Protein Crystallography through NKN M. V. Hosur Advanced Centre for - PowerPoint PPT Presentation

Protein Crystallography through NKN • M. V. Hosur • Advanced Centre for Treatmentmt, Research, and Education in Cancer (ACTREC) NaviMumbai, India. • Collaboration with Computer Division BARC at HBNI.

Protein Crystallography • Is a technique for determining accurately spatial positions of all atoms in any given biological molecule(of any size), to understand structure-function relationships. • Is an interdisciplinary field requiring participation of physicists, chemists, biologists, computer engineers etc. • Has been recognised as an important field through award of many Nobel Prizes.

Molecular size is no limitation From Structures of inorganic salts, NaCl, to Atomic Resolution structure of Viruses . Complete Ribosome structure.

Crystallography -based design: FDA approved drugs • Cimetidine – H2-receptor antagonist. • NSAIDs – inhibitors of COX-2 as pain killers. • Dorzolamide – inhibitor of CA to treat glaucoma. • Enfuvirtide – HIV entry inhibitor. • Zolpidem, Zopiclone, Probenecid – bone disease. • SSRIs – antidepressants, Nutilin – anticancer. • vemurafenib – anticancer, an inhibitor of BRAF kinase. • Gleevec – drug against leukemia. • Rupintrivir – an antiviral drug against human rhinovirus. • Relenza – anti-flu drug inhibits neuraminidase. • Isentress – HIV integrase inhibitor. • Ritonavir, Indinavir, Nelfinavir, Azatanavir, Tiprinavir, Darunavir etc. – HIV protease inhibitors. • Drastically cuts down the time required to pass clinical trials. • Helps vaccine development.

The X-ray Diffraction Method - repetitiveness X-rays Crystal Diffracted X-rays A protein crystal ρ( x,y,z ) = Σ|F hkl |cos2Π( hx+ky+lz- φ hkl ). Maxima in ρ represent atomic positions thereby giving molecular structure and molecular interactions in three dimensions. MAD method enables estimation of φ hkl A typical oscillation pattern. Each dark spot is a data-point, in the Fourier experimentally. summation. Many to be collected .

3 rd generation Synchrotrons as Source of X-rays APS – 7 GeV ESRF – 6 GeV Spring8 – 8 GeV Chicago, USA Grenoble, France Harima, Japan In all three, light charged particles travel in ‘circular’ orbits at speeds nearing that of light. Result is emission of: 1) very brilliant and 2) highly collimated continuous spectrum radiation , allowing use of small crystals and experimental phasing .

A Beamlines Different types of equipment are installed along the tangential path to create beamlines that deliver X-ray beams tailor made for carrying out different types of experiments. Generate about thousand times more intense X-ray beams for research. Expensive to build and maintain.

Equipment automation for PX beamline Sample Protein Crystal sample Optical elements (M1, DCM, M2) adjusted for X- held flash frozen in a ray wavelength selection and focussing. cryo-loop. The adjustment is completely automated Sample manipulation also enabling remote operation. completely automated.

Remote operation is feasible because of automation. • A critical parameter is the latency. Internet speeds higher than 512 kBPS, required for remote control and real-time operation. More the better. • Mega Facilities ($trillion?) (beamlines and experimental stations) available only in few countries. Remote operation is a Boon to rest of the world.

Statistics of remote usage at Stanford Synchrotron Radiation Laboratory (a) The total number of groups with active proposals at SSRL (blue bars) and the number of research groups using remote access since its release in 2005 (purple bars). (b) The total number of remote starts (user groups starting a remote data- collection run) since 2005. Remote usage is picking up and soon all synchrotrons will provide this capability.

Remote-enabled beamlines at APS for protein crystallography Sr. No. Beamline ID Supported technique Energy range 1. 23-ID-B MAD/SAD, Microbeam 3.5-20 keV 2. 17-BM-B MAD/SAD, Microbeam 7.5-14 keV 3. 17-ID-B MAD/SAD, Microbeam 6.0 – 20 keV 4. 19-ID-D MAD/SAD, Microbeam 6.5-19.5 keV 5. 21-ID-D MAD/SAD 6.5-20 keV 6. 21-ID-F Fixed Wave length 12.7 keV 7. 22-BM-D MAD/SAD 8.0 – 20 keV 8. 22-ID-D MAD/SAD, Microbeam 6.0 – 20 keV 9. 23-ID-B MAD/SAD, Microbeam 3.5-20.0 keV 10. 23-ID-D MAD/SAD, Microbeam 5.0-20 keV Comparable beamlines also on NSLS I & II and ALS.

National Knowledge Network- India • The NKN is a state-of-the-art multi-gigabit pan-India network. • The NKN seamlessly integrates with the global scientific community at multiple gigabits per second speed. • NKN is connected to GEANT, TEIN3 etc. to operate at speeds exceeding 1000 Mbps. • Exploring Remote Operation of beamlines at ESRF, ALS, APS through use of NKN.

Remote Data collection control room at BARC(HBNI), Anushaktinagar, Mumbai- 94 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

Collaboratory: Working Together Apart ESRF (FIP), Grenoble, HBNI, Mumbai France X-Ray Diffraction Pattern Libraries Atomic level Structure Organic A Laboratory without walls Synthesis

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 operations in PX • 1. Conditioning X-ray beam and wavelength by aligning optical elements. Matching beam and sample sizes. • 2. Quick mounting 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. Exposure to X-ray beam by opening shutter. • 6. Reading the detector and data transfer. • 7. Check crystal quality, if found good, then setting the data collection parameters: detector distance, oscillation angle and number of frames to collect. If not, go to next sample, step 2.

Movie1

Movie 2

Movie 3

International remote operation Crystals of cancer-related proteins Complex 1 Complex 2 Remote operation of FIP beamline on ESRF in Grenoble, France, 9 th September 2013 (from ACTREC), ( Collaborator, Dr. Ashok Varma).

G-ROB – Robotic Goniometer for in-situ data collection, (National) G-rob to play dual role of goniometer and sample changer Crystallisation plate Collaboration with DRHR

Grabber holding plate (BARC-Robot)

In-situ data collection with G-ROB 96-well crystallisation plate mounted on the robot 96-well crystallisation plate In Standard goniometers cannot handle plates and also crystal selection.

Summary • NKN has come-in as a great boon to protein crystallographers of India. • Can use any synchrotron in the world simultaneously! • All members of the group can participate in the data collection process. • The quality of data collected remotely using NKN is outstanding.

Thank You