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

A typical oscillation pattern. Each dark spot is a data-point, in the Fourier

• summation. Many to be collected.

A protein crystal

X-rays

Crystal Diffracted X-rays

ρ(x,y,z) = Σ|Fhkl|cos2Π(hx+ky+lz-φhkl). Maxima in ρ represent atomic positions thereby giving molecular structure and molecular interactions in three dimensions. MAD method enables estimation of φhkl experimentally.

3rd generation Synchrotrons as Source of

X-rays

ESRF – 6 GeV Grenoble, France APS – 7 GeV Chicago, USA Spring8 – 8 GeV 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

• f experiments.

Generate about thousand times more intense X-ray beams for research. Expensive to build and maintain.

Equipment automation for PX beamline

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

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
• perate 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

Libraries

A Laboratory without walls HBNI, Mumbai ESRF (FIP), Grenoble, France

Organic Synthesis X-Ray Diffraction Pattern Atomic level Structure

Experimental Station – FIP beamline

CATS robot Sample changer Sample storage cryo-dewar CCD X-ray detector X-ray beam Cryo-jet Crystal Single axis diffractometer

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

Remote operation of FIP beamline on ESRF in Grenoble, France, 9th September 2013 (from ACTREC), ( Collaborator, Dr. Ashok Varma).

Complex 1 Complex 2

G-ROB – Robotic Goniometer for in-situ data collection, (National)

Collaboration with DRHR

G-rob to play dual role

• f goniometer and

sample changer Crystallisation plate

Grabber holding plate (BARC-Robot)

In

In-situ data collection with G-ROB

96-well crystallisation plate 96-well crystallisation plate mounted on the robot 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
• utstanding.

Thank You