Cristalografía y Biología Estructural = CBE
Some most recent results      (See also our remaining publications & projects)
SEBBM Prizes
Successful participation of our Department at the 44th SEBBM Congress celebrated in Málaga:

Our colleague and "Ramón y Cajal" Fellow, Dr. Rafael Molina (left), presented his work developed in Denmark (NNF-CPR-University of Copenhagen) and received the 2nd MDPI-SEBBM Best poster Prize.

Our PhD student Daniel Muñoz-Reyes (right), working under the supervision of Dr. María José Sánchez-Barrena was awarded with the IUBMB Best Short Talk Presentation Prize at the Protein Structure & Function Meeting celebrated in the Congress.

Well done guys!

Lena Caruso
Lena Caruso joined us for a three months stay. She will be working on multicomponent crystallization on the supervision of Dr. Lourdes Infantes San Mateo

CBE news...

Pce, a neumococcal lysine 

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CBE = Cristalografía y Biología Estructural

The first crystallographic article from the Department
The very first Acta Cryst. article by our predecessors...
Acta Cryst. (1948) 1, 3-4

About our origins...

See some CBE videos

PBP1. Click on it to get a larger image
Dissecting the role of PBP1 from S. aureus, a Key Component of the Divisome Machinery. The Penicillin-Binding Proteins (PBPs) are transpeptidases that catalyze crosslinking in the bacterial cell wall and the molecular targets of the penicillin antibiotics. Here, we report an integrative structural biology study of PBP1 from Staphylococcus aureus (saPBP1), providing mechanistic clues about its function and regulation during cell division. A significant result provided in this work is the unexpected arrangement of the PASTA domains respect to the rest PBP1 domains, pointing to profound functional implications. Thus, although PBP1-PASTA domains preserve their compact fold, SAXS and molecular-dynamics simulations revealed that they are conformationally mobile and separated of transpeptidase domain in contrast with their homologues in PBP2x from Streptococcus pneumoniae and Streptococcus thermophilus. A series of crystallographic complexes with β-lactam antibiotics (as inhibitors) and penta-Glycine (as a substrate mimetic) allowed the molecular characterization of both inhibition by antibiotics and binding for the donor and acceptor peptidoglycan strands. Mass-spectrometry experiments with synthetic peptidoglycan fragments revealed binding by PASTA domains in coordination with the remaining domains. Collectively, our structural and modelling studies allowed us, for the first time, to decipher key structural features and differences between saPBP1 and other PBPs. The observed mobility of the PASTA domains in saPBP1 could play a crucial role for in vivo interaction with its glycosyltransferase partner in the membrane or with other components of the divisome machinery, as well as for coordination of transpeptidation and polymerization processes in the bacterial divisome.
Computational and Structural Biotechnology Journal (2021) 19, 5392-5405  (doi: 10.1016/j.csbj.2021.09.018)

Synaptotagmin-1. Click on it to get a larger image
The molecular players maintaining plant cell membrane to face abiotic stress. Cold, Salinity and other abiotic stresses cause crop productivity loss at worldwide scale. Many of the physiological changes motivated by the plant adaptation to these stresses unbalance lipid homeostasis at the plasma membrane (PM) and induce membrane instability. To counteract this situation, a highly controlled system drives lipid transfer from the endoplasmic reticulum (ER) to PM through the so-called contact sites. There, the Synaptotagmin protein family (SYTs) plays a central role displaying a dual function to tether ER and PM and to transport lipids from one membrane to the other. Our analysis explains how SYTs function is activated by changes in the lipid composition at PM and Ca2+ concentration at cytosol.
Life Science Alliance (2021) published on line 18 August  (doi: 10.26508/lsa.202101152)
Plant Cell (2021) 33, 2431-2453  (doi: 10.1093/plcell/koab122)

Xyn11. Click on it to get a larger image
Molecular mechanism behind extremophilicity. Endoxylanases active under extreme conditions of temperature and alkalinity can replace the use of highly pollutant chemicals in the pulp and paper industry. A comprehensive bioinformatics study of the GH10 family identified Xyn11, with extraordinary xylanolytic activity under extreme conditions. Addition of a carbohydrate binding domain at the C-terminus of the protein sequence further improved the activity of the enzyme at high pH. The crystallographic analysis of Xyn11 structure provides an explanation of its function at extreme conditions. Not surprisingly, the atomic interactions responsible for Xyn11 resistance are the same known to sustains protein stability, namely hydrogen bonds, ion pairs, hydrophobic and aromatic interactions. However, the number and distribution in which these interactions appear and its extremophilic enzyme properties makes Xyn11 an outstanding case for study.
Computational and Structural Biotechnology Journal (2021)  (doi: 10.1016/j.csbj.2021.05.004)

TOC-3. Click on it to get a larger image
A collaborative effort between scientist from the IQFR (Dept. of Cristalografía y Biología Estructural and Dept. of Sistemas de Baja Dimensionalidad, Superficies y Materia Condensada) and Dept. of Química Orgánica, Facultad de Ciencias Químicas, Universidad Complutense of Madrid, allowed design, synthesis and structural characterization of the first phasing agent designed for solving native protein structures by X-ray crystallography at the wavelength corresponding to the maximum intensity of the synchrotron facilities applied in biomolecular crystallography. The agent consists of a neutral ytterbium (III)-caged complex that exhibits higher anomalous signals at shorter wavelengths when compared to the best, currently applied lanthanide-based phasing agents. As a proof of principle, the complex allows determining the 3D structure of a 36 kDa protein without setting the incident beam wavelength at the metal absorption edge, the strategy followed to date to gain the strongest anomalous signal even at the expense of crystallographic resolution. The agent becomes non-disruptive to the diffraction quality of the marked crystals and allows determining accurate phases, both leading to high-quality electron density maps that enable the full tracing of the protein structure only with one agent unit bound to the protein. The high-phasing power, efficient binding to the protein, low metal−macromolecule ratio, and easy handling support the developed Yb (III) complex as the best phasing agent for X-ray crystallography of a complex biomacromolecule without using modified analogues.
ACS Applied Bio Materials (2021) in press  (doi: 10.1021/acsabm.1c00305)

eEF1A2 moonlighting - cover
Structural cues for understanding eEF1A2 moonlighting. Spontaneous mutations in the EEF1A2 gene are increasingly being recognized as a source of epilepsy and severe neurological disabilities in children. The crystal structure of eEF1A2 protein purified from rabbit skeletal muscle reveals the spatial locations of the side‐chain carboxylates of Glu301 and Glu374, to which phosphatidylethanolamine is uniquely attached via an amide bond, thereby defining the anchoring points of an eEF1A2 dimer to cellular membranes. In this new light, the role of eEF1A2 as an ancient, multifaceted, and articulated G protein at the crossroads of autophagy, oncogenesis, and viral replication appears very distant from the “canonical” one of delivering aminoacyl‐tRNAs to the ribosome that has dominated the scene and much of the thinking for many decades.
Chembiochem (2021) 22, 374 –391  (doi: 10.1002/cbic.202000516)

Chitinase. Click on it to get a larger image
The molecular machinery behind processive fungal chitinases. Chitinases degrade chitin, one of the most widespread polysaccharides in nature, into low molecular weight chitooligomers (COS), which have a broad range of pharmaceutical and medicinal applications. We report a detailed picture of the molecular events behind substrate/products binding in a fungal chitinase, giving full insight into its donor and acceptor subsites. Our crystallographic analysis revealed a previously unobserved dynamic on-off ligand binding process associated with motion of its insertion CID domain. This might represent a molecular mechanism complementary to the crucial role ascribed to aromatics at the catalytic site in processivity, which is an essential property modulating the bioconversion of chitin.  Furthermore, our analysis elucidates the implication of some highly flexible residues in activity and suggested new targets to address engineering of these biotechnologically important enzymes.
Computational and Structural Biotechnology Journal (2021) 19, 5466-5478  (doi: 10.1016/j.csbj.2021.09.027)

Click on it to get a larger image
EuXFEL can be used as an important tool for biomedically relevant research. Here, we illustrate what happens inside the catalytic cleft of an enzyme when substrate or ligand binds on single-millisecond timescales. The initial phase of the enzymatic cycle is observed with near-atomic resolution using the most advanced X-ray source currently available: the European XFEL (EuXFEL). The high repetition rate of the EuXFEL combined with our mix-and-inject technology enables the initial phase of ceftriaxone binding to the Mycobacterium tuberculosis β-lactamase to be followed using time-resolved crystallography in real time. It is shown how a diffusion coefficient in enzyme crystals can be derived directly from the X-ray data, enabling the determination of ligand and enzyme–ligand concentrations at any position in the crystal volume as a function of time. In addition, the structure of the irreversible inhibitor sulbactam bound to the enzyme at a 66 ms time delay after mixing is described.
IUCrJ (2021) 8(6), published on line (doi: 10.1107/S2052252521008125)

DDP1. Click on it to get a larger image
DDP1, a bridge between inositide and polyphosphate metabolisms. DDP1 is a yeast inositol pyrophosphatase able to connect the inositide (InsP) and polyphosphate (polyPs) metabolisms. In this work, recently published in Science Advances and in collaboration with the University of Oxford, the structure of DDP1 is analysed in complex with its multiple substrates or analogues which belongs to three different type of compounds. Particularly, this work uncovers the structural basis for inositide pyrophosphates (PP-InsPs), dicucleotide phosphates (ApnAs) and inorganic polyphosphates (polyPs) recognition. This protein is able to clamp the phosphate groups of these substrates whereas the pyrophosphate moiety to be cleaved remains near to the “Nudix motif” responsible of the hydrolysis. This research revealed a full view of DDP1 ligand binding and stability through a complete analysis of DDP1 crystallographic structures, mutagenesis, thermal shift and activity assays. The results of this work go beyond the inositide signalling and reveal the molecular basis to design inhibitors against a pathway strictly regulated in fungi, with putative application in fungal infections treatment.

EH. Click on it to get a larger image
A single residue swapping the substrate ambiguity and chiral specificity in an esterase. Carboxylic ester hydrolases are among the most important biocatalysts in the field of biotechnology because of their capacity to catalyze hydrolysis with exquisite enantio-, regio-, and stereospecificity. We previously identified EH3, which has remarkable multi-specificity, with sequence positions that modulate both substrate ambiguity and chiral specificity. Here, combined analyses of specificity through evolutionary trace, structure determination and mutagenesis reveal that substrate ambiguity and chiral specificity in a single hydrolase can be modulated by a single residue. In this way, it is feasible to engineer prominent substrate-promiscuous yet stereospecific hydrolases that are relevant to the field of organic synthesis. The work is a collaborative study with the ICP (CSIC), with the participation  of scientist from USA and UK.
Computational and Structural Biotechnology Journal (2021)  (doi: 10.1016/j.csbj.2021.04.041)

EstD11. Click on it to get a larger image
Collaboration between IQFR scientists, CBM Severo Ochoa center and the Vigo and A Coruña Universities, together with the synthetic chemical company Galchimia, reveals unprecedent characteristics in a thermophile esterase, obtained from a hot spring metagenomic library.
Esterases and lipases count with keen industrial interest due to its capacity to accept a wide range of non-natural substrates and stability in organic solvents, and, in the case of extremophile organisms, its ability to tolerate extreme temperatures and pH. In this work, published in the Computational and Structural Biotechnology Journal, a novel esterase, EstD11, has been biochemically and structurally characterized. The tridimensional structure of EstD11 and the inactive mutant has been determined together with 8 crystallographic complexes with different substrates and products of the esterase reaction, revealing the molecular basis underlying enzyme reactivity and specificity. Interestingly, a unique methionine zipper has been identified lining the active site and cap domains that may be responsible for the thermostability and catalytic promiscuity of the protein. Furthermore, the success of high-temperature crystallization experiments has been key in obtaining singular information on cap dynamics.

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