Crystallization Buffers Research Articles

Analysis of protein structures containing HEPES and MES molecules

X‐ray crystallography is the main experimental method behind ligand–macromolecule complexes found in the Protein Data Bank (PDB). Applying bioinformatics methods to such structural data can fuel drug discovery, albeit under the condition that the information is correct. Regrettably, a small number of structures in the PDB are of suboptimal quality due to incorrectly identified and modeled ligands in protein–ligand complexes. In this paper, we combine a theoretical‐graph approach, nuclear density estimates, bioinformatics methods, and prior chemical knowledge to analyze two non‐physiological ligands, HEPES and MES, that are frequent components of crystallization and purifications buffers. Our analysis includes quantum mechanics calculations and Cambridge Structure Database (CSD) queries to define the ideal conformation of these ligands, geometry analysis of PDB deposits regarding several quality factors, and a search for homologous structures to identify other small molecules that could bind in place of the parasitic ligand. Our results highlight the need for careful refinement of macromolecule–ligand complexes and better validation tools that integrate results from all relevant resources.

An In-Depth Look at DNA Crystals through the Prism of Molecular Dynamics Simulations

Summary X-ray crystallography is the primary tool for biomolecular structural determination. However, contacts formed through the crystal lattice are known to affect structures, especially for small and flexible molecules such as DNA oligomers, by introducing significant structural changes in comparison to solution. Furthermore, why molecules crystallize in certain symmetry groups, which role crystallization additives play, and whether they are just innocuous and unspecific crystallization catalysts remain unclear. By using one of the currently best-performing DNA force fields and applying significant computational effort, we described the nature of intermolecular forces that stabilize B-DNA crystals in various symmetry groups and solvent environments with an unprecedented level of detail. We showed a tight coupling between the lattice stability and the type of crystallization additives and that certain symmetry groups are stable only in the presence of a specific additive. Additives and crystal contacts induce small but non-negligible changes in the physical properties of DNA.

Mg2+ ions: do they bind to nucleobase nitrogens?

Given the many roles proposed for Mg2+ in nucleic acids, it is essential to accurately determine their binding modes. Here, we surveyed the PDB to classify Mg2+ inner-sphere binding patterns to nucleobase imine N1/N3/N7 atoms. Among those, purine N7 atoms are considered to be the best nucleobase binding sites for divalent metals. Further, Mg2+ coordination to N7 has been implied in several ribozyme catalytic mechanisms. We report that Mg2+ assigned near imine nitrogens derive mostly from poor interpretations of electron density patterns and are most often misidentified Na+, K+, NH4+ ions, water molecules or spurious density peaks. Consequently, apart from few documented exceptions, Mg2+ ions do not bind to N7 atoms. Without much of a surprise, Mn2+, Zn2+ and Cd2+, which have a higher affinity for nitrogens, may contact N7 atoms when present in crystallization buffers. In this respect, we describe for the first time a potential Zn2+ ribosomal binding site involving two purine N7 atoms. Further, we provide a set of guidelines to help in the assignment of Mg2+ in crystallographic, cryo-EM, NMR and model building practices and discuss implications of our findings related to ion substitution experiments.

Anions in Nucleic Acid Crystallography.

Nucleic acid crystallization buffers contain a large variety of chemicals fitting specific needs. Among them, anions are often solely considered for pH-regulating purposes and as cationic co-salts while their ability to directly bind to nucleic acid structures is rarely taken into account. Here we review current knowledge related to the use of anions in crystallization buffers along with data on their biological prevalence. Chloride ions are frequently identified in crystal structures but display low cytosolic concentrations. Hence, they are thought to be distant from nucleic acid structures in the cell. Sulfate ions are also frequently identified in crystal structures but their localization in the cell remains elusive. Nevertheless, the characterization of the binding properties of these ions is essential for better interpreting the solvent structure in crystals and consequently, avoiding mislabeling of electron densities. Furthermore, understanding the binding properties of these anions should help to get clues related to their potential effects in crowded cellular environments.

CD spectroscopy: an essential tool for quality control of protein folding.

The production of diffraction quality protein crystals for X-ray crystallography has been greatly accelerated by the development of high-throughput protein (HTP) methods, which enable a large number of crystallization conditions to be rapidly investigated. Monitoring sample quality and the effect of crystallization buffers on protein behavior in solution should be considered as part of the crystallization experiment. Circular Dichroism (CD) spectroscopy is the ideal technique for these tasks as it can be operated in a high-throughput mode. Using CD to screen ligand binding interactions could show whether protein function/activity is retained, altered, or lost under different crystallization conditions. In this chapter, several methods for high-throughput CD (HTCD) applied to the preparation of proteins for crystallization will be presented. Quality control (QC) of protein batches in terms of conformational folding is often disregarded in protein production. Examples of batch-to-batch variation in the local tertiary structure of aromatic side chain residues revealed by CD will be discussed. In some of the examples, the fact that ligand binding properties were affected by changes in folding clearly shows that the characterization of folding of recombinant protein batches should not be ignored but be implemented as an important part of protein quality control.

Molecular Structure of the Brucella abortus Metalloprotein RicA, a Rab2-Binding Virulence Effector

The Gram-negative intracellular pathogen Brucella abortus is the causative agent of brucellosis, which is among the most common zoonoses globally. The B. abortus RicA protein binds the host-expressed guanosine nucleotide-binding protein, Rab2, and modulates B. abortus infection biology. We have solved the first X-ray crystal structure of RicA to 2.7 Å resolution and have quantified the affinity of RicA binding to human Rab2 in its GDP-bound and nucleotide-free forms. RicA adopts a classic γ-carbonic anhydrase (γ-CA) fold containing a left-handed β-helix followed by a C-terminal α-helix. Two homotrimers of RicA occupy the crystallographic asymmetric unit. Though no zinc was included in the purification or crystallization buffers, zinc is contained within the RicA crystals, as demonstrated by X-ray fluorescence spectroscopy. Electron density for a Zn(2+) ion coordinated by three histidine residues is evident in the putative active site of RicA. However, purified RicA preparations do not exhibit carbonic anhydrase activity, suggesting that Zn(2+) may not be the physiologically relevant metal cofactor or that RicA is not a bona fide carbonic anhydrase enzyme. Isothermal titration calorimetry (ITC) measurements of purified RicA binding to purified human Rab2 and GDP-Rab2 revealed similar equilibrium affinities (Kd ≈ 35 and 40 μM, respectively). This study thus defines RicA as a Zn(2+)-binding γ-carbonic anhydrase-like protein that binds the human membrane fusion/trafficking protein Rab2 with low micromolar affinity in vitro. These results support a model in which γ-CA family proteins may evolve unique cellular functions while retaining many of the structural hallmarks of archetypal γ-CA enzymes.

Direct Observation of Protein Microcrystals in Crystallization Buffer by Atmospheric Scanning Electron Microscopy

X-ray crystallography requires high quality crystals above a given size. This requirement not only limits the proteins to be analyzed, but also reduces the speed of the structure determination. Indeed, the tertiary structures of many physiologically important proteins remain elusive because of the so-called “crystallization bottleneck”. Once microcrystals have been obtained, crystallization conditions can be optimized to produce bigger and better crystals. However, the identification of microcrystals can be difficult due to the resolution limit of optical microscopy. Electron microscopy has sometimes been utilized instead, with the disadvantage that the microcrystals usually must be observed in vacuum, which precludes the usage for crystal screening. The atmospheric scanning electron microscope (ASEM) allows samples to be observed in solution. Here, we report the use of this instrument in combination with a special thin-membrane dish with a crystallization well. It was possible to observe protein crystals of lysozyme, lipase B and a histone chaperone TAF-Iβ in crystallization buffers, without the use of staining procedures. The smallest crystals observed with ASEM were a few μm in width, and ASEM can be used with non-transparent solutions. Furthermore, the growth of salt crystals could be monitored in the ASEM, and the difference in contrast between salt and protein crystals made it easy to distinguish between these two types of microcrystals. These results indicate that the ASEM could be an important new tool for the screening of protein microcrystals.

Structure and Proposed Mechanism for the pH-Sensing Helicobacter pylori Chemoreceptor TlpB

pH sensing is crucial for survival of most organisms, yet the molecular basis of such sensing is poorly understood. Here, we present an atomic resolution structure of the periplasmic portion of the acid-sensing chemoreceptor, TlpB, from the gastric pathogen Helicobacter pylori. The structure reveals a universal signaling fold, a PAS domain, with a molecule of urea bound with high affinity. Through biophysical, biochemical, and invivo mutagenesis studies, we show that urea and the urea-binding site residues play critical roles in the ability of H.pylori to sense acid. Our signaling model predicts that protonation events at Asp114, affected by changes in pH, dictate the stability of TlpB through urea binding.

Chemical composition of spinel from Uralian-Alaskan-type Mafic–Ultramafic complexes and its petrogenetic significance

Uralian-Alaskan-type mafic–ultramafic complexes are recognized as a distinct class of intrusions regarding lithologic assemblage, mineral chemistry and petrogenetic setting. In the present study, we discuss new data on the distribution of major elements in minerals of the spinel group in rocks from Uralian-Alaskan-type complexes in the Ural Mountains, Russia. Cr-rich spinel (Cr2O3 = 20–53 wt%) in dunite with interstitial clinopyroxene and in wehrlite cumulates indicate that it reacted with interstitial liquid resulting in the progressive substitution of Al2O2 and Cr2O3 by Fe2O3 and TiO2. A distinct change in the spinel chemistry in dunite (Cr2O3 = 47–53 wt%), towards Al2O3- and Cr2O3-poor but Fe2O3-rich compositions monitors the onset of clinopyroxene fractionation in wehrlite (Cr2O3 = 15–35 wt%, Al2O3 = 1–8 wt%, Fe2O3 = 25–55 wt%). In more fractionated mafic rocks, the calculated initial composition of exsolved spinel traces the sustained crystallization of clinopyroxene by decreasing Cr2O3 and increasing FeO, Fe2O3 and fO2. Finally, the initiation of feldspar crystallization buffers the Al2O3 content in most of the spinels in mafic rocks at very low Cr2O3 contents (<5 wt%). The fractionation path all along and the reaction with interstitial liquid are accompanied by increasing Fe2O3 contents in the spinel. This likely is caused by a significant increase in the oxygen fugacity, which suggests closed system fractionation processes. Spinel with Cr2O3 < 27 wt% is exsolved into a Fe2O3-rich and an Al2O3-rich phase forming a variety of textures. Remarkably, exsolved spinel in different lithologies from complexes 200 km apart follows one distinct solvus line defining a temperature of ca. 600°C. This indicates that the parental magmas were emplaced and eventually cooled at similar levels in the lithosphere, likely near the crust–mantle boundary. Eventually, these 600°C hot bodies were rapidly transported into colder regions of the upper crust during a regional tectonic event, probably during the major active phase of the Main Uralian Fault.

An Asymmetric Model for Na+-translocating Glutaconyl-CoA Decarboxylases

Glutaconyl-CoA decarboxylase (Gcd) couples the biotin-dependent decarboxylation of glutaconyl-CoA with the generation of an electrochemical Na(+) gradient. Sequencing of the genes encoding all subunits of the Clostridium symbiosum decarboxylase membrane complex revealed that it comprises two distinct biotin carrier subunits, GcdC(1) and GcdC(2), which differ in the length of a central alanine- and proline-rich linker domain. Co-crystallization of the decarboxylase subunit GcdA with the substrate glutaconyl-CoA, the product crotonyl-CoA, and the substrate analogue glutaryl-CoA, respectively, resulted in a high resolution model for substrate binding and catalysis revealing remarkable structural changes upon substrate binding. Unlike the GcdA structure from Acidaminococcus fermentans, these data suggest that in intact Gcd complexes, GcdA is associated as a tetramer crisscrossed by a network of solvent-filled tunnels.

Crystallographic Insights into the Autocatalytic Assembly Mechanism of a Bacteriophage Tail Spike

The tailed bacteriophage phi29 has 12 "appendages" (gene product 12, gp12) attached to its neck region that participate in host cell recognition and entry. In the cell, monomeric gp12 undergoes proteolytic processing that releases the C-terminal domain during assembly into trimers. We report here crystal structures of the protein before and after catalytic processing and show that the C-terminal domain of gp12 is an "autochaperone" that aids trimerization. We also show that autocleavage of the C-terminal domain is a posttrimerization event that is followed by a unique ATP-dependent release. The posttranslationally modified N-terminal part has three domains that function to attach the appendages to the phage, digest the cell wall teichoic acids, and bind irreversibly to the host, respectively. Structural and sequence comparisons suggest that some eukaryotic and bacterial viruses as well as bacterial adhesins might have a similar maturation mechanism as is performed by phi29 gp12 for Bacillus subtilis.

Ruffling of Metalloporphyrins Bound to IsdG and IsdI, Two Heme-degrading Enzymes in Staphylococcus aureus

IsdG and IsdI are paralogous proteins that are intracellular components of a complex heme uptake system in Staphylococcus aureus. IsdG and IsdI were shown previously to reductively degrade hemin. Crystal structures of the apoproteins show that these proteins belong to a newly identified heme degradation family distinct from canonical eukaryotic and prokaryotic heme oxygenases. Here we report the crystal structures of an inactive N7A variant of IsdG in complex with Fe(3+)-protoporphyrin IX (IsdG-hemin) and of IsdI in complex with cobalt protoporphyrin IX (IsdI-CoPPIX) to 1.8 A or better resolution. These structures show that the metalloporphyrins are buried into similar deep clefts such that the propionic acids form salt bridges to two Arg residues. His(77) (IsdG) or His(76) (IsdI), a critical residue required for activity, is coordinated to the Fe(3+) or Co(3+) atoms, respectively. The bound porphyrin rings form extensive steric interactions in the binding cleft such that the rings are highly distorted from the plane. This distortion is best described as ruffled and places the beta- and delta-meso carbons proximal to the distal oxygen-binding site. In the IsdG-hemin structure, Fe(3+) is pentacoordinate, and the distal side is occluded by the side chain of Ile(55). However, in the structure of IsdI-CoPPIX, the distal side of the CoPPIX accommodates a chloride ion in a cavity formed through a conformational change in Ile(55). The chloride ion participates in a hydrogen bond to the side chain amide of Asn(6). Together the structures suggest a reaction mechanism in which a reactive peroxide intermediate proceeds with nucleophilic oxidation at the beta- or delta-meso carbon of the hemin.

Crystal Structure of the BARD1 Ankyrin Repeat Domain and Its Functional Consequences

BARD1 is the constitutive nuclear partner to the breast and ovarian cancer-specific tumor suppressor BRCA1. Together, they form a heterodimeric complex responsible for maintaining genomic stability through nuclear functions involving DNA damage signaling and repair, transcriptional regulation, and cell cycle control. We report the 2.0A structure of the BARD1 ankyrin repeat domain. The structure includes four ankyrin repeats with a non-canonical C-terminal capping ankyrin repeat and a well ordered extended loop preceding the first repeat. Conserved surface features show an acidic patch and an acidic pocket along the surface typically used by ankyrin repeat domains for binding cognate proteins. We also demonstrate that two reported mutations, N470S and V507M, in the ankyrin repeat domain do not result in observable structural defects. These results provide a structural basis for exploring the biological function of the ankyrin repeat domain and for modeling BARD1 isoforms.

Macromolecular structures without crystals

Electron microscopy has many attractive capabilities as a tool for experimentally visualizing the structures of biological cells and macromolecules. Particles can be imaged while freely suspended in biochemically “native” buffers, and electrons can be focused to resolutions exceeding anything that a biochemist's heart might desire. To be sure, biological electron microscopy also is subject to certain physical limitations, perhaps the most problematic of which arise from the fact that short-wavelength electrons also are a form of ionizing radiation. However, among the goals for which technology rather than physical principles currently appears to be the limitation, many regard the most desirable to be the production of 3D density maps that show the positions and rotamer conformations of every amino acid by using specimens in which the proteins are in the form of well dispersed, single particles. The structures of such proteins would not only be free of crystal-packing constraints, they would also be free of the influences of the associated crystallization buffers, which often have unphysiological pH, ionic strength, ionic composition, or water activity. With the publication of the structure of the icosahedral “inner capsid particle” of a human rotavirus in this issue of PNAS, Zhang et al. (1) have come remarkably close to that goal. The technological frontier now moves to accomplishing the same results with macromolecular complexes that are significantly smaller than viruses and with structures for which a high degree of symmetry cannot be exploited to ease the task.

Use of layer silicate for protein crystallization: Effects of Micromica and chlorite powders in hanging drops

Two kinds of layer silicate powder, Micromica and chlorite, were used to aid protein crystallization by the addition to hanging drops. Using appropriate crystallization buffers, Micromica powder facilitated crystal growth speed for most proteins tested in this study. Furthermore, the addition of Micromica powder to hanging drops allowed the successful crystallization of lysozyme, catalase, concanavalin A, and trypsin even at low protein concentrations and under buffer conditions that otherwise would not generate protein crystals. Except for threonine synthase and apoferritin, the presence of chlorite delayed crystallization but induced the formation of large crystals. X-ray analysis of thaumatin crystals generated by our novel procedure gave better quality data than did that of crystals obtained by a conventional hanging drop method. Our results suggest that the speed of crystal growth and the quality of the corresponding X-ray data may be inversely related, at least for the formation of thaumatin crystals. The effect of Micromica and chlorite powders and the application of layer silicate powder for protein crystallization are discussed.

High-Throughput Protein Crystallography

An automated platform for low-volume, highthroughput protein crystallization by the sitting drop vapor diffusion has been developed. This platform consists of a novel Society of Biomolecular Screening standard microplate technology, standard automation liquid handling equipment, a software to design customizable, random crystallization buffers, an in-house designed scanner, a crystal recognition software, and Laboratory Information Management Systems. These technologies are designed to eliminate obstacles to the production of protein crystals and to address a number of critical issues faced by crystallography laboratories, namely precision and accuracy, ease of integration and use, speed, and cost.

On the purification and preliminary crystallographic analysis of isoquinoline 1-oxidoreductase from Brevundimonas diminuta 7.

Isoquinoline 1-oxidoreductase (IOR) from Brevundimonas diminuta is a mononuclear molybdoenzyme of the xanthine-dehydrogenase family of proteins and catalyzes the conversion of isoquinoline to isoquinoline-1-one. Its primary sequence and behaviour, specifically in its substrate specificity and lipophilicity, differ from other members of the family. A crystal structure of the enzyme is expected to provide an explanation for these differences. This paper describes the crystallization and preliminary X-ray diffraction experiments as well as an optimized purification protocol for IOR. Crystallization of IOR was achieved using two different crystallization buffers. Streak-seeding and cross-linking were essential to obtain well diffracting crystals. Suitable cryo-conditions were found and a structure solution was obtained by molecular replacement. However, phases need to be improved in order to obtain a more interpretable electron-density map.

Crystal structure of a putative oxalate decarboxylase (TM1287) from Thermotoga maritima at 1.95 A resolution.

Crystal structure of a putative oxalate decarboxylase (TM1287) from Thermotoga maritima at 1.95 A resolution.

Spectroscopic evidence that osmolytes used in crystallization buffers inhibit a conformation change in a membrane protein.

BtuB is a bacterial outer-membrane protein that transports vitamin B(12). Spectroscopic studies using site-directed spin labeling (SDSL) indicate that the N-terminus of BtuB undergoes a dramatic structural change from a docked (folded) to an undocked (unfolded) configuration upon substrate binding. However, this dramatic conformational change is not observed in the crystal structures of BtuB. Here, we make an attempt to resolve this discrepancy and find that the effects of solutes can explain the discrepancy between the results obtained using these two methods. Specifically, if SDSL is performed with the buffers used for the crystallization of BtuB, the substrate-induced order-disorder transition of the N-terminal Ton box observed in intact membranes is blocked. Moreover, poly(ethylene glycol) 3350, which is a component of the crystallization and soaking buffers, is shown to inhibit this structural transition. It is likely that the crystal structure of BtuB in its holo form represents an osmotically trapped conformation. Conformational changes involving relatively modest energy differences and significant hydration changes may be sensitive to solutes used during crystallization, and this example demonstrates the value of combining multiple structural methods in the examination of protein structure and function.

Introduction of Fluorometry to the Screening of Protein Crystallization Buffers

A system to use fluorometry for the search of protein crystallization buffers was developed. The screening of candidates was done with a use of short gel-filtration column (Sephacryl S-100 HR) linked to the fluorometer. Protein elution was monitored by intrinsic fluorescence or emission from its labels. This method was applied to actin and actin complexes. When nuclei were formed in actin solution, preceding the peak of actin, a new peak appeared on the elution curve. It was found that the fluorescence allows detection of even small amount of nuclei formed in the buffer. Using the screened buffers, crystal growths were attempted. The images of crystals were captured by fluorescence microscope. The monitoring of nuclei with this method will accelerate the screening of crystallization buffers. This system is applicable to the crystallization of other proteins and their complexes.