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Villigen, 6 Sep 2016. The MX group at the Swiss Light Source has just published a paper that describes the best way of collecting macromolecular diffraction data with EIGER. In collaborative work with scientists at DECTRIS, Meitian Wang and colleagues show that measuring with EIGER leads to higher-quality data than with PILATUS, as long as the data are finely enough sliced in phi. Whereas an oscillation angle of 1/2 the XDS mosaicity was recommended for PILATUS, EIGER data benefit from collection at 1/10 the XDS mosaicity. Regardless of phi-slicing, the smaller pixel size of EIGER further increases data quality in the highest resolution shells because less background is measured with weak reflections.
The paper, published in the September 2016 issue of Acta Crystallographica Section D and freely available under a Creative Commons Attribution License, marks another milestone in the progress of macromolecular crystallography. In 2006, PILATUS introduced Hybrid Photon Counting and revolutionized data collection, both in terms of speed and in terms of quality. Now, ten years later, EIGER takes science further yet. Ultrafine phi-slicing provides data of unprecedented quality, collected even more quickly than before. Like those researchers that recently solved structures of Zika virus envelope protein in complex with a neutralizing antibody and of CRISPR-Cpf1 in complex with guide RNA and target DNA from EIGER data, crystallographers will determine structures from ever more challenging crystals and increase our understanding of complex biological processes in health and disease.
Baden, 4 Aug 2016. As the Zika epidemic spreads through the American continent and threatens not only this month's Olympic Games in Rio de Janeiro but also, more insidiously, the health of millions, exceptional efforts go towards finding treatments or prevention of Zika infection. Today, the journal Nature published an article that reports a breakthrough on the way towards a vaccine against Zika (Barba-Spaeth et al.). The results critically depended on X-ray crystallographic data collected at Synchrotron SOLEIL with DECTRIS detectors. For beamline Proxima-2A, it is the first publication with data from their new EIGER X 9M detector, but it is also the first structure in the Protein Data Bank solved with EIGER X 9M data.
DECTRIS is excited that EIGER X 9M premieres as part of a research project of such significance and hopes that the findings are quickly translated into medical solutions. We congratulate Bill Shephard and his team at the beamline as well as the group around Felix Rey at Institut Pasteur, Paris, on this accomplishment.
Zika virus is a flavivirus closely related to the causative agents of dengue and yellow fever and like those, it can be communicated sexually or transmitted by Aedes mosquitoes. The virus is linked to blindness, deafness, seizures and a form of temporary paralysis called Guillain-Barré syndrome, but the majority of infected adults experience no symptoms. Children born to infected mothers, on the other hand, show a high incidence of microcephaly, a birth defect characterized by abnormally small heads and irreversible brain damage. The connection between Zika infection and microcephaly was recently shown experimentally in flies (Cugola et al.) and is now generally accepted to be true in humans as well.
Since its appearance in Brazil a year ago, Zika has radiated outward on the American continent to such an extent that the World Health Organization has declared it a Public Health Emergency of International Concern. The W.H.O. expects the virus to become established in areas ranging from northern Argentina to the southern United States by the end of 2016, infecting millions of people.
There is currently no treatment of Zika infection and no vaccine exists to prevent it. Indeed, the study of Zika, despite the identification of the virus in Africa nearly 70 years ago, is still in its infancy. Only this year and undoubtedly driven by the threat of vast numbers of new cases has structural information of the virus become available. These recent results dramatically advance our understanding of the virus and open possibilities for the treatment and prevention of infection.
To start with, two structures of the intact virus, solved by electron cryo-microscopy (Sirohi et al., Kostyuchenko et al.), gave a first detailed view of the entire virus. Two more papers reported structures of individual protein components of Zika, envelope protein (Dai et al.) and a fragment of non-structural protein 1 (NS1, Song et al.). The first crystal structure revealed the position of a protective antibody and identified a loop in the protein that might serve as an epitope for future therapeutic antibodies, though their development is a long way off. The structure of full-length NS1, recently solved from PILATUS3 6M data at Advanced Photon Source (APS), showed marked differences in epitopes among flaviviruses – with ramifications for vaccine design (Brown et al.).
Crystal structures of two enzymes essential for the Zika virus life cycle lay the foundation for the rational design of antiviral agents. The structure of NS3 helicase was determined independently at Shanghai Synchrotron Radiation Facility, with data collected on a PILATUS3 6M detector, and at APS (Tian et al. and Jain et al.). Helicases from other viruses have already been used as targets for the development of antiviral agents. The structures of NS3 helicase pave the way for similar work in Zika. Of comparable importance is the structure of Zika protease in complex with an inhibitor, solved from data collected with a PILATUS 6M at PETRA III (Lei et al.).
The intense worldwide structural work on Zika reached a provisional conclusion today with a paper published in Nature that reports the structures of Zika virus E protein, the main target of neutralizing antibodies in flaviviruses, in complexes with antibodies cross-reactive against Zika and a close relative, Dengue (Barba-Spaeth et al.). The antibodies were isolated from dengue patients and shown to neutralize Zika virus. Knowledge of the structure of the epitope-antibody interaction raises the prospect of epitope-focused design of a vaccine that is active against both Zika and dengue viruses simultaneously. Of more immediate impact is the suggestion that one of the two antibodies under study could be used for immune prophylaxis in pregnant women at risk of contracting Zika.
This breakthrough is the result of a broad international collaboration. Clinicians, virologists, immunologists and infectious disease specialists from Paris, London and Vienna and as far away as Thailand and Tahiti combined their efforts, but at the heart of the study are the three crystal structures. The data leading to these structures were acquired at Synchrotron SOLEIL with PILATUS 6M and EIGER X 9M detectors.
While PILATUS 6M has been at the heart of X-ray crystallography for nearly ten years, EIGER X 9M makes its debut in the Protein Data Bank (PDB) with the Zika E protein structure. With this, it follows hot on the heels of EIGER X 4M and EIGER X 16M whose first output was released by the PDB in April and May. EIGER detectors are now firmly established as powerful tools for structural biology and important players in the progress of medicine.
In only three years, the PILATUS@SNBL project has come a long way: from an idea over construction and commissioning to a heavily overbooked platform, used by a broad user community . Chemists, physicists and material scientists employ the diffractometer at the BM01A branch in various fields of basic and applied research. This success is not a coincidence.
The SNBL is a split beamline (BM01A and BM01B branch lines) that has been operating for over 20 years. Recently, in the scope of the PILATUS@SNBL project, the two diffractometers of BM01A, equipped with two detectors, were replaced with a flexible goniometer and one PILATUS 2M detector. This combination, supported by a user-friendly software suite for data collection and processing, resulted in a multi-purpose beamline that supports various types of X-ray crystallographic experiments including single crystal measurements, in situ powder diffraction, high pressure and thin film characterization.
The PILATUS 2M detector was chosen for this diffraction platform as the ideal balance between large active area and moderate weight. Its size allows not only flexibility in the movements of the mechanical parts (detector support, kappa goniometer, rotary tables) but also collection of a large angular range in a single exposure. These two parameters are crucial for the operation of any multi-purpose beamline.
The diversity of users and their requirements are supported by a single program, Pylatus, developed for the user-friendly and versatile control of the diffractometer and ancillary equipment. All data obtained by PILATUS are processed by one of the programs that are integrated in the in-house package SNBL-ToolBox. Crysis and Esperanto translate PILATUS single crystal data to suitable input format for Crysalis, and Converter deals with powder data, preparing it for processing in Fit2D. Pylatus and SNBL-ToolBox made small molecule crystallography and diffuse scattering measurements very comfortable at BM01A.
A powerful feature of the SNBL-software support is Bubble, a common project of SNBL and DUBBLE (Dutch-Belgium Beamlines). This is a software project dedicated to online integration of powder diffraction data. The development of Bubble reflects the needs of the ever-growing powder community, which has adopted PILATUS as a fast and convenient tool for sample characterization. The combination of Bubble’s online integration, the speed of PILATUS, and Pylatus’ advanced features has resulted in a much improved quality and range of dynamic experiments. Time-resolved measurements are now easily accessible, and in situ diffraction techniques are routinely combined with Raman, UV or VIS spectroscopy.
Synergistic approaches are often employed in investigations of new functional materials, where collecting a full diffraction pattern in real time or the local environment relies on superior data statistics (Delgado, T. et al. (2015), Hino, S. et al. (2015), Humphries, T.D. et al. (2015)). Gigli and co-workers took this approach one step further and exploited the noise-free performance and high dynamic range of PILATUS for a detailed structural analysis of a zeolite material (Gigli, L. et al. (2014), Gigli, L. et al. (2015)), a difficult task that is usually performed at high-resolution powder beamlines. These results should encourage the powder community to take full advantage of the potential of 2D HPC detectors for their research.
The flexible setup and full user-support have resulted in a wide range and quantity of proposals for the PILATUS@SNBL platform. We congratulate the BM01A staff on their great work and wish them loads of interesting data!
For the full description of the beamline setup the reader is referred to the paper by Vadim Dyadkin et al. A short overview of single-crystal applications at BM01A can be found in the recent article by Dmitry Chernyshov, and for details on in situ and structural analysis studies on powder samples visit the DECTRIS Applications section.
 Dyadkin, V., Pattison, Ph., Dmitriev, V., Chernyshov, D. A new multipurpose diffractometer PILATUS@SNBL J. Synchrotron Rad., 23 (2016), 825.
Baden, 19 May 2016. Over the last six months, the first EIGER detectors have been delivered to various synchrotrons in Europe, Asia and North America. They have smoothly entered user operation and are producing high-quality data. The first structures have now been published by the PDB and the accompanying research papers in high-profile journals.
With more systems on the way to eager customers, DECTRIS has just published a White Paper that focuses on the features and control of EIGER detectors and its performance in biological crystallography. The White Paper can be downloaded using the button to the right.
Baden, 6 May 2016. Targeted cleavage of foreign DNA by CRISPR-Cas plays a key role in adaptive immunity in certain bacteria. The Cas9 endonuclease introduces double-stranded breaks in viral DNA to protect the bacterium from infection. It gets its specificity from short RNA sequences derived from viral DNA obtained during earlier infections.
CRISPR-Cas is currently one of the hottest topics in biology because of its promise as an easily programmed and highly specific genome-editing tool that works in a wide range of target organisms. In contrast to earlier tools like Zn-finger nucleases, CRISPR splits recognition and cleavage in two separate units. Recognition by the nuclease is mediated by RNA sequences that can be tuned to provide exquisite precision at a fraction of the cost of earlier approaches.
The major weakness of Cas9 nuclease is its requirement for two different RNA molecules, a guiding crRNA and a trans-activating crRNA. Last October, a related nuclease that relies on one RNA molecule only was identified in a number of bacterial species. Called Cpf1, it has been subject to frenzied study since then. Now, the first major results have been published.
Last week, a paper in Nature showed that Cpf1 excises crRNAs from CRISPR repeats and can use them as specificity signal to introduce double-stranded breaks in target DNA. This makes it the most minimal CRISPR-Cas system known to date and an exciting prospect for biotechnological and medical applications. In the same issue of Nature, the crystal structure of CRISRP-Cpf1 in complex with crRNA was published - solved from data collected with a PILATUS3 6M detector.
Today, a paper in Cell reported another milestone, the structure of CRISPR-Cpf1 in complex with crRNA and target DNA. The data were collected at Swiss Light Source in February using an EIGER X 16M detector that had been operational for only a few months. Takanori Nakane, a computational crystallographer and one of the coauthors of the paper, processed the data using DIALS, a software package specifically developed for the processing of low-background data from HPC detectors and the first package that can read EIGER data directly. "At first, I encountered several problems", said Nakane, "but the developers swiftly fixed them. DECTRIS was also very helpful and quick with technical information."
The structure was solved by experimental phasing by single-wavelength anomalous dispersion, a method whose success critically depends on the accuracy of the measured intensities. With smaller pixels than PILATUS and more flexibility when it comes to fine-phi slicing, EIGER records data of superior quality. Another advantage of the detector is the high frame rate. "This enabled us to collect datasets from a large number of crystals and choose the best one later", said Hiroshi Nishimasu, another coauthor of the paper.
Nishimasu, Nakane and other members of Osamu Nureki's laboratory at the University of Tokyo are excited that the Japanese synchrotron SPring-8 has installed an EIGER X 9M detector at beamline BL32XU. "Most of our data will be collected on DECTRIS detectors, both for membrane proteins and soluble proteins", concludes Nakane.
DECTRIS is proud that EIGER contributes to a better understanding of CRISPR-Cas. The technology has already been used to attenuate human pathogens and genetically engineer crops and livestock in the laboratory but, says Clemens Schulze-Briese, CSO of DECTRIS, "more structural and functional work is required before the technology can be deployed safely for what is arguably its greatest promise, gene therapy of devastating human diseases." With EIGER detectors being installed at beamlines all over the world, that goal is now within closer reach.
Baden, 22 Apr 2016. The first protein structure solved from data collected with a DECTRIS EIGER detector was released by the PDB this week. The crystal structure of PsoE (PDB code 5fhi), an enzyme in the biosynthetic pathway of pseurotin, a fungal secondary metabolite with wide-ranging biological activities of medicinal importance, was solved from data collected at beamline BL-1A of Photon Factory, an endstation designed for low-energy experiments. The goniometer and two EIGER X 4M detectors are housed in a helium-filled enclosure to avoid errors due to absorption and scattering by air. The structure is part of a paper published in Angew Chem Int Ed Engl.
Baden, 2 Feb 2016. X-ray crystallography has long been the most potent method for obtaining high-resolution structural information on proteins, nucleic acids and their complexes. In 2014, 9628 entries were added to the Protein Data Bank (PDB) to bring the total count of released structures above 100,000. In 2015 this success continued, with again more than 9000 structures released throughout the year. But it was not just the quantity of the structures. The increasing quality is something to celebrate as well.
In 2015, 148 papers in the three journals with the broadest scientific reach (Cell, Science and Nature) presented results obtained with contributions from macromolecular x-ray crystallography. These papers contained 440 crystal structures of biologically important systems, from antibodies against human pathogenic viruses (Hashiguchi et al., Impagliazzo et al., Robinson et al., Rouvinski et al., Scharf et al., Wu et al., Zhou et al.) to important trans-membrane transporters (Arakawa et al., Deng et al., Kato et al., Leung et al., Lin et al., Moser von Filseck et al., Nomura et al., Perez et al., Tao et al., Wang et al.) and proteins involved in membrane fusion (Diao et al., Rostislavleva et al., Zhou et al.). We would like to congratulate all involved researchers on their achievements.
At DECTRIS, we are proud that data from PILATUS detectors is over-represented among the highest-impact research compared to the PDB overall. Whereas 23% of the coordinate sets released in 2015 were obtained from data collected on PILATUS detectors, 37% of PDB entries published in Cell, Science and Nature were based on PILATUS data. Forty-seven of the structures reported last year in these three high-impact journals were solved by experimental phasing. In this subset where the highest data quality is essential, the share of structures solved from PILATUS data is nearly 43%. Our customers know that better detectors will help them obtain better data.
It is thus not surprising that we ship increasing numbers of PILATUS3 and now also its successor, EIGER, to beamlines at synchrotrons worldwide. In 2015, we installed a PILATUS3 S 2M at beamline 14-2 of Bessy II in Berlin, Germany, and a PILATUS3 S 6M at sector 5 of ALS in Berkeley, California. We also delivered one PILATUS3 X 6M detector each to SBC-CAT at APS in Argonne, Illinois, and to beamline 11C of PLS in Pohang, Korea, but the big news for crystallographers was the arrival of EIGER at the first synchrotron facilities.
In May, two EIGER X 4M detectors were installed at beamline BL-1A of Photon Factory in Tsukuba, Japan, in a V-shaped conformation inside a helium-filled chamber. This unusual setup will facilitate the collection of high-quality anomalous diffraction data at x-ray energies as low as 3.7 keV. The first EIGER X 16M, our new flagship detector, entered service at beamline X06SA of SLS in Villigen, Switzerland, in October.
In the two months that remained of the year, an EIGER X 16M was installed at beamline FMX of NSLS II in Brookhaven, New York, an EIGER X 9M at beamline Proxima-2A of Soleil in Saclay, France, and another EIGER X 9M at LS-CAT at APS. The EIGER X 9M at Proxima-2A produced data to solve the first novel protein structure less than a month after arrival of the detector onsite.
The future is bright for structural biology. Cryo-electron microscopy has just been named Method of the Year by the editors of the Nature journals. Electron microscopy and crystallography will be increasingly synergistic in resolving challenging targets, but crystallography is still growing and will dominate structural biology for most biological systems for the foreseeable future.
Villigen, November 2015: The macromolecular crystallography beamline X06SA at the Swiss Light Source (SLS), a synchrotron operated by Paul Scherrer Institute (PSI), is the first one in the world to upgrade its detector to an EIGER X 16M. On 14 Oct 2015, the new detector entered service, and it is already a success. Meitian Wang, group leader and beamline responsible, is delighted with the new detector's stability. "Since its installation last month, the EIGER has operated without any problems", he said.
The EIGER X 16M succeeds a PILATUS 6M, an instrument that has led to the determination of many important protein structures and contributed to the reputation of beamline X06SA as one of the world's best. In addition to the technical specs, Wang is also excited about is the application programming interface that simplifies detector operation. The transition from the camserver of the PILATUS to the new API was a happy one.
Firmware version 1.5 delivered with the EIGER represents a very stable and high-performance release. "The system installed at SLS exceeds the specifications that we promised during the Synchrotron Radiation Instrumentation conference in July 2015", said Clemens Schulze-Briese, CSO of DECTRIS.
In tests before EIGER went online, Wang and colleagues collected data from crystals whose longest unit cell dimension exceeded 1000 Å. Thanks to EIGER's pixel size of only (75 µm)2, data could have been collected to well beyond the resolution limit of the crystal of 3 Å. More relevant for regular users will be the continuous read-out feature, which ensures duty cycles of above 99% even when the detector runs at full speed and makes breathtakingly fast experiments possible. In separate tests, Wang was able to successfully collect an entire S-SAD dataset within one second.
Among the first users of the EIGER X 16M was Hauke Hillen, a PhD student from the Max Planck Institute for Biophysical Chemistry in Göttingen. On a recent afternoon, he was busy collecting data for experimental phasing of a crystal structure based on the sulfur signal. Between datasets, he stopped for a quick chat: "I'm very impressed with the EIGER", he said. "It's so fast." With this, the conversation was over, as he had to rush back to his experiment.
The high frame rate and large number of pixels of the EIGER X 16M lead to unprecedented amounts of raw data. To make online data processing, speedy transfer to the users' home labs and reliable archiving possible, bit-shuffle filtered LZ4 compression was introduced. This compression strategy is not only fast enough to keep up with the generated data but also more efficient than CBF compression. Vincent Olieric, beamline scientist at the PSI, was impressed that the new detector does not generate much more data on disk than the previous PILATUS 6M. "It's great for users", he said.
The development of the EIGER X 16M now operating at SLS was accelerated by a grant by the Swiss Commission for Technology and Innovation (CTI) to promote collaboration between academia and business. The detector could thus be tested under realistic experimental conditions from an early stage. Moving from prototype to final product, DECTRIS engineers and scientists have developed an instrument that meets the needs of the most exacting X-ray crystallographers. Eventually, all EIGER customers will benefit from that.
About DECTRIS Ltd.
DECTRIS Ltd. is the technologically leading company in Hybrid Photon Counting X-ray detection. DECTRIS Hybrid Photon Counting detectors have transformed basic research at synchrotron light sources, as well as in the laboratory and with industrial X-ray applications. DECTRIS aims to continuously improve the measurement quality, thereby enabling new scientific findings. A broad range of products is based on this pioneering technology; all scaled to meet the needs of various applications. DECTRIS also develops bespoke solutions for scientific and industrial X-ray detection.
The Paul Scherrer Institute (PSI) develops, builds and operates large, complex research facilities and makes them available to the Swiss and international research community. The institute's own key research priorities are in the fields of matter and materials, energy and environment and human health. PSI is committed to the training of future generations. Therefore about one quarter of the staff are post-docs, post-graduates or apprentices. Altogether PSI employs 1900 people, thus being the largest research institute in Switzerland. The annual budget amounts to approximately CHF 350 million.EIGER X 16M @ SLS
EIGER X 16M at Swiss Light Source beamline X06SA by Oliver Bunk, Paul Scherrer Institute.
Individual diffraction spots resolved for crystal with unit cell of 1000 Å by Arnau Casañas, Paul Scherrer Institute.
Karlsruhe, Germany and Baden, Switzerland, September 2, 2015 — Bruker AXS and DECTRIS Ltd. today announced an agreement for the supply of Hybrid Photon Counting (HPC) pixel detectors for Bruker’s X-ray diffraction (XRD) business.“We have chosen DECTRIS as a supplier because of its high-performance, reliable, and proven HPC detector technology and high-volume production capacity”, says Frank Burgaezy, President of Bruker AXS. “The combination of our world-class technologies will benefit customers in a wide range of demanding applications such as micro-high-resolution diffraction (HRXRD), grazing incidence small angle X-ray scattering (GISAXS) and kinetic studies’’“DECTRIS is pleased to supply state-of-the-art HPC detectors to Bruker, a global leader in X-ray technology with a long tradition of innovation”, states Christian Broennimann, CEO of DECTRIS. “We are excited by the performance of our noise-free PILATUS3 detector combined with the cutting-edge D8 DISCOVER diffraction solutions.” The PILATUS3 R 100K-A 2D X-ray detector for the D8 DISCOVER diffraction solutions was announced today at the Japan Analytical & Scientific Instrument Show 2015 (JASIS). About Bruker AXS:Bruker AXS, a division of the Bruker Corporation (BRKR), is a global market and technology leader in materials research, life science and quality control instrumentation for elemental and crystalline structure investigations. The solutions cover bulk material and surface sensitive X-ray diffraction, biological and chemical crystallography, wavelength and energy dispersive X-ray fluorescence analysis, optical emission spectroscopy, and combustion analysis. Learn more about Bruker AXS at www.bruker.com.About DECTRIS Ltd.DECTRIS is the technology leader in Hybrid Photon Counting X-ray detection. Based in Baden, Switzerland, the company has all the required experiences and resources for the design, production and global distribution of its detectors. The DECTRIS photon counting detectors have transformed basic research at synchrotron light sources, as well as in the laboratory and with industrial X-ray applications. DECTRIS aims to continuously improve the measurement quality, thereby enabling new scientific findings. This pioneering technology is the basis of a broad range of products, all scaled to meet the needs of various applications. DECTRIS also provides solutions for customer developments in scientific and industrial X-ray detection.
Villigen, March 2015: Researchers at the Swiss Light Source, a synchrotron radiation facility at the Paul Scherrer Institute (PSI) in Villigen, have developed a new approach to solving crystal structures from unmodified protein and nucleic acid crystals. Traditionally, the determination of a new protein structure depended on the calculation of the positions of exogenous heavy atoms whose incorporation is often laborious or inefficient.
Using a DECTRIS PILATUS 2M detector, PSI researchers improved a method known as “native SAD” (single-wavelength anomalous diffraction) where only atoms present in the protein are used to determine the structure. Until now, this method was limited to structures of small, well-diffracting proteins. With the new approach of collecting low-dose data at high redundancy, in multiple sweeps with the crystal in multiple orientations, the weak signal inherent in light atoms naturally occurring in protein crystals (e.g. sulfur, calcium, potassium) can be extracted reliably, in particular since radiation damage is minimized.
The high sensitivity of the PILATUS detectors produced by DECTRIS was critical to the success of the new approach. “These detectors can record signals with low noise with a low intensity of X-ray light, which means one can determine the protein structure with a low X-ray dose,” explains Vincent Olieric, a scientist at PSI who was involved in the project. With the new, broadly applicable technique, five new structures, among them challenging targets like a human membrane protein, a protein-DNA complex and a large multiprotein-ligand complex, were solved from native protein crystals (Weinert et al., Nature Methods 12, 131–133, 2015).
About DECTRIS Ltd.DECTRIS Ltd. is the technologically leading company in Hybrid Photon Counting X-ray detection. DECTRIS Hybrid Photon Counting detectors have transformed basic research at synchrotron light sources, as well as in the laboratory and with industrial X-Ray applications. DECTRIS aims to continuously improve the measurement quality, thereby enabling new scientific findings. The broad range of products is based on this pioneering technology; all scaled to meet the needs of various applications. DECTRIS also provides solutions for customer developments in scientific and industrial X-Ray detection.
About PSIThe Paul Scherrer Institute PSI develops, builds and operates large, complex research facilities and makes them available to the national and international research community. The institute's own key research priorities are in the fields of matter and materials, energy and environment and human health. PSI is committed to the training of future generations. Therefore about one quarter of our staff are post-docs, post-graduates or apprentices. Altogether PSI employs 1900 people, thus being the largest research institute in Switzerland. The annual budget amounts to approximately CHF 350 million.