Protein Crystallography - Remote Operation & Control of BARC - - PowerPoint PPT Presentation

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

• f 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

A typical oscillation pattern. Thousands

• f frames to be collected.

At different wave lengths for MAD. Repetitive nature of method.

A protein crystal(Many screened before selecting one)

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 in three dimensions. MAD method enables estimation of φhkl experimentally.

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, (I=1 million)

Higher intensity enables usage of small crystals, even nano-crystals.

RAG – BARC, (I=1) LCLS Stanford (I=1 trillion)

Getting big crystals not always possible. Crystals as small as few microns can be used.

Microfocus beamlines at ESRF & APS

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

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

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,
• scillation 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 1σ. 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 (shown in yellow carbons) fits the density perfectly.

M36I

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

• rientations.

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
• ther 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