The evolution of the Collaborative Computational Project No. 4 (CCP4) has been described in a new article by Agirre et al. [(2023). Acta Cryst. D79, 449-461] that should provide the definitive reference for the CCP4 suite of programs.

Climate disruption is an increasing pressure on food supply worldwide. Consequently, it is required to develop strategies to improve the yield of crops under abiotic stress as plants have to endure novel adverse environmental conditions. Among them, drought and salinity constrain agricultural productivity most dramatically. Many of the plant adaptive responses occur at the cell membrane. There, the communication between those processes that are disrupted as a consequence of the adverse environmental stimuli and those involved in the plant adaptive response is established. The available data show that this communication is achieved by a regulated localisation of different signalling molecules to the vicinity of ion channels and transporters. The structural characterisation of those complexes constitutes a major challenge to understand the mechanism to confer plant resistance to stress and to implement novel biotechnological approaches to ensure food security. Key Concepts The understanding at the molecular level of the mechanisms for plant adaptation to drought and salinity helps to implement novel biotechnological approaches to ensure food security. The cytosolic levels of the phytohormone abscisic acid (ABA) and the Ca 2+ ion (Ca 2+ ) act as a molecular switch to plant adaptive response to stress. Plant cell response to abiotic stress is mediated by supramolecular complexes that are formed in the vicinity or at the cell membrane. Structural biology has provided the bases for ABA and Ca 2+ roles as they affect the molecular architecture of the signalling complexes regulating plant response. The structural information can be used to drive a genetic approach and for the identification of small molecules that can be used as new agrochemicals for plant improvement.

Abscisic acid (ABA) is the main phytohormone involved in adaptive crop responses to drought. ABA signaling relies on the family of pyrabactin resistance 1/PYR1-like/regulatory components of ABA receptors (PYR/PYL/RCAR), which upon ABA binding form high-affinity ternary complexes with clade A protein phosphatases type 2C (PP2Cs) and inhibit them (Cutler et al., 2010Cutler S.R. Rodriguez P.L. Finkelstein R.R. Abrams S.R. Abscisic acid: emergence of a core signaling network.Annu. Rev. Plant Biol. 2010; 61: 651-679Crossref PubMed Scopus (1964) Google Scholar). This results in the activation of an SnRK2-dependent phosphorylation cascade affecting a high number of targets in the plant cells, regulating stomatal aperture and the expression of stress-responsive genes (Cutler et al., 2010Cutler S.R. Rodriguez P.L. Finkelstein R.R. Abrams S.R. Abscisic acid: emergence of a core signaling network.Annu. Rev. Plant Biol. 2010; 61: 651-679Crossref PubMed Scopus (1964) Google Scholar). Our current understanding of the structural mechanism of ABA signaling relies exclusively on crystallographic analyses performed with Arabidopsis thaliana ABA receptors (reviewed by Santiago et al., 2012Santiago J. Dupeux F. Betz K. Antoni R. Gonzalez-Guzman M. Rodriguez L. Marquez J.A. Rodriguez P.L. Structural insights into PYR/PYL/RCAR ABA receptors and PP2Cs.Plant Sci. 2012; 182: 3-11Crossref PubMed Scopus (83) Google Scholar). Although mechanistic insights obtained in Arabidopsis are usually translated to other plant species, studies in crops might overturn or update the dogma established in Arabidopsis. As a result of structural studies performed with Citrus sinensis (sweet orange) and Solanum lycopersicum (tomato) ABA receptors, we have identified a novel latch-closed gate-open ABA-bound intermediate that provides novel mechanistic insight into ABA signaling. We propose an updated model that highlights the role of PP2C as a necessary co-receptor to increase ABA binding affinity by facilitating interconversion among the different conformations adopted by the receptor toward the formation of the Receptor-ABA-PP2C complex. To understand the structural basis of ABA recognition by ABA receptors in crop species, we have solved the crystal structures of the apo form of CsPYL1 and SlPYL1, their binary complexes with ABA, and the ternary complex CsPYL1-ABA-AtHAB1ΔN (amino acids 179–511) (Figure 1, Supplemental Figures 1 and 2, Supplemental Table 1, and Supplemental Methods). Comparison of the apo forms and the ternary complex reveals the well-reported conformational changes of the latch and gate loops surrounding the ABA binding pocket that are required for productive PP2C binding (Figure 1A and 1C) (Melcher et al., 2009Melcher K. Ng L.M. Zhou X.E. Soon F.F. Xu Y. Suino-Powell K.M. Park S.Y. Weiner J.J. Fujii H. Chinnusamy V. et al.A gate-latch-lock mechanism for hormone signalling by abscisic acid receptors.Nature. 2009; 462: 602-608Crossref PubMed Scopus (508) Google Scholar). ABA binding in the presence of PP2C induces a structural reorganization of these loops that generates a solvent-accessible surface complementary to the PP2C active site. Intriguingly, unlike other Arabidopsis ABA-bound receptor complexes that display identical closed conformation in the binary ABA-receptor form and in the ternary complex with PP2C (Melcher et al., 2009Melcher K. Ng L.M. Zhou X.E. Soon F.F. Xu Y. Suino-Powell K.M. Park S.Y. Weiner J.J. Fujii H. Chinnusamy V. et al.A gate-latch-lock mechanism for hormone signalling by abscisic acid receptors.Nature. 2009; 462: 602-608Crossref PubMed Scopus (508) Google Scholar), the crystal structures of the ABA-bound CsPYL1 and SlPYL1 complexes show a closed conformation of the latch while the gate displays a nonproductive open conformation incompatible with PP2C binding (Figure 1C and Supplemental Figure 3). In addition to these structural features, the binding of ABA in the binary complex does not induce the intermonomer swiveling that is reported to favor dissociation of homodimeric receptors by a reduction of the dimeric interface buried area (Supplemental Figure 4). The fact that the apo and ABA-bound forms of SlPYL1 and CsPYL1 display largely identical structures, although they were crystallized in different space groups with different crystal packing contacts, indicates that their conformations and oligomeric state are unlikely to be affected by crystal packing (Supplemental Table 1). This indicates that while ABA binding promotes the dissociation of AtPYL1, the dimeric nature of SlPYL1 remains unaltered despite ABA binds into the pocket as observed in the crystal structures of SlPYL1 and CsPYL1 (Supplemental Figure 5). Hence, all these data suggest that the crystallization of ABA-bound SlPYL1 and CsPYL1 has selected a stable intermediate between the apo and the ternary receptor complex with ABA and PP2C. The isolation of a gate-open latch-closed ABA-bound intermediate makes it possible to perform a deeper structural analysis of how receptors interconvert between different folded states to develop their function. Previous structural analyses assumed that ABA binding, gate and latch closure, and dimer dissociation were coupled events (Melcher et al., 2009Melcher K. Ng L.M. Zhou X.E. Soon F.F. Xu Y. Suino-Powell K.M. Park S.Y. Weiner J.J. Fujii H. Chinnusamy V. et al.A gate-latch-lock mechanism for hormone signalling by abscisic acid receptors.Nature. 2009; 462: 602-608Crossref PubMed Scopus (508) Google Scholar, Weiner et al., 2010Weiner J.J. Peterson F.C. Volkman B.F. Cutler S.R. Structural and functional insights into core ABA signaling.Curr. Opin. Plant Biol. 2010; 13: 495-502Crossref PubMed Scopus (176) Google Scholar). However, our crystallographic data clearly show that closure of the latch and the gate are independent events that could be studied separately. To investigate the bases of the latch closure, we compared the apo and ABA-bound SlPYL1 structures. The analysis highlights the role of the conserved Glu/Asp-His-Arg motif at the latch, as its conformation changes drastically (Figure 1D). In particular, the transition from the open to the closed structure involves the coordinated movement of the Glu150 and Arg152 pair to make the binding site accessible and His151 reorientation to define the ABA binding pocket. This means that the so-called open and closed conformations of the latch (Melcher et al., 2009Melcher K. Ng L.M. Zhou X.E. Soon F.F. Xu Y. Suino-Powell K.M. Park S.Y. Weiner J.J. Fujii H. Chinnusamy V. et al.A gate-latch-lock mechanism for hormone signalling by abscisic acid receptors.Nature. 2009; 462: 602-608Crossref PubMed Scopus (508) Google Scholar) should not be referred to as accessibility of the ABA binding site; open is an incompetent conformation for ABA binding and closed is the conformation of the latch in the ternary complex, competent for ABA and PP2C binding. The analysis also shows that the His151 side chain should move to a closed conformation before ABA enters the pocket, otherwise the hormone would hinder the structural rearrangement required for receptor activation (Figure 1D). To explore further whether these rearrangements precede or follow the ABA binding step, we analyzed the crystal structures of the apo forms of the ABA receptors available in the literature (Figure 1E and Supplemental Methods). These data show that while the structures of Arabidopsis PYR1, PYL1, and PYL2 display a variable latch-open conformation incompetent for ABA binding, the structures of PYL3, PYL5, and PYL10 adopt a latch-closed conformation competent for ABA binding. This indicates that ABA receptors are in an equilibrium state between latch-open and latch-closed in the absence of ABA in the pocket and consequently, the hormone either induces the shift of this equilibrium to the closed form or will selectively bind to the closed conformation (Vogt et al., 2014Vogt A.D. Pozzi N. Chen Z.W. Di Cera E. Essential role of conformational selection in ligand binding.Biophys. Chem. 2014; 186: 13-21Crossref PubMed Scopus (86) Google Scholar, Agafonov et al., 2015Agafonov R.V. Wilson C. Kern D. Evolution and intelligent design in drug development.Front. Mol. Biosci. 2015; 2: 27Crossref PubMed Scopus (6) Google Scholar) (Figure 1I). Once ABA is in the binding pocket, the receptor should undergo a structural rearrangement leading to gate closing for a successful PP2C interaction. However, the analyses of the crystal structures of the binary complexes of CsPYL1 and SlPYL1 with ABA show that this is not always the case; rather it indicates that ABA receptors are able to adopt several stable conformations including gate-open latch-closed or gate-closed latch-closed. To understand the bases of this behavior, we compared the gate loop structures of CsPYL1 and SlPYL1 receptor in complex with ABA with that of the active form of CsPYL1 in complex with PP2C. This analysis shows that the conserved Pro117/124 (for orange/tomato, respectively) undergoes a cis to trans isomerization upon gate closure (Figure 1F and 1G). Proline cis to trans isomerization has been reported to occur from the apo to the ABA-bound forms of AtPYR1 and AtPYL1 (Melcher et al., 2009Melcher K. Ng L.M. Zhou X.E. Soon F.F. Xu Y. Suino-Powell K.M. Park S.Y. Weiner J.J. Fujii H. Chinnusamy V. et al.A gate-latch-lock mechanism for hormone signalling by abscisic acid receptors.Nature. 2009; 462: 602-608Crossref PubMed Scopus (508) Google Scholar). This transition occurs in the slow microsecond to millisecond timescales and constitutes a rate-determining step in protein folding (Wedemeyer et al., 2002Wedemeyer W.J. Welker E. Scheraga H.A. Proline cis-trans isomerization and protein folding.Biochemistry. 2002; 41: 14637-14644Crossref PubMed Scopus (355) Google Scholar). Indeed, NMR-HSQC experiments suggest that, under ABA saturated conditions, the PYR1 receptor displays equilibrium between the open and closed forms of the gate within these timescales (Peterson et al., 2010Peterson F.C. Burgie E.S. Park S.Y. Jensen D.R. Weiner J.J. Bingman C.A. Chang C.E. Cutler S.R. Phillips Jr., G.N. Volkman B.F. Structural basis for selective activation of ABA receptors.Nat. Struct. Mol. Biol. 2010; 17: 1109-1113Crossref PubMed Scopus (90) Google Scholar). Hence, this suggests that this equilibrium has been shifted to the open conformation in the ABA-bound SlPYL1 and CsPYL1 crystal structures (Figure 1I). Although the ABA-bound forms of CsPYL1 and SlPYL1 are not compatible with PP2C binding, it has been shown that these receptors are able to interact and inhibit PP2C activity under physiological conditions (Gonzalez-Guzman et al., 2014Gonzalez-Guzman M. Rodriguez L. Lorenzo-Orts L. Pons C. Sarrion-Perdigones A. Fernandez M.A. Peirats-Llobet M. Forment J. Moreno-Alvero M. Cutler S.R. et al.Tomato PYR/PYL/RCAR abscisic acid receptors show high expression in root, differential sensitivity to the abscisic acid agonist quinabactin, and the capability to enhance plant drought resistance.J. Exp. Bot. 2014; 65: 4451-4464Crossref PubMed Scopus (124) Google Scholar, Arbona et al., 2017Arbona V. Zandalinas S.I. Manzi M. Gonzalez-Guzman M. Rodriguez P.L. Gomez-Cadenas A. Depletion of abscisic acid levels in roots of flooded Carrizo citrange (Poncirus trifoliata L. Raf. x Citrus sinensis L. Osb.) plants is a stress-specific response associated to the differential expression of PYR/PYL/RCAR receptors.Plant Mol. Biol. 2017; 93: 623-640Crossref PubMed Scopus (31) Google Scholar). Moreover, we provide structural data characterizing this interaction (Figure 1C) and biochemical data showing that CsPYL1 and SlPYL1 are able to interact and inhibit PP2C activity with IC50 values similar to those measured for AtPYL1 (Supplemental Figure 6A). This highlights the role of the PP2C as ABA co-receptor by selecting the gate-closed latch-closed receptor form. The phosphatase will solely interact with the ABA-bound gate-closed form of the receptor, stabilizing its conformation by the insertion of the gate loop into the PP2C active site and the formation of a water-mediated conserved network of hydrogen bonds that links the carbonyl of Arg 145/152 at the latch, the carbonyl of Pro 117/124 at the gate, the ketone group of ABA, and the side chain of Trp385 from AtHAB1ΔN (Figure 1H). Moreover, the HAB1W385A phosphatase was markedly refractory to inhibition by either Arabidopsis or crop receptors (Dupeux et al., 2011aDupeux F. Antoni R. Betz K. Santiago J. Gonzalez-Guzman M. Rodriguez L. Rubio S. Park S.Y. Cutler S.R. Rodriguez P.L. et al.Modulation of abscisic acid signaling in vivo by an engineered receptor-insensitive protein phosphatase type 2C allele.Plant Physiol. 2011; 156: 106-116Crossref PubMed Scopus (91) Google Scholar) (Supplemental Figure 6B and 6C), highlighting the crucial role played by the hydrogen bond network that connects PP2C with both ABA and the gate/latch loops of the receptor. To provide additional biochemical evidence for the role of PP2C as co-receptor, we have quantified the ABA binding properties of SlPYL1 in the presence or absence of phosphatase using a fluorescence emission assay (Supplemental Figure 7). According to our data, PP2C can be considered as co-receptor in the ternary complex as the equimolar presence of phosphatase and SlPYL1 results in an apparent ABA Kd (dissociation constant) of 158 ± 60 nM, whereas the ABA Kd for SlPYL1 in absence of phosphatase is approximately three orders of magnitude higher (90 ± 10 μM). Thus, the receptor-ABA-phosphatase complex behaves as a high-affinity system for ABA binding able to detect physiological changes in ABA concentration, whereas the dimeric receptor alone could not perform a biologically relevant function. Therefore, both biochemical and structural evidence support the role of PP2C as ABA co-receptor. In our model, PP2C has a proactive role by shifting the gate-open gate-closed equilibrium of the binary complex toward the formation of the ternary complex (Figure 1I). This provides an updated model to explain why the receptor-ABA-phosphatase complex behaves as a high-affinity sensor for ABA binding rather than the receptor itself (Ma et al., 2009Ma Y. Szostkiewicz I. Korte A. Moes D. Yang Y. Christmann A. Grill E. Regulators of PP2C phosphatase activity function as abscisic acid sensors.Science. 2009; 324: 1064-1068Crossref PubMed Scopus (1625) Google Scholar, Santiago et al., 2009Santiago J. Dupeux F. Round A. Antoni R. Park S.Y. Jamin M. Cutler S.R. Rodriguez P.L. Marquez J.A. The abscisic acid receptor PYR1 in complex with abscisic acid.Nature. 2009; 462: 665-668Crossref PubMed Scopus (390) Google Scholar). Altogether, our data suggest that ABA perception involves three steps (Figure 1I): first, the physical binding of the hormone to a latch-closed competent state; second, a conformational transition to activate the receptor; and third, the binding of PP2C to the receptor in the latch- and gate-closed conformation. As has been observed for the Arabidopsis dimeric receptors, our structural data show that the dimerization interface of CsPYL1 and SlPYL1 overlaps with that formed between CsPYL1 and the PP2C (Figure 1). Therefore, for PP2C binding of dimeric receptors, the ABA perception mechanism would include an additional dissociation step between the gate-open ABA-bound state and the latch- and gate-closed state (Supplemental Figure 4). This work was funded by grants from MINECO (BFU2014-59796-R to A.A. and BIO2014-52537-R to P.L.R.). J.L.-J was supported by a Juan de la Cierva contract from MINECO and by the Marie Skłodowska-Curie Action H2020-MSCA-IF-2015-707477.

Crystallography, that underpins nearly all of the sciences today, remains relatively unknown to the general public.The International Year of Crystallography (IYCr2014) has greatly helped to raise awareness of the role that this science plays in our world, and many efforts have been made to spread the message of crystallography in various countries where scientific activity is not as well developed or as well organized as elsewhere.However, crystallography still seems to remain an unresolved matter in academic environments of even some developed countries.As an example, the American Crystallographic Association and the US National Committee for Crystallography suggested in 2006 that "perhaps due to rapid technological advances in the field of modern crystallography, there appears to be a declining number of professional crystallographers, as well as a lack of sufficient education and training in crystallography...".In this context, the Department of Crystallography and Structural Biology of the Institute of Physical-Chemistry "Rocasolano" (Spanish National Research Council, CSIC) offers a website entitled "Crystallography-Cristalografia" that occupies a space internationally recognized to learn Crystallography: http://www.xtal.iqfr.csic.es/Cristalografia/

Separation of daughter cells during bacterial cell division requires splitting of the septal cross wall by peptidoglycan hydrolases. In Streptococcus pneumoniae, PcsB is predicted to perform this operation. Recent evidence shows that PcsB is recruited to the septum by the transmembrane FtsEX complex, and that this complex is required for cell division. However, PcsB lacks detectable catalytic activity in vitro, and while it has been proposed that FtsEX activates PcsB, evidence for this is lacking. Here we demonstrate that PcsB has muralytic activity, and report the crystal structure of full-length PcsB. The protein adopts a dimeric structure in which the V-shaped coiled-coil (CC) domain of each monomer acts as a pair of molecular tweezers locking the catalytic domain of each dimeric partner in an inactive configuration. This suggests that the release of the catalytic domains likely requires an ATP-driven conformational change in the FtsEX complex, conveyed towards the catalytic domains through coordinated movements of the CC domain.

Plant cells have developed specific protective molecular machinery against environmental stresses. The family of CBL-interacting protein kinases (CIPK) and their interacting activators, the calcium sensors calcineurin B-like (CBLs), work together to decode calcium signals elicited by stress situations. The molecular basis of biological activation of CIPKs relies on the calcium-dependent interaction of a self-inhibitory NAF motif with a particular CBL, the phosphorylation of the activation loop by upstream kinases, and the subsequent phosphorylation of the CBL by the CIPK. We present the crystal structures of the NAF-truncated and pseudophosphorylated kinase domains of CIPK23 and CIPK24/SOS2. In addition, we provide biochemical data showing that although CIPK23 is intrinsically inactive and requires an external stimulation, CIPK24/SOS2 displays basal activity. This data correlates well with the observed conformation of the respective activation loops: Although the loop of CIPK23 is folded into a well-ordered structure that blocks the active site access to substrates, the loop of CIPK24/SOS2 protrudes out of the active site and allows catalysis. These structures together with biochemical and biophysical data show that CIPK kinase activity necessarily requires the coordinated releases of the activation loop from the active site and of the NAF motif from the nucleotide-binding site. Taken all together, we postulate the basis for a conserved calcium-dependent NAF-mediated regulation of CIPKs and a variable regulation by upstream kinases.

The Arabidopsis SOS2 family of twenty-six protein kinases (CIPKs), their interacting activators, the SOS3 family of ten calcium-binding proteins (CBLs) and protein phosphatases type 2C (PP2C), function together in decoding calcium signals elicited by different environmental stimuli. Biochemical data suggest that stable CBL-CIPK or CIPK-PP2C complexes may be regulating the activity of various substrates controlling ion homeostasis. The available structural information provides a general regulatory mechanism in which calcium perception by CBLs and kinase activation is coupled. The structural basis of this molecular mechanism and the specificity of the network is reviewed and discussed in detail.

Resumen del Poster presentado en Environment Workshop 2013: Genomic, Physiological and Breeding Approaches for Enhancing Drought Resistance in Crops. Baeza (Spain), 23-25 September (2013)

ABSTRACT Fosfomycin targets the first step of peptidoglycan biosynthesis in Streptococcus pneumoniae catalyzed by UDP- N -acetylglucosamine enolpyruvyltransferase (MurA1). We investigated whether heteroresistance to fosfomycin occurs in S. pneumoniae . We found that of 11 strains tested, all but 1 (Hungary 19A ) displayed heteroresistance and that deletion of murA1 abolished heteroresistance. Hungary 19A differs from the other strains by a single amino acid substitution in MurA1 (Ala 364 Thr). To test whether this substitution is responsible for the lack of heteroresistance, it was introduced into strain D39. The heteroresistance phenotype of strain D39 was not changed. Furthermore, no relevant structural differences between the MurA1 crystal structures of heteroresistant strain D39 and nonheteroresistant strain Hungary 19A were found. Our results reveal that heteroresistance to fosfomycin is the predominant phenotype of S. pneumoniae and that MurA1 is required for heteroresistance to fosfomycin but is not the only factor involved. The findings provide a caveat for any future use of fosfomycin in the treatment of pneumococcal infections.

The SnRK2.6 (SNF1-related kinase 2.6) gene from Arabidopsis thaliana encodes the serine/threonine protein kinase SnRK2.6/OST1 (OPEN STOMATA 1). It plays a central role in the drought-tolerance mechanism. OST1 is in fact the main positive effector in the hydric stress response. The SnRK2.6 gene was cloned into the pGEX4T1 plasmid, mutated and expressed in Escherichia coli, allowing purification to homogeneity in two chromatographic steps. Various OST1 mutants yielded crystals using vapour-diffusion techniques, but only one mutant showed a good diffraction pattern. Its crystals diffracted to 2.8 Å resolution and belonged to space group P222(1), with unit-cell parameters a=77.7, b=99.4, c=108.4 Å. A promising molecular-replacement solution was found using the structure of the kinase domain of the yeast AMP-activated protein kinase SNF1 (PDB entry 3hyh) as the search model.

AmpD is a cytoplasmic peptidoglycan (PG) amidase involved in bacterial cell-wall recycling and in induction of β-lactamase, a key enzyme of β-lactam antibiotic resistance. AmpD belongs to the amidase_2 family that includes zinc-dependent amidases and the peptidoglycan-recognition proteins (PGRPs), highly conserved pattern-recognition molecules of the immune system. Crystal structures of Citrobacter freundii AmpD were solved in this study for the apoenzyme, for the holoenzyme at two different pH values, and for the complex with the reaction products, providing insights into the PG recognition and the catalytic process. These structures are significantly different compared with the previously reported NMR structure for the same protein. The NMR structure does not possess an accessible active site and shows the protein in what is proposed herein as an inactive “closed” conformation. The transition of the protein from this inactive conformation to the active “open” conformation, as seen in the x-ray structures, was studied by targeted molecular dynamics simulations, which revealed large conformational rearrangements (as much as 17 Å) in four specific regions representing one-third of the entire protein. It is proposed that the large conformational change that would take the inactive NMR structure to the active x-ray structure represents an unprecedented mechanism for activation of AmpD. Analysis is presented to argue that this activation mechanism might be representative of a regulatory process for other intracellular members of the bacterial amidase_2 family of enzymes.

Sessions C342fusion partners that play important roles in this stem cell maintenance.The protein consists of a BTB domain, an intrinsically unfolded linker region and 9 zinc fingers.It is known that the BTB domain mediates the dimerization of the PLZF protein.Present theories suggest that a chromosomal fusion occurs with RARα and that PLZF dimer or oligomer binding causes the transcriptional repression responsible for APL.One hypothesis concerns the potential oligomerization of PLZF during DNA interaction, it is however not known which part of the protein is responsible for the protein:protein contacts.The aim of the present SAXS study is to shed light on the dimerization and oligomerization, thus understanding the mechanisms of PLZF mediated DNA transcriptional repression.So far most studies have been concentrated on the BTB domain, since the unstructured linker region is inherently difficult to study.Using SAXS, we have the great advantage to work with proteins in solution, thus it was possible to collect data on the native-like condition of the linker region.The analysis however, is demanding and not much experience exists on data from intrinsically un-folded regions.We have collected data on three PLZF constructs, one containing the BTB domain only, one consisting of the linker part only, and one spanning the BTB and linker part.The oligomerization process has been analyzed in a pH series spanning five pH values.Complementary analysis has been carried out using Dynamic Light Scattering and Circular Dichroism spectroscopy.The fact that our data set is very extensive was very important for the interpretation of the data.We have successfully identified the involved species in the oligomerization process and have analysed the change of behavior of the intrinsically un-folded linker region during this process.