X-Ray Crystallography Facility Back

The goal of X-ray Crystallography Facility is to provide state-of-the-art resources to study and analyze the 3-D structures of biological macromolecules and their constituents at atomic level. This advanced facility provides:

  1. High-throughput automated crystallization set up, incubators for crystal growth and screening procedures to identify potential crystallization conditions. 

  2. X-ray source that is considered the most intense home laboratory source available today for macromolecular crystallography, with brightness exceeding certain second generation bending magnet synchrotron beamlines. The X-ray generators operational at the facility are:

    (i) MicroMax™ 007 HF microfocused rotating Cu anode generator using Mar345-dtb image plate detector and Oxford cryosystems 

    (ii) Dual wavelength Rigaku FR-E+ SuperBright System with Cu and Cr anode generators using R-axis IV++ image plate detector and X-stream cryosystem. 

  3. Crystallographic Computing: The raw data collected from diffraction source is processed using crystallographic computational software. For this purpose, the facility provides Network-attached storage(NAS-server) with Silicon Graphics (SGI Fuel) Workstations and Intel Quad-Core Linux-based Systems for molecular modeling. 

High Throughput (HT) Crystallization Facility:

Protein crystallography is a powerful technique to determine the structure of proteins and other macromolecules at their atomic resolution. It helps to elucidate the mechanisms by which these molecules carry out their function in living cells.  

Under controlled conditions, proteins can form crystals, made of many copies of the molecule arranged in a regular 3-D lattice. This technique relies on the fact that wavelengths of X-rays are of the same order of magnitude as the distances between atoms in molecules (~1Å).  

Protein crystal serves as a 3-D diffraction grating to a beam of X-ray. X-rays scattered by atoms in the crystal lattice produce intense spots giving rise to diffraction pattern. By analyzing the intensities and positions of diffraction spots using various crystallographic computational software, the interatomic distances and angles can be determined leading to structure solution of the macromolecule. Obtaining diffraction-quality single crystals remains the primary bottleneck in protein crystallography. 

The High-Throughput Facility provides automation of the complete crystallization set-up starting from Crystal screen preparation to Experimental drop set-up, along with an integrated system for temperature controlled incubation for Crystal growth, inspection and imaging crystal drops. Main components are I) Alchemist for liquid handling, II) Crystallization Robotic systems for crystallization drop set up and III) Minstrel III with two incubators for storage of plates and imaging. Automated crystallization process is easier, faster and repeatable.


 

I. Liquid handling system : 

   Alchemist (Rigaku Corporation) 

  • Alchemist provides a highly accurate way of creating coarse and optimized protein crystallization screens. 

  • Dispenses 1 µl to 10 ml in different types of plates or tubes (e.g. 96 well deep well blocks, crystallization trays or 15 ml tubes). 

  • Uses patented bird feeder technology using disposable, multiuse syringes to dispense up to 70 different stock solutions. 

  • Can prepare a deep well plate containing complicated crystallization solutions in about 1 hour.

  • Screen optimizations using the CrystalTrak™ software.

II. Crystallization Robotic Systems : Automated crystallization drop set up

    1. Mosquito (TTP LabTech) : Crystallization method-Hanging Drop


  • Mosquito dispenses drop volume of 50 nl to 1200 nl. 

  • Automates plate set-up by aliquoting protein solution from a single source column to 96 windows on a hanging drop plate seal. 

  • The linear drives and optical sensors of Mosquito provide positional accuracy of 0.05 mm in X,Y and Z axes on flat surface of plate seal for perfect imaging.

    2. Oryx 4 (Douglas Instruments) : Crystallization method-Sitting Drop

  • Oryx 4 robot is used for screening (finding new crystallization conditions) using the microbatch crystallization method

  • Accurately dispenses 0.2 to 10µl volume of drops from pre-dispensed reservoirs into 96x3 sitting drop wells.

  • Works fast and uses very little protein for screening experiments.

3.Hydra II-eDrop (Matrix Technologies) : Crystallization method-Sitting Drop

  • Hydra-eDrop is composed of 96 channels that can dispense 100 nl to 100 µl solutions in different plate wells and subwells. 

  • eDrop uses patented EMB technology to dispense 100 nl to 100 µl protein solution in different subwells. 

  • Combined system can prepare a crystallization plate with three subwells in less than 5 minutes and the protein requirement is less than 40 ml for 288 drops.

III. Storage of Crystallization plates and imaging:

     1. Minstrel III (Robodesign / Rigaku Corporation) with two Robo-Incubators:

  • Minstrel III is a plate-handling and machine vision system for protein crystal imaging. 

  • It is integrated with two incubators (4ºC and 20ºC) automated for plate incubation, storage (768 Linbro plates) and retrieval for inspection of protein crystal growth. 

  • Software tools are integrated that can automatically,

    • identify each plate by reading its unique barcode 

    • locate the drop within each well

    • zoom in on the drop and auto-focuses on it 

    • image the drop 

    • store each image to a database for later viewing and classification

2. Nano Biochem Technology (NaBiTech) UV imager

For rapid screening of protein crystals.

Throughput :

  • 50 nanolitres to several microlitre size crystallization drops

  • Sitting drop, hanging drop, microbatch crystallizations, micro seeding

  • Barcoded SBS as well as Linbro crystallization plates

  • Crystallization at two different temperatures (4ºC and 20ºC)

  • High resolution imaging without manual interventions. 

  • Initial conditions could be expanded using Alchemist 

  • Software for overall control of the crystallization operations: CrystalTrak 

List of HT crystallization screens:

    1. Hampton Research

  • CrystalScreenHT (96 conditions)

  • IndexHT (96 conditions)

  • SaltRxHT (96 conditions)

    2. JBS

  • JBS Classic 1-10 Bulk ( 240 conditions)

    3. Emerald Biosystems

  • Wizard I, II, III (120 conditions)

  • Precipitant Synergy (3 sets x 64 conditions)

  • Ozma 1K & 4K (48 conditions)

  • Ozma 8K & 10K (48 conditions)

Recipes for expansions from any combination

Contact:

Scientists In-charge

Dr. Rajan Sankaranarayanan sankar@ccmb.res.in Ph: 27192832 
Dr. Biswajit Pal pal@ccmb.res.in Ph: 27192831

Technical In-charge

Ms. P. Sambhavi sambavi@ccmb.res.in Ph: 27192835

Instrumentation In-Charge

Mr. U S T R B Bapi Raju bapiraju@ccmb.res.in Ph: 27192583
Mr. Sudatt T Tambe sudatt@ccmb.res.in Ph: 27192625

Software Links :

  1. CrystalTrak - http://www.rigaku.com/products/automation/ 
  2. Alchemist - http://www.rigaku.com/products/automation/alchemistht
  3. Mosquito - http://www.ttplabtech.com/products/mosquito/crystallography.html
  4. Oryx 4 - http://www.douglas.co.uk/oryx4.htm
  5. Hydra-II-eDrop - http://www.matrixtechcorp.com/automated/pipetting.aspx?id=25
  6. Minstrel III with Robo-incubators - http://www.rigaku.com/products/automation/minstrelht
  7. Hampton Research - http://hamptonresearch.com/menus.aspx?id=1
  8. JBS Screen - http://www.jenabioscience.com/cms/en/1/browse/632_screens.html
  9. Emrald Biosystems - http://www.emeraldbiosystems.com/c-1-crystallography-screens.aspx

Forum Links :

  1. Hampton Research - http://hamptonresearch.com/links.aspx
  2. CCP4 Bulletin Board - https://www.jiscmail.ac.uk/cgi-bin/webadmin?A1=ind1203&L=ccp4bb
  3. Protein purification for crystallization: http://www.proteincrystallography.org/protein-purification/
  4. Microseeding : http://www.douglas.co.uk/mms.htm

X-Ray Source at the Crystallography Facility:

MicroMax™ 007 HF: Cu anode generator

MicroMax™ 007 HF is the home-lab X-ray source for macromolecular crystallography. Features include:

  • Rotating Cu anode generator, generating X-rays of 1.54 Å wavelength

  • Power: 1.2 kW provides high flux density for small samples (crystals)

  • Flux intensity = 8×1010 X-rays/mm²/sec 

  • High brilliance with microfocused beam; focal spot size = 70 µm diameter

  • VariMax HF Optics : Beam Size at sample = 208 µm

  • Automated beam alignment system

  • Mar345-dtb image plate detector for recording diffraction patterns

  • Mar-dtb goniometer for mounting crystals

  • Oxford cryosystems (700 series) with temperature range: 80K - 400K

  • Chiller for the X-ray machine.

  • Software for Data collection and processing: automar


FR-E+DW SuperBright System: (i) Cu and (ii) Cr anode generator

 

  • Microfocus rotating anode generator with dual wavelengths: copper radiation(1.54 Å) for traditional data collection and chromium radiation (2.29Å) for single wavelength anomalous dispersion (SAD) phasing.
  • Synchrotron-class generator producing a high flux intensity: 16x1010 X-rays/mm²/sec 

  • Maximum power: 2.475 kW

  • High brilliance: 7.37x109 X-rays/mm²/sec/mR²

  • Microfocused beam; smallest focal spot size = 70 µm diameter

  • Optics: VariMax HF (left-port) VariMax HR (left port) 

  • Beam Size at sample = 100 µm

  • Shorter exposure times and faster data collection.

  • R-axis IV++ image plate detector

  • Automated beam alignment system

  • Goniometer for mounting crystals

  • X-stream cryosystem 

  • Chiller for the X-ray machine.

Software for Data collection and processing: d’TREK

Both the generators are shielded from users by glass enclosures. The facility temperature is maintained at 22 °C

Throughput:

X-Ray systems

  • Work on small and poor quality crystals at home source

  • Single wavelength anomalous diffraction (SAD)

  • Single/multiple isomorphous replacement (SIR/MIR)

  • Molecular replacement (MR)

  • Combination of either two or more methods, i.e., SIRAS, MIRAS, MRSAD

  • Rapid screening of the samples 

  • In-house structure determination of macromolecules

Contact:

Scientist In-charge 
Dr. Rajan Sankaranarayanan sankar@ccmb.res.in Ph: 27192832

Technical In-Charge
Ms. R. Rukmini ruks@ccmb.res.in Ph:  27192953 (R-100, R & D Building) ; 27192835 (Lab, Main Building)
Mr. K. Mallesham malleshk@ccmb.res.in Ph:27192921


Instrumentation In-Charge 
Mr. R. E. C. Johnson johnson@ccmb.res.in Ph: 27192581
Mr. Venkatnarayana venkat@ccmb.res.in Ph : 27192591

FR-E+ Lab [New R & D Structural Biology Facility] : Room:108 Ph.:27192920
MicroMax Lab [New R & D Structural Biology Facility] : Room:109 Ph.:27192921

 

Resource Links 

Software Links :

1. Automar : http://www.marresearch.com/products.automar.html

Forum Links :

1. X-ray Detectors : http://www.proteincrystallography.org/detectors/

2. MicroMax : http://www.rigaku.com/products/protein/micromax007

3. FR-E+ SuperBright : http://www.rigaku.com/products/protein/fre

 

Crystallographic Computing:

  • The computing and model-building facility provides a platform to address biological problems through computing tools.

  • The facility houses network-attached storage(NAS-server) linked to Silicon Graphics (SGI Fuel) Workstations and Intel Quad-Core Linux-based Systems for computationally intensive applications in biology.

  • The main objective is to process the raw data (image files) generated from the diffraction source using various crystallographic software (licence-based, web-based and open-source).

  • It provides a computing environment for bioinformatics, biomolecular simulation, molecular modeling, protein visualization and 3-D structure analysis. 

  • A complete suite of crystallographic, structural and bioinformatic application software is maintained and updated from time-to-time.

System Administrator: 

Ms. R. Rukmini ruks@ccmb.res.in Ph: 27192953 (R-100, R & D Building) ; 27192835 (Lab, Main Building)

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