Institut de Biologie StructuraleGrenoble / France

Contact person(s) related to this article / ROYANT Antoine

IBS-GSY webpage

Group Leader : Antoine Royant


The Synchrotron Group of the IBS develops and operates X-ray-crystallography-related platforms, all located at the ESRF. Besides, we tackle a number of structural biology projects benefiting from these instruments.

Team members

IBS location

  • Franck Borel (CEA scientist)
  • Martin Byrdin (CEA scientist, 30%)
  • David Cobessi (CNRS scientist)
  • Jérôme Dupuy (UGA assistant professor)
  • Antoine Royant (CNRS scientist)
  • Monika Spano (UGA assistant professor)
  • Caroline Barathon (UGA administrative officer)
  • Lucia d’Auria (PhD student)
  • Soumiya Muthukumar (PhD student)

ESRF location

  • Antoine Royant (CNRS scientist)
  • Eric Mathieu (CNRS engineer)
  • Yoann Sallaz-Damaz (CNRS engineer)
  • Christophe Berzin (CEA technician)
  • Pascale Israel-Gouy (CEA technician)
  • Philippe Jacquet (CNRS technician)
  • Sylvain Engilberge (ESRF postdoc)
  • Nicolas Caramello (PhD student)
  • Sofia Jaho (PhD student)
  • Elham Vahdatar (PhD student)


The macromolecular crystallography beamline BM07-FIP2 (contact : Eric Mathieu, Yoann Sallaz-Damaz & Antoine Royant)

The BM30A-FIP beamline was operated between 1999 and 2018 to serve the French and international macromolecular crystallography user community. Built for the MAD phasing technique, FIP has constantly been at the forefront of technological developments in automation based on the use of robotics, most notably for in situ diffraction. In the context of the EBS upgrade of the ESRF, the beamline has been transferred onto port BM07 of the storage ring and will open to users in 2021 as BM07-FIP2. Link to the FIP2 website

- WIFIP : a web-based user interface for automated synchrotron beamlines. Sallaz-Damaz Y, Ferrer J-L (2017) J. Synchrotron Radiat. 24, 1105-1111

- Combining ’dry’ co-crystallization and in situ diffraction to facilitate ligand screening by X-ray crystallography. Gelin M, Delfosse V, Allemand F, Hoh F, Sallaz-Damaz Y, Pirocchi M, Bourguet W, Ferrer J-L, Labesse G, Guichou JF (2015) Acta Crystallogr. D71, 1777-1787.

- A geometrical approach for semi-automated crystal centering and in situ X-ray diffraction data collection. Heidari Khajepour MY, Lebrette H, Vernede X, Rogues P, Ferrer J-L (2013) J. Appl. Crystallogr. 46, 740-745

The in crystallo optical spectroscopy laboratory icOS (contact : Antoine Royant)

The icOS Lab (formerly known as the Cryobench) is a facility dedicated to performing various complementary optical spectroscopic analyses on protein crystals under conditions (mini-diffractometer with an on-axis camera, sample changer, 100 K or room temperature) similar to those used during diffraction data collection on the Structural Biology Group beamlines of the ESRF. Available spectroscopic setups include UV-visible absorption, emission fluorescence and Raman. Lasers can also be used as actinic light sources to induce photochemical changes within samples. Samples are usually crystals, but nanolitre volumes of solution can be used. The icOS Lab is often used as an ‘offline’ facility (i.e. experiments are not performed at the same time as the corresponding X-ray diffraction experiments). However, an ‘online’ mode of operation allows the interleaved measurement of diffraction and spectroscopic data using either a UV/visible microspectrophotometer (‘online microspec’) or a Raman setup (‘online Raman’). As such, the icOS Lab provides complementary information that cab be crucial to the correct interpretation of crystal structures. Applications include identifying the physiological state of the protein (e.g. the oxidation state of metal centres), monitoring the extent of radiation damage, and characterizing reaction intermediates in kinetic crystallography experiments. Link to the icOS website

- Online Raman spectroscopy for structural biology on beamline ID29 of the ESRF. von Stetten D, Giraud, T, Bui S, Steiner RA, Fihman F, de Sanctis D, Royant A. (2017) J. Struct. Biol. 200, 124-127

- In crystallo optical spectroscopy (icOS) as a complementary tool on the macromolecular crystallography beamlines of the ESRF. von Stetten D, Giraud T, Carpentier P, Sever F, Terrien M, Dobias F, Juers D H, Flot D, Mueller-Dieckmann C, Leonard G A, de Sanctis D, Royant A (2015) Acta Crystallogr. D71, 15-26

The crystallisation platforms OptiCrys/MicroCrys (contact : Monika Spano)

The so-called crystallization bench (OptiCrys) is a prototype of an integrated apparatus for the rational optimization of crystal growth by mapping and manipulating temperature–precipitant–concentration phase diagrams. It comprises a flow cell dialysis set-up (typical volume >20 μL) to exchange conditions and control temperature during the experiments. Based on this macro scale instrument, a miniaturized device that allows precise control of the experimental parameters exploiting the advantages of microfluidics such as the control of transport phenomena and the reduced sample volume needed per experiment (typical volume <1 μL) is also available (MicroCrys). The novelty is the use of the dialysis method on a unique versatile microfluidic device, designed to meet the requirements of both on-chip protein crystallization and in situ X-ray serial crystallography. Precipitant concentration is controlled via both platforms (OptiCrys/MicroCrys) for fluid handling and an interactive software interface designed for the preparation of the crystallization solution. Salts, solvents, buffers and other solutions can be premixed and subsequently pumped into the (macro/micro)fluidic system in a fully automated way. Uncontrolled concentration variations observed in traditional crystallization techniques due to random mixing are eliminated. Temperature is controlled within the (macro/micro)fluidic devices via a prototype using external heating through Peltier modules and copper elements. Therefore, both, the homogeneous temperature distribution or temperature gradients, are achieved. Physico-chemical parameters such as temperature, concentration of crystallizing agents and pH can be controlled over time during the crystallization, so that the state of the substance studied moves along a well defined kinetic trajectory in the phase diagram. The dialysis membrane allows for adjusting the mass transfer through the membrane during the crystallization process. As a result, the gradients created can be controlled and affect the size and quality of generated crystals.

- A crystallization apparatus for temperature-controlled flow-cell dialysis with real-time visualization. Junius N, Oksanen E, Terrien M, Berzin C, Ferrer J-L, Budayova-Spano M (2016) J. Applied Crystallogr. 49, 806-813.

- A microfluidic device for both on-chip dialysis protein crystallization and in situ X-ray diffraction. Junius N, Jaho S, Sallaz-Damaz Y, Borel F, Salmon J-B, Budayova-Spano M (2020) Lab Chip 20, 296-310.


Rationalisation of protein crystallization (contact : Monika Spano)

A rational way to find the appropriate conditions to grow crystal samples for bio- crystallography is to explore the crystallization phase diagram allowing the precise control of the parameters affecting the crystal growth process. First, the nucleation is induced at supersaturated conditions close to the solubility boundary between the nucleation and metastable regions. Then, crystal growth is further achieved in the metastable zone – which is the optimal location for slow and ordered crystal expansion – by modulation of specific physical parameters. A thorough knowledge of the phase diagram is vital in any crystallization experiment. The relevance of the selection of the starting position and the kinetic pathway undertaken in controlling most of the final properties of the synthesized crystals has been shown.
The OptiCrys/MicroCrys platforms enable users to benefit from established rational strategies for the optimization of crystal growth using precise in situ control of the temperature and chemical composition of the crystallization solution through dialysis (and microdialysis). Systematic phase diagrams in multidimensional space can be investigated using far less protein material than previously. With the serial approach proposed by developed instruments, a break with the current paradigm of parallel experiments is marked.
Established rational crystallization strategies can be beneficial to provide sufficient scattering volumes for neutron protein crystallography that require large-volume well ordered single crystals as well as to generate homogeneous populations of uniformly sized protein crystals required for use by other advanced serial diffraction techniques.

- Large crystal growth for neutron protein crystallography. Budayova-Spano M, Koruza K, Fisher Z (2020) Methods Enzymol. 634, 21-46.

- Optimization of crystallization of biological macromolecules using dialysis combined with temperature control. Junius N, Vahdatahar E, Oksanen E, Ferrer J-L, Budayova-Spano M (2020) J. Appl. Crystallogr. 53, 686-698.

- Mixing Salts and Poly(ethylene glycol) into Protein Solutions : The Effects of Diffusion across Semipermeable Membranes and of Convection. Apostolopoulou V, Junius N, Sear RP, M. Budayova-Spano (2020) Cryst. Growth Des. 20, 3927-3936.

- Optimization of crystal growth for neutron macromolecular crystallography. E. Vahdatahar E, Junius N, Budayova-Spano M (2021) JoVE (in press)

- Crystallization of proteins on chip by microdialysis for in situ X-ray diffraction studies. Jaho S, Junius N, Borel F, Sallaz-Damaz Y, Salmon J-B, Budayova-Spano M (2021) JoVE (in press)

(Time-resolved) room temperature crystallography (contact : Martin Byrdin & Antoine Royant)

Time-resolved macromolecular crystallography (TR-MX) at synchrotrons has long been limited to the use of the Laue diffraction technique and was only successful for a handful of biological systems. The recent advent of X-ray free-electron lasers has rejuvenated the field by fostering the development of serial crystallography, which consists in composing a complete data set from single diffraction images obtained from tens of thousands of microcrystals passing through a pulsed X-ray beam. The associated development of crystal injection techniques, coupled to the advent of faster, noiseless X- ray detectors, has paved the way for the revival of TR-MX at synchrotrons, starting with the generalisation of room temperature MX. We use and develop methods in TR-MX at synchrotrons and XFELs, including the development of time-resolved spectroscopy at the icOS Lab.

- Ultrafast structural changes within a photosynthetic reaction centre. Dods R, Båth P, Morozov D, Gagnér VA, Arnlund D, Luk HL, Kübel J, Maj M, Vallejos A, Wickstrand C, Bosman R, Beyerlein KR, Nelson G, Liang M, Milathianaki D, Robinson J, Harimoorthy R, Berntsen P, Malmerberg E, Johansson L, Andersson R, Carbajo S, Claesson E, Conrad CE, Dahl P, Hammarin G, Hunter MS, Li C, Lisova S, Royant A, Safari C, Sharma A, Williams GJ, Yefanov O, Westenhoff S, Davidsson J, DePonte DP, Boutet S, Barty A, Katona G, Groenhof G, Brändén G, Neutze R (2021) Nature 589, 310-314, doi : 10.1038/s41586-020-3000-7

- Millisecond time-resolved serial oscillation crystallography of a blue-light photoreceptor at a synchrotron. Aumonier S, Santoni G, Gotthard G, von Stetten D, Leonard GA, Royant A (2020) IUCrJ 7, 728-736

- Specific radiation damage is a lesser concern at room temperature. Gotthard G, Aumonier S, De Sanctis D, Leonard G, von Stetten D, Royant A (2019) IUCrJ 6, 665-680

- A three-dimensional movie of structural changes in bacteriorhodopsin. Nango E, Royant A, Kubo M, Nakane T, Wickstrand C, Kimura T, Tanaka T, Tono K, Song C, Tanaka R, Arima T, Yamashita A, Kobayashi J, Hosaka T, Mizohata E, Nogly P, Sugahara M, Nam D, Nomura T, Shimamura T, Im D, Fujiwara T, Yamanaka Y, Jeon B, Nishizawa T, Oda K, Fukuda M, Andersson R, Båth P, Dods R, Davidsson J, Matsuoka S, Kawatake S, Murata M, Nureki O, Owada S, Kameshima T, Hatsui T, Joti Y, Schertler G, Yabashi M, Bondar AN, Standfuss J, Neutze R, Iwata S (2016) Science 354, 1552-1557

Structural biology research topics

Biochemical and structural studies of the plastid-encoded RNA-polymerase and its associated proteins involved in chloroplast biogenesis (contact : David Cobessi)

The health of our biosphere is maintained by the green linage’s ability to perform photosynthesis within its chloroplasts, which are essential for the cycles of carbon and oxygen. The assembly of the photosynthetic apparatus in plastids requires transcription of the photosynthesis associated plastid genes (PhAPGs) and of photosynthesis associated nuclear genes (PhANGs). Upon perception of the first rays of light, plastids differentiate into chloroplasts with strong coordination of PhAPGs and PhANGs transcription. The PhAPGs are transcribed by the plastidial RNA-polymerase (PEP), a machinery of unknown 3D structure of 1 MDa that contains 4 catalytic chloroplastic rpo subunits, and 12 PEP associated proteins (PAPs) essential for an in vitro activity encoded by the nuclear genome and transported into the plastid. Several PAPs are dually localized into the plastid and nucleus where they could regulate the transcription of PhANGs. In collaboration with LPCV (, using a multi-scale integrated approach we investigate the nuclear and plastid transcription by chloroplast proteins and the 3D structure of the proteins and complexes involved.

- Nucleo-plastidic PAP8/pTAC6 couples chloroplast formation with photomorphogenesis. Liebers M, Gillet FX, Israel A, Pounot K, Chambon L, Chieb M, Chevalier F, Ruedas R, Favier A, Gans P, Boeri Erba E, Cobessi D, Pfannschmidt T, Blanvillain R (2020) EMBO J. 39:e104941.

Inhibition of synthesis of terpenoids to fight antibiotic resistance in human pathogens (contact : Franck Borel)

Terpenoids are key metabolites present in all kingdoms of life. They are produced by condensation of two building blocks : Isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Two pathways produce these building blocks : the mevalonate (MVA) and methylerythritol phosphate (MEP) pathways. The MEP pathway, found in many pathogens, is built on seven consecutive enzymes - namely DXS, IspC, IspD, IspE, IspF, IspG and IspH - the last one producing both IPP and DMAPP. At the opposite, the MVA pathway produces IPP that is transformed into DMAPP using Idi, a specific isomerase. Human uses exclusively the MVA pathway and a type 1 Idi, making the MEP pathway and type 2 Idi (Idi-2) valuable therapeutic bacterial targets, especially for multi-drug resistant (MDR) pathogens.
The goal of the project is the inhibition of the biosynthesis of DMAPP in resistant pathogens that were listed as critical or high priority by WHO. For species depending solely on the MEP pathway to produce DMAPP, we will specifically target the IspD, IspE, and IspF enzymes (steps 3 to 5 of the MEP pathway). To address those that use the MVA pathway (S. aureus, E. faecium and E. faecalis) we will target Idi-2.
The design of specific inhibitors against these enzymes is carried out by combining fragment-based crystallography screening at both room and cryogenic temperatures, and in silico virtual screening supplemented by activity assays.

- Synthesis and Kinetic evaluation of an azido analogue of methylerythritol phospate : a Novel Inhibitor of E. coli YgbP/IspD. Baatarkhuu Z, Chaignon P, Borel F, Ferrer J-L, Wagner A, Seemann M (2018) Sci. Rep. 8:17892.

- Further insight into crystal structures of E. coli IspH/LytB in complex with two potent inhibitors of the MEP pathway : a starting point for rational design of new antimicrobials. Borel F, Barbier E, Krasutsky S, Janthawornpong K, Chaignon P, Dale Poulter C, Ferrer J-L, Seemann M (2017) ChemBioChem 18, 2137-2144.

Structural understanding of fluorescence (or Reactive Oxygen Species generation) efficiency in fluorescent proteins (contact : Jérôme Dupuy & Antoine Royant)

As an application of the development of the icOS Lab, our group has specialized in the coupled structural and spectroscopic characterization of fluorescent proteins of various types (GFP-like, phytochrome-derived) and hues (cyan, green, yellow, red, near-infrared) to decipher the structural determinants controlling fluorescence properties in order to be able to improve them. We are also interested in understanding how certain fluorescent flavoproteins can act as photosensitizers and thus serve in various cell imaging techniques used for functional studies.

- Aequorea’s secrets revealed : New fluorescent proteins with unique properties for bioimaging and biosensing. Lambert GG, Depernet H, Gotthard G, Schultz DT, Navizet I, Lambert T, Adams SR, Torreblanca-Zanca A, Chu M, Bindels DS, Levesque V, Nero Moffatt J, Salih A, Royant A, Shaner NC (2020) PLoS Biol. 18:e3000936

- Tailing miniSOG : structural bases of the complex photophysics of a flavin-binding singlet oxygen photosensitizing protein. Torra J, Lafaye C, Signor L, Aumonier S, Flors C, Shu X, Nonell S, Gotthard G, Royant A (2019) Sci. Rep. 9:2428

- mScarlet : a bright monomeric red fluorescent protein for cellular imaging. Bindels D S, Haarbosch L, van Weeren L, Postma M, Wiese K E, Mastop M, Aumonier S, Gotthard G, Royant A, Hink M & Gadella T W Jr (2017) Nat. Methods 14, 53-56


A list of all publications since the year 2014 can be consulted here