ from JBScreen Thermofluor SPECIFIC.

Protein Crystallization: Simply grab the needle from the haystack

Talk held at HEC19 in Warberg, September 29th, 2016

References

• Ericsson et al. (2006) Thermofluor-based high-throughput stability optimization of proteins for structural studies. Anal. Biochem. 357(2):289.
• Reinhard et al. (2013) Optimization of protein buffer cocktails using Thermofluor. Acta Cryst. F 69:209.
• Niesen et al. (2007) The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability. Nat. Protoc. 2(9):2212.
• http://www.rcsb.org/pdb/home/home.do

JBScreen Detergents contain 4 x 24 unique detergents that are compatible with most common crystallization reagents and are therefore perfectly suited for membrane protein solubilization:

• Ionic detergents
• Non-ionic detergents
• Zwitterionic detergents
• Non-detergent Sulfobetaines
• Synthetic Lipids

JBScreen Detergents can be used throughout the protein purification process or can be added afterwards by dialysis or ion-exchange chromatography (detergent exchange). Detergent exchange can be vital for obtaining well-diffracting membrane-protein crystals [1].
JBScreen Detergents is also valuable for additive screening with detergents and detergent mixtures [2,3] in combination with the JBScreen Membrane. This combination will enable you to screen a broad range of detergents, while concentrating on the most successful crystallization conditions, making crystallization screening of membrane proteins much more efficient and less time consuming.

JBScreen Detergents & Prometheus: A winning team

Screen for stabilizing detergents by combining JBScreen Detergents with Prometheus NT.48, a label-free nanoDSF technology developed by NanoTemper Technologies, ideally suited for membrane protein stabilization.

References

[1] Rosenow et al. (2003) The influence of detergents and amphiphiles on the solubility of the light harvesting complex. Acta Cryst. D59:1422
[2] Adir (1999) Crystallization of the oxygen-evolving reaction centre of photosystem II in nine different detergent mixtures. Acta Cryst. D55:891
[3] Koronakis et al. (2000) Crystal structure of the bacterial membrane protein TolC central to multidrug efflux and protein export. Nature 405:914

• Hofmann et al. (2020) High-Level Expression, Purification and Initial Characterization of Recombinant Arabidopsis Histidine Kinase AHK1. Plants 9(3):304.
• Delle Bovi et al. (2017) Expression and purification of functional insulin and insulin-like growth factor 1 holoreceptors from mammalian cells. Anal. Biochem. DOI 10.1016/j.ab.2017.08.011.

JBScreen Plus is an additive screen most useful in the optimization of preliminary crystallization conditions. The selection of the additives is based on the Hofmeister series, which reflects the ability of ions to stabilize the structure of proteins. Thus ions can be classified as either kosmotropic or chaotropic. The first having structure stabilizing properties, thus they may assist in, e.g. crystallizing proteins with a high proportion of flexible loop regions. The latter show structure disturbing properties which may assist in the crystallization of large complexes allowing them to re-arrange to form favorable crystal contacts.

JBScreen Plus consists of 5 individual kits, JBScreen Plus Kosmotropic, JBScreen Plus Chaotropic, JBScreen Plus Salts, JBScreen Plus Additives and JBScreen Plus Volatiles, containing 24 different additives each. The ready-to-use reagents are supplied in 1 ml aliquots.

The 96 solutions of JBScreen Plus HTS, comprising the reagents of the kosmotropic, chaotropic, salts and additive kit, are supplied in a sterile deep well block containing 1 ml per well.

Recommended reading

• Herberhold et al. (2004) Effects of Chaotropic and Kosmotropic Cosolvents on the Pressure-Induced Unfolding and Denaturation of Proteins: An FT-IR Study on Staphylococcal Nuclease. Biochemistry 43:3336.
• Batchelor et al. (2004) Impact of protein denaturants and stabilizers on water structure. J. Am. Chem. Soc. 126:1958.
• Boström et al. (2003) Specific ion effects: Why the properties of lysozyme in salt solutions follow a Hofmeister series. Biophys. J. 85:686.
• Uedaira et al. (2001) Role of hydration of polyhydroxy compounds in biological systems. Cell. Mol. Biol. 47:823.
• http://www.lsbu.ac.uk/water/kosmos.html
• Cacace et al. (1997): The Hofmeister series: salt and solvent effects on interfacial phenomena. Quarterly Reviews of Biophysics 30:241.
• Von Hippel et al. (1965) On the Conformational Stability of Globular Proteins: The Effects of Various Electrolytes and Non-electrolytes on the Thermal Ribonuclease Transition. J. Biol. Chem. 240:3909.