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Protein Crystallography

MEMBERS

  • Alejandro Buschiazzo, PhD (Head)
  • Joaquín Dalla Rizza (Technician)
  • Nicole Larrieux (Technician)
  • Felipe Trajtenberg, PhD (Research Scientist)

Our group has a double mission:

  1. Perform scientific research (for details see Laboratory of Molecular & Structural Microbiology)
  2. Maintain a state-of-the-art core facility to perform protein crystallography studies
  1. Our research lines tackle molecular studies of key proteins in cell regulation and pathogenicity of microorganisms.We focus on unravelling the molecular structure and biologic function of proteins involved in signal transduction processes and response regulation, with special attention on human and animal pathogens. The structure/function relationship of these macromolecular species not only allows to understand the biological processes, but also opens the way to molecular intervention strategies (protein engineering, design/optimization of vaccines). We study different models, in particular bacterial (Bacillus, Leptospira).
  2. The purpose of the Protein Crystallography Facility (PXF) is to provide equipment, training, assistance, and technological innovations for determining three-dimensional structures of protein and other macromolecules and macromolecular assemblies.X-ray crystallography is one of the most powerful techniques to study the three-dimensional structures of macromolecules and it has transformed our understanding of biological processes. This facility allows users to crystallize macromolecules and solve their 3D structures by X ray diffraction.

MAIN EQUIPMENT

Crystallization robot – Honeybee963®

The Honeybee963® (Digilab) robot is a bench-top system for the automation and miniaturization of vapor diffusion in sitting-drop protein crystallization experiments. Proprietary Cartesian synQUAD® dispensers couple high-speed micro-solenoid valves with high resolution syringe pumps, dispensing volumes down to 100 nL. We currently use 200-300 nL nanodrops to maximize precision and crystallizability.
The 96-needle arm allows for very fast dispensing of reservoir solutions on a 96-well setup. Three independent protein synQUAD® needles then proceed to dispense up to three different proteins, variable volumes are defined using the robot’s software. Automation enables the assay of typically hundreds of different potential crystallogenesis conditions in a matter of minutes, allowing to greatly increase the search space, a known requisite to raising the probability of finding hits.

Screen Production and Optimization robot – Alchemist DT®

The Alchemist DT ® (Rigaku) is a bench-top liquid handling robot for the screen production and optimization of crystallization conditions. It provides consistent, precise and accurate liquid dispensing in a volume range of 1 μl to 10 ml into SBS, Linbro ® and Nextal® footprint plates
Due to its technology, elimination of tubing means no waste and removes the possibility of cross-contamination.
CrystalTrak™, the integrated software package, is designed specifically for protein crystallography. Once the screen is designed, CrystalTrak™ automatically calculates the recipe and defines the necessary stock solutions for use with the Alchemist. 26 different stock solutions can be stored on the deck at one time. Stock management tools and barcode tracking ensure that the correct stock solutions and necessary volumes of solutions are available on the deck before any plate generation begins.

X ray generator – Rigaku MicroMax-007HF®

Micromax007-HF® (Rigaku) is an X-ray generator with a 0.07 mm diameter effective focal size at the source. Equipped with a Cu rotating anode, it provides an output of 1200 W and a brightness of 31 kW/mm2. In combination with the installed optics (Varimax-HF®, Rigaku) which consists of confocal multilayer mirrors, the resulting X rays focused on the crystalline sample are ultra-bright, and can be used effectively for various measurement purposes. We can solve structures with atoms that scatter anomalously at 1.5418Å wavelength (S, I, Cs, lanthanides, among the most used). Molecular replacement problems can be tackled, as well as data collection for high-resolution refinement (including ligands, inhibitors, drugs, point-mutation protein variants, etc).

Image plate area detector – MAR345®

The MAR345® (Mar Research) detector installed on a MAR345dtb®table is an  image plate detector that enables us to collect data up to 1.2Å resolution on our geometric setup (taking full advantage of the 2θ angle). It is a single Φ-angle oscillation setup, equipped with a convenient χ-motor that facilitates crystal mounting under cryogenic conditions. Read-out cycles range from 108 to 34 seconds, depending on pixel size and effectively scanned plate diameter.
The read-out system of the Mar345 is unique in its use of a single high performance 85mW laser which delivers more than 0.8 µJ/pixel at the plate. This ensures that an extremely high percentage (>95%) of trapped F-centers are transformed into photostimulated luminiscence.

X ray cryosystem – 700 series Cryostream®

The Cryostream® (Oxford Cryosystems) allows a continuous laminar flow of gas nitrogen at cryogenic temperatures during single crystal data collection. Fast cool-down to 100 Kelvin is achievable in 20 minutes. It has a fairly low liquid nitrogen consumption with a variable flow from 5 to 10 litre/minute.
We have an accessory auto-fill system that uses a level probe in the cryosystem’s Dewar, that automates the topping-up of the Dewar basically during data collections that last for several hours/days.

Liquid nitrogen generator – LN40®

The LN40® (Rigaku) is a helium compressor-based machine able to produce up to 40lts/day of highly pure (>98%). liquid nitrogen. The gas input comes from the air pumped by the included PSA (Pressure Swing Adsorption) system.

The operation of the cryogenic refrigeration system is based on a closed-loop Gifford-McMahon (GM) helium expansion cycle. The PSA system consists of two basic components: a vessel(s) containing “carbon molecular sieve“(CMS) and an air compressor(s) or source of clean dry air.
services

SERVICES

The Protein Crystallography Facility (PXF) is up and running, with a sustained number of users and trainees, from Argentina and Uruguay.

The web page is kept up to date, and for the last 8 years now, our facility has become fully operational to receive and process all the users’ requests (mainly from IPMontevideo, from the Uruguayan community and from Argentina). The web page informs on the detailed specifics of the available equipment and ways of using the platform.

Experimental approaches currently available for users

  • Protein crystallization screenings (manual and robotic [Honeybee963® 96-well robot)
  • Follow-up and optimization of initial crystallization hits (manually and robot-assisted with an Alchemist® instrument)
  • X ray Diffraction – Testing & Crystal Characterization
  • X ray Diffraction – single crystal data collection
  • Crystal Structure Determination & Refinement

 

To access the facility users should write to pxf@pasteur.edu.uy making a short request. A form will be provided, which includes information about the project and specifications on the macromolecule and other details. This will be evaluated by the Facility staff in terms of technical feasibility and scientific relevance. All requests will have a due response.

It was X ray diffraction that provided the first clues to the structure of the DNA double helix 60 years ago, giving profound insights into how DNA is replicated. Much more recently, the whole ribosome, a huge RNA-protein assembly, has been solved at atomic resolution. Prokaryotic and eukaryotic whole ribosome structures have neatly revealed molecular details of the protein synthesis mechanism, translation inhibition by certain antibiotics, and even how certain bacteria develop resistance to these compounds.

It is now very clear that the knowledge of molecular structure frequently discloses the mechanism of action of biological molecules, eventually establishing a rational framework for protein engineering or the design of specific inhibitors and drugs.

The PXF, given its own experimental approach, is intimately linked to basic and applied research goals. We are strongly convinced that technology is most fruitful in the context of a stimulating scientific environment. The very concept of facility is being built, wherein we believe the facility shall be integrated and contributing intra- and extra-mural scientific research projects.

The PXF is potentially open both for users from the IP Montevideo, as well as for external users from Uruguay and the region. In all these scenarios, a great flexibility is pursued, in order to agree on a case-by-case basis on the most convenient way to use the facility, both in terms of using the available instruments as well as with regards to technical support from our staff. The level of support ultimately provided by the Unit’s personnel will thus depend on the users’ autonomy.

PXF is not a provider of services, as the experimental approach cannot be thought as a routine technical procedure, each protocol heavily depending on each project’s specificities. Since there is no national or regional program to support the use of facilities, eventual financial support will be arranged with the users, in order to cover running costs and according to each project’s particulars.

Finally, it is important to highlight that the PXF works in tight coordination with the Recombinant Proteins and the Protein Biophysics facilities, expanding the capabilities to approach any given project.

 

AVAILABLE APPROACHES FOR THE USE OF THE FACILITY

Two sets of approaches are available: the first includes crystallogenesis screenings, optimization of initial hits, preliminary X ray diffraction tests and full data collection. The second goes further beyond, including analyses involving diffraction data processing, structure determination and refinement, and potentially, preliminary structural analyses.

The first set will be available on a freely accessible facility mode, in the event that the user is autonomous In all cases, the PXF staff will guarantee the correct operation of equipment, and technical advice for each and every step. Running heavy equipment (crystallization and optimization robots, X ray diffraction) will always be supervised by our personnel.

Fees to cover costs (reagents, plastic consumables, use of diffraction, etc.) will always be agreed upon with the users, according to the outline of the project and the users’ preferences for crystallization assays. These assays may include:

  • Set-up of crystallization screens, both manually or robot-assisted, using commercial or customized conditions.
  • Crystal optimization (to improve crystal size and/or quality), both manual or robotic
  • Preliminary X ray diffraction tests to determine cell parameters, point group and overall crystal quality.
  • Full data set collection with our home X ray source, in the case X ray diffraction-quality crystals are obtained; if synchrotron radiation is judged necessary, the facility can assess the user to access appropriate facilities (direct contact with biology lines in LNLS, Campinas; Soleil, Paris; ALS, Berkeley; NSLS, Brookhaven).

The full approach to structure determination and analisys, owing to its higher demand, both in time as well as in technical assistance, will be discussed for each project with the users to agree in terms of costs, eventual scientific collaboration perspectives and timing schedules. These approaches include:

  • Data processing using XDS (and AutoProc), MOSFLM.
  • Structure determination using MR, MIR, MAD, SAD techniques and software such as BnP, SHARP, CCP4, CNS, SHELX, and PHENIX.
  • Structure refinement and fitting using  REFMAC5, PHENIX, BUSTER/TNT, CCP4, COOT.
  • Structure visualization and analysis using PYMOL, COOT, CCP4.

EDUCATION-COURSES

  • Organization of several courses and workshops :

    • Course “Macromolecular Crystallography: introduction and applications” – 2010 (Institut Pasteur de Montevideo)
    • School “Macromolecular Crystallography School – From data processing to structure refinement and beyond”. Course co-organized with CCP4 (UK), with annual periodicity since 2013, foundational year when the workshop took place at the Institut Pasteur de Montevideo. Every other year (2014, 2016) the course was held at the Instituto de Fisica de Sao Carlos (Univ de Sao Paulo, Sao Carlos, Brazil), alternating with the Inst Pasteur de Montevideo venue (in 2015, next edition planned for 2017).
    • Hands-on workshop: “Isolation of Leptospira spp. strains from field cases of bovine leptospirosis”. 2014 (Institut Pasteur de Montevideo, INIA (estacion La Estanzuela); Universidad de la Republica, Instituto de Higiene, Fac de Medicina; and DILAVE (Min de Ganaderia, Agricutlura y Pesca). With participation of invited professors from Massey University (New Zealand).
    • Workshop “Modern Approaches in Drug Discovery for Neglected Infectious Diseases”. 2014 Institut Pasteur de Montevideo.
    • Hands-on workshop: “Integrative methods in Structural Biology to enhance high impact research in health and disease” 2016 Institut Pasteur de Montevideo, co-organized with the University of Oxford and the European Structural Biology network Instruct. 

    Training and supervision of students :

    • 3 undergraduate students

    • 3 MSc students 

    • 3 PhD students

    • 3 postdoctoral fellows

    • Training of >10 students and interns from Uruguay and abroad

PUBLICATIONS

(last 5 years)

Trajtenberg FImelio JA, Machado MR, Larrieux N, Marti MA, Obal G, Mechaly AEBuschiazzo A. Regulation of signaling directionality revealed by 3D snapshots of a kinase:regulator complex in action. Elife. 2016 Dec 12;5. pii: e21422. doi: 10.7554/eLife.21422.

Morán-Barrio J*, Lisa MN*Larrieux N, Drusin SI, Viale AM, Moreno DM, Buschiazzo A¶, Vila AJ¶. Crystal structure of the metallo-β-lactamase GOB in the periplasmic dizinc form reveals an unusual metal site. Antimicrob Agents Chemother. 2016 Sep 23;60(10):6013-22. doi: 10.1128/AAC.01067-16. *[These authors contributed equally to this work]   ¶[Corresponding authors]

Wunder EA, Figueira CP, Benaroudj N, Hu B, Tong BA, Trajtenberg F, Liu J, Reis MG, Charon NW, Buschiazzo A, Picardeau M, Ko AI. A novel flagellar sheath protein, FcpA, determines filament coiling, translational motility and virulence for the Leptospira spirochete. Mol Microbiol. 2016 Aug;101(3):457-70. doi: 10.1111/mmi.13403.

Meyer PA, Socias S, Key J, Ransey E, Tjon EC, Buschiazzo A, et al. Data publication with the structural biology data grid supports live analysis. Nat Commun. 2016 Mar 7;7:10882. doi: 10.1038/ncomms10882

Fouts DE, Matthias MA, Adhikarla H, Adler B, Amorim-Santos L, Berg DE, Bulach D, Buschiazzo A, Chang YF, Galloway RL, Haake DA, Haft DH, Hartskeerl R, Ko AI, Levett PN, Matsunaga J, Mechaly AE, Monk JM, Nascimento AL, Nelson KE, Palsson B, Peacock SJ, Picardeau M, Ricaldi JN, Thaipandungpanit J, Wunder EA Jr, Yang XF, Zhang JJ, Vinetz JM. What Makes a Bacterial Species Pathogenic?:Comparative Genomic Analysis of the Genus Leptospira. PLoS Negl Trop Dis. 2016 Feb 18;10(2):e0004403.

East A, Mechaly AE, Huysmans GH, Bernarde C, Tello-Manigne D, Nadeau N, Pugsley AP, Buschiazzo A, Alzari PM, Bond PJ, Francetic O. Structural Basis of Pullulanase Membrane Binding and Secretion Revealed by X-Ray Crystallography, Molecular Dynamics and Biochemical Analysis. Structure. 2016 Jan 5;24(1):92-104.

Saita E, Abriata LA, Tsai YT, Trajtenberg F, Lemmin T, Buschiazzo A, Dal Peraro M, de Mendoza D, Albanesi D. A coiled coil switch mediates cold sensing by the thermosensory protein DesK. Mol Microbiol. 2015 Oct;98(2):258-71.

Obal G*, Trajtenberg F*, Carrión F, Tomé L, Larrieux N, Zhang X, Pritsch O¶, Buschiazzo A¶. Conformational plasticity of a native retroviral capsid revealed by x-ray crystallography. Science. 2015 Jul 3;349::95-8. *[These authors contributed equally to this work] ¶[Corresponding authors]

Methot SP, Litzler LC, Trajtenberg F, Zahn A, Robert F, Pelletier J, Buschiazzo A, Magor BG, Di Noia JM. Consecutive interactions with HSP90 and eEF1A underlie a functional maturation and storage pathway of AID in the cytoplasm. J Exp Med. 2015 Apr 6;212(4):581-96.

Trajtenberg F, Albanesi D, Ruétalo N, Botti H, Mechaly AE, Nieves M, Aguilar PS, Cybulski L, Larrieux N, Mendoza D, Buschiazzo A. Allosteric activation of bacterial response regulators: the role of the cognate histidine kinase beyond phosphorylation. (2014) mBio 5:e02105-14. 

Morero NRBotti H, Nitta KR, Carrión F, Obal G, Picardeau M, Buschiazzo A. HemR is an OmpR/PhoB-like response regulator from Leptospira, which simultaneously effects transcriptional activation and repression of key haem metabolism genes. Mol Microbiol. 2014 Oct;94:340-52.

Trajtenberg F*, Altabe S*, Larrieux N, Ficarra F, de Mendoza D, Buschiazzo A, Schujman GE. Structural insights into bacterial resistance to cerulenin. FEBS J. 2014 May;281(10):2324-38. *[These authors contributed equally to this work] 

Martinez A, Peluffo G, Petruk AA, Hugo M, Piñeyro D, Demicheli V, Moreno DM, Lima A, Batthyány C, Durán R, Robello C, Martí MA, Larrieux N, Buschiazzo A, Trujillo M, Radi R, Piacenza L. Structural and molecular basis of the peroxynitrite-mediated nitration and inactivation of Trypanosoma cruzi iron-superoxide dismutases (Fe-SODs) A and B: disparate susceptibilities due to the repair of Tyr35 radical by Cys83 in Fe-SODB through intramolecular electron transfer. J Biol Chem. 2014 May 2;289(18):12760-78.

Correa A*, Trajtenberg F*, Obal G, Pritsch O, Dighiero G, Oppezzo P, Buschiazzo A. Structure of a human IgA1 Fab fragment at 1.55 Å resolution: potential effect of the constant domains on antigen-affinity modulation. Acta Crystallogr D Biol Crystallogr. 2013 Mar;69(Pt 3):388-97. *[These authors contributed equally to this work] 

Albanesi D, Reh G, Guerin ME, Schaeffer F, Debarbouille M, Buschiazzo A, Schujman GE, de Mendoza D, Alzari PM. Structural basis for feed-forward transcriptional regulation of membrane lipid homeostasis in Staphylococcus aureus. PLoS Pathog. 2013 Jan;9(1):e1003108. 

Horjales S, Schmidt-Arras D, Limardo RR, Leclercq O, Obal G, Prina E, Turjanski AG, Späth GF, Buschiazzo A. The crystal structure of the MAP kinase LmaMPK10 from Leishmania major reveals parasite-specific features and regulatory mechanisms. Structure. 2012 Oct 10;20(10):1649-60. 

Buschiazzo A*, Muiá R, Larrieux N, Pitcovsky T, Mucci J, Campetella O*. Trypanosoma cruzi trans-sialidase in complex with a neutralizing antibody: structure/function studies towards the rational design of inhibitors. PLoS Pathog. 2012 Jan;8(1):e1002474. *[Corresponding authors]. 

CONTACT

pxf@pasteur.edu.uy