Institut de Biologie StructuraleGrenoble / France

Contact person(s) related to this article / BYRDIN Martin


Serial nanocrystallography

(PI Colletier)

Research summery
The SNaX team focuses on serial nano-crystallography, with aim to develop expertise pertaining to all of its aspects, from nanocrystal production and evaluation, to sample presentation, data collection, data processing and fine structural analysis. Our long term goal is to establish recombinant in vivo crystallization as a new method to obtain nanocrystals of proteins — some elusive to in vitro crystallisation — with view to perform static and time-resolved serial crystallography experiments at XFELs and at 3rd and 4th generation synchrotrons.

Jacques-Philippe Colletier (researcher, CNRS – DR2)
Ninon Zala (technician, CEA, 70%)
Guillaume Tetreau (postdoc)
Julie Lopes (PhD student)
Elena Andreeva (PhD student)

Manfred Burghammer (ESRF, Grenoble, France)
Marco Cammarata (Rennes University, France)
Nicolas Coquelle (ILL, Grenoble, France)
David Eisenberg (UC Los Angeles, USA)
Brian Federici (UC Riverside, USA)
Stanislas Gobec (University of Ljubljana, Slovenia)
Diana Kirilovsky (IB2C, Paris, France)
Meytal Landau (Israel Institute of Technology (Technion), Haifa Israel)
Pierre-Yves Renard (University Rouen, France)
Michael Sawaya (UC Los Angeles, USA)
Ilme Schlichting (MPI, Heidelberg, Germany)
Israel Silman (Weizmann Institute, Israel)
Joel Sussman (Weizmann Institute, Israel)
Michel Sliwa (LASIR, Lille, France)
Mathias Winterhalter (Jacobs University, Bremen, Germany)

Research activity
The advent of X-ray free electron lasers (XFELs) has revolutionized structural biology, opening doors to macromolecular structure determination from nanocrystals and to time-resolved experiments on ultra-fast time-scales (fs-ps). The SNaX team aims at developing an in-vivo crystallization method to obtain nanocrystals of proteins with view to perform static and time-resolved serial crystallography experiments at XFELs and at 3rd and 4th generation synchrotrons. Nanometer sized crystals are indeed perfectly adapted for time-resolved studies on light-driven proteins. Our strategy is to use nature-evolved crystalliferous bacterium Bacillus thuringiensis (Bt) as a crystallisation vessel, and its naturally crystalline Cry/Cyt proteins as crystallisation chaperones, respectively. We focus on two cargo proteins, which differ in function, stability and proneness to crystallise namely : i) the fluorescent protein rsFolder, designed in previous work to promptly mature and stably fold, and to be crystallization prone (El-Khatib et al. 2016, Sci Rep) ; ii) the orange carotenoid protein (OCP), a two-domain protein encasing a ketocarotenoid pigment and whose function is to enable non-photochemical quenching in cyanobacteria, protecting them against light-induced damage under strong irradiance.

Our motivation to obtain nanocrystals of OCP is thus two-fold. First, a success with the in vivo crystallisation of OCP would demonstrate the reaches of recombinant nanocrystallization in Bt, establishing that not only an exogenous protein – such as BinAB – but also its pigment can be recombinantly co-produced and co-crystallised. Our long-term goal is indeed to establish in vivo nanocrystallisation as a method of choice for investigators seeking to produce high amount of nanocrystalline material (e.g. TR-SFX), or to solve structures of fragile protein and protein-complexes which — alike BinAB — do not easily crystallize and/or degrade following extraction from the cell. Second, the SNaX team works in collaboration with the groups of Diana Kirilovsky (IB2C, Saclay), Michel Sliwa (Lasir, Lilles) and Ilme Schlichting (Max Planck Institute for Biomedical Research, Heidelberg, Germany) towards developing more active OCP variants to be used as regulators of light uptake in artificial photosynthetic systems.

Key publications

Nass Kovacs G, Colletier JP, Grünbein ML, Yang Y, Stensitzki T, Batyuk A, Carbajo S, Doak RB, Ehrenberg D, Foucar L, Gasper R, Gorel A, Hilpert M, Kloos M, Koglin JE, Reinstein J, Roome CM, Schlesinger R, Seaberg M, Shoeman RL, Stricker M, Boutet S, Haacke S, Heberle J, Heyne K, Domratcheva T1, Barends TRM & Schlichting I (2019) Three-dimensional view of ultrafast dynamics in photoexcited bacteriorhodopsin Nat. Comm. 10 : 3177

El-Khatib M, Nasrallah C, Lopes J, Tran QT, Tetreau G, Basbous H, Fenel D, Gallet B, Lethier M, Bolla JM, Pages JM, Vivaudou M, Weik M, Winterhalter M, Colletier JP (2018) Porin self-association enables cell-to-cell contact in Providencia stuartii floating communities. Proc Natl Acad Sci U S A 115 : E2220-E2228

Tayeb-Fligelman E, Tabachnikov O, Moshe M, Goldshmidt-Tran O, Sawaya MR, Coquelle N, Colletier J-P, Landau M (2016) The Cytotoxic Staphylococcus aureus PSMα3 Reveals a Novel Cross-alpha Amyloid-like Fibril. Science 355 : 831-833.

Colletier JP, Sawaya MR, Gingery M, Rodriguez JA, Cascio D, Brewster AS, Michels-Clark T, Hice RH, Coquelle N, Boutet S, Williams GJ, Messerschmidt M, DePonte DP, Sierra RG, Laksmono H, Koglin JE, Hunter MS, Park HW, Uervirojnangkoorn M, Bideshi DK, Brunger AT, Federici BA, Sauter NK, Eisenberg DS. (2016) A potent binary mosquito larvicide revealed by de novo phasing with an X-ray free-electron laser. Nature 539 : 43-47.

Coquelle N, Brewster AS, Kapp U, Shilova A, Weinhausen B, Burghammer M, Colletier J-P (2015) Raster-scanning serial protein crystallography using micro- and nano-focused synchrotron beams. Acta Crystallogr D Biol Crystallogr 71 : 1184–1196.

Tetreau G, Banneville AS, Andreeva E, Brewster AS, Hunter M, Sierra R, Teulon JM, Young I, Burke N, Grünewald T, Beaudouin J, Snigireva I, Fernandez-Luna MT, Burt A, Park HW, Signor L, Bafna J, Sadir R, Fenel D, Boeri-Erba E, Bacia M, Zala N, Laporte F, Després L, Weik M, Boutet S, Rosenthal M, Coquelle N, Burghammer M, Cascio D, Sawaya M, Winterhalter M, Gratton E, Gutsche I, Federici B, Pellequer JL, Sauter N, Colletier JP (2020)
Serial femtosecond crystallography on in vivo-grown crystals drives elucidation of mosquitocidal Cyt1Aa bioactivation cascade. Nature Communications, 11 (1), ⟨10.1038/s41467-020-14894-w⟩.

De la Mora E, Coquelle N, Bury C, Rosenthal R, Holton J, Carmichael I, Garman E, Burghammer M, Colletier JP, Weik M (2020)
Radiation damage and dose limits in serial synchrotron crystallography at cryo- and room temperatures. PNAS 117 : 4142-4151.