Application of e-infrastructure in Protein Crystallography - Remote Operation & Control of BARC Robot & ESRF- beamline, FIP M. V. Hosur Bhabha Atomic Research Centre, Mumbai, India. Collaboration: Computer Division and DRHR BARC, Jean-Luc Ferrer, IBS/ESRF, Grenoble.
Outline of the talk 1 . Protein crystallography ( repetitive nature) 2. Application of e-infrastructure and automation in Protein Crystallography
Crystallography • Is a technique for determining accurately spatial positions of all atoms in any given molecule to understand structure- function relationships. • Is applied in all branches of science such as physics, chemistry and biology. • Significant contributions from physicists, chemists, biologists, instrumentation and computer engineers.
Molecular size is not a limitation Atomic level structure of Viruses Understand difference between plant and animal viruses. Design anti-viral compounds and vaccines.
Bio-Crystallography has won many Nobel Prizes • F. H. C. Crick, J. D. Watson, M. H. C. Wilkins (Physiology or Medicine, 1962) • for their discoveries concerning the molecular structure of nucleic acids and its significance for information transfer in living material. • M. F. Perutz, Sir J. C. Kendrew (Chemistry, 1962) • for their studies of the structures of globular proteins. • D. Crowfoot Hodgkin (Chemistry, 1964) • for her determinations by X-ray techniques of the structures of important biochemical substances. • Sir A. Klug (Chemistry, 1982) • for his development of crystallographic electron microscopy and his structural elucidation of biologically important nucleic acid-protein complexes. • J. Deisenhofer, R. Huber, H. Michel (Chemistry, 1988) • for the determination of the three-dimensional structure of a photosynthetic reaction centre. • P. D. Boyer, J. E. Walker, J. C. Skou (Chemistry, 1997) • for their elucidation of the enzymatic mechanism underlying the synthesis of adenosine triphosphate (ATP) [Boyer, Walker]. • for the first discovery of an ion-transporting enzyme, Na+, K+ -ATPase [Skou]. • J. B. Fenn, K. Tanaka, K. Wüthrich (Chemistry, 2002) • for the development of methods for identification and structure analyses of biological macromolecules. • for their development of soft desorption ionisation methods for mass spectrometric analyses of biological macromolecules [Fenn, Tanaka]. • for his development of nuclear magnetic resonance spectroscopy for determining the three-dimensional structure of biological macromolecules in solution[Wüthrich]. • R. D. Kornberg (Chemistry, 2006) • for his studies of the molecular basis of eukaryotic transcription. • V. Ramakrishnan, T.A. Steitz, A.E. Yonath (Chemistry 2009) • for studies of the structure and function of the ribosome. • Robert Lefkowitz and Brian Kobilka (Chemistry 2012) • For studies on GPCR proteins.
Crystallography - key component of Biomedical Research and drug-design Dramatic reduction in deaths due to AIDS is due to development of inhibitors of HIV enzymes through Structure-based drug-design approach. Has become a key component of Modern Biomedical Research. The Congressional Joint Economic Committee of USA has estimated a net annual gain of $2.4 trillion as a result of increased life expectancy alone through BR.
E-infrastructure effectively used in PX method Crystal X-rays Diffracted X-rays A protein crystal(Many screened before selecting one) ρ(x,y,z) = Σ|F hkl |cos2Π(hx+ky+lz - φ hkl ). Maxima in ρ represent atomic positions thereby giving molecular structure in three dimensions. MAD method enables estimation of φ hkl A typical oscillation pattern. Thousands of frames to be collected. experimentally. At different wave lengths for MAD. Repetitive nature of method.
In-situ diffraction to improve crystal growth conditions • In-situ screening Commercial instruments do not provide for plate mounting
Protein Vs Salt, and quality of protein crystal
ROBOTIC Goniometer at BARC Normal Goniometer Can be transported to INDUS-II beamlines
BARC- Robot • LIVE
E-infrastructure to access Sources of X- rays ESRF – 6 GeV Grenoble, France, RAG – BARC, (I=1) LCLS Stanford (I=1 million) (I=1 trillion) Higher intensity enables usage of small crystals, even nano-crystals.
Microfocus beamlines at ESRF & APS Getting big crystals not always possible. Crystals as small as few microns can be used.
E-infrastructure to collect diffraction data • Protein Crystallography beamlines at synchrotrons are fully automated. So remotely usable, thanks to NKN and other e-networks. • Mega Facilities ($trillion?) (beamlines and experimental stations) available only in few countries. Remote operation is a Boon to rest of the world.
Experimental Station – FIP beamline Single axis Cryo-jet diffractometer Crystal X-ray beam CCD X-ray detector CATS robot Sample changer Sample storage cryo-dewar
Remote Data collection Facility at BARC, Trombay, Mumbai-85 Softwares – usage for Nxclient – export desktop Visu – view exptal station Xnemo - Detector control, Input Data collection and storage parameters Gonio2 - Crystal mounting through CATS sample changer, crystal centering, mounting other tools Adxv - View diffraction image
Remote operations in PX • 1. Conditioning X-ray beam and wavelength by aligning optical elements. • 2. Quick mounting/dismounting of frozen samples onto the diffractometer. • 3. Location of sample crystals within the loop. • 4. Centering of crystal in the X-ray beam path. • 5. setting the data collection parameters: detector distance, oscillation angle and number of frames to collect. • 6. Exposure to X-ray beam by opening shutter. • 7. Reading the detector and data transfer. • ( Live demo of coloured steps)
Play movie before LIVE demo Beamtime upto 6:30 PM. Welcome to try operation till then
Xnemo Detector movement • LIVE
Gonio2 - Crystal Mounting/Dismounting • LIVE
Results of data collection on FIP - remotely from HBNI Electron density map is contoured at 1 σ . The four stranded β -sheet in HIV-1 protease/ritonavir drug complex is clearly visible. The inter-strand hydrogen bond lengths are marked.
Drug-resistant HIV-1 protease enzyme Data collected by operating FIP beamline from HBNI, India. Resolution 1.6 Å. The data is of good quality , because, structure refined to low R (= 16.2%), and mutation is clearly seen. SA-OMIT map clearly shows electron density appropriate for the mutation M36I. The original amino acid Met (shown in purple carbons) doesn ’ t fit density. The changed amino acid Ile M36I (shown in yellow carbons) fits the density perfectly.
ED for drug in Nelfinavir – N88S HIV-1 PR complex N88S is resistant toward Nelfinavir. Data collected by operating FIP beamline from HBNI, India. Resolution 1.9 Å. structure refined to R (= 18.2%), Drug density is clearly seen. The drug is bound in two orientations. Structure gives insight into drug- resistance mechanism. Electron density for Nelfinavir
Advantages of remote usage 1. Financial savings on travel costs 2. No hassels of travel and visa-procedures 3. More people can be trained on synchrotron usage 4. More efficient use of beamtime 5. Possibility to use simultaneously more than one synchrotron 6. Use the beamline collaboratively with experts in other labs
SUMMARY • Remote usage of PX beamlines on synchrotrons provides excellent quality data even on challenging crystals. • With NKN, protein crystallographers are in for a quantum jump in the availability of resources for research.
Thank You
Linear Coherent Light Source – Stanford (October 2009) 15 – 50 GeV. X-ray beam a billion times more intense than ESRF beams. Sample injected as 3 – 4 µl drops containing nano-crystals of proteins. Data collection at Room Temperature
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