Title: Making the most of your protein crystals
1(No Transcript)
2Making the most of your protein
crystals Ashley Deacon Stanford Synchrotron
Radiation Laboratory adeacon_at_slac.stanford.edu
3Overview
- Single crystal diffraction experiment
- Braggs law
- Rotation geometry
- Synchrotron beam line
- Beam line hardware instrumentation
- Beam line control software
- Step-by-step data collection
- Future developments at SSRL
- Example experiments
- Greater atomic detail
- Faster structures determination
- More challenging systems
- Questions and discussion
4A unique index for each diffracting plane
Molecules in crystal
Indices determine resolution
Low resolution (larger spacing)
1, 1
1, 2
1, 3
High resolution (smaller spacing)
5Braggs law
Position on detectordiffracting angle 2?
Crystal lattice planeresolution d
X-ray beam wavelength ?
2?
?
n ? 2 d sin?
Braggs law
6Rotation geometry
Rotate crystal to satisfy Braggs law for all
lattice planes
X-ray beam
7Synchrotron light sources in the USA
Advanced Light Source (ALS), Lawrence Berkeley
National Laboratory
Advanced Photon Source (APS), Argonne National
Laboratory
National Synchrotron Light Source (NSLS),
Brookhaven National Laboratory
Stanford Synchrotron Radiation Laboratory (SSRL),
Stanford Linear Accelerator Center
8Structural biology at SSRL
- Small Angle Scattering
- protein folding, conformational change, oligomer
assembly and low resolution virus structures - X-ray Absorption Spectroscopy
- metalloproteins and their reaction intermediates,
compare solution vs. crystalline state - Macromolecular Crystallography
9Insertion devices beam line 11 wiggler
10Station 11-1 optics
Focusing mirror
Monochromator
11Outside station 9-2
12Control electronics
Sample viewingscreen
Storage ringmonitor
Personal protectionsystem
Beam line controlterminal
Cold-stream control rack
13Computational facilities
Graphics computers provide a common interface to
boththe beam line operation and CPU servers for
analysis
14Inside the hutch
Sample viewingscreen
Detectorxyz-positioner
Beam line controlvia X-terminal
Microscope
15Beam conditioning system
Metal foils forenergy calibrationand beam
attenuation
Two horizontal and vertical beam collimation
slits
Ionchambers
16Beam definition and monitoring
17Sample position and detectors
Fluorescencedetector
Samplelighting
ADSC Quantum 42x2 CCD detector
Sampleenvironment
18Detectors for crystallography
- Image plate
- Large area (345mm diameter)
- Large dynamic range
- Reliable technology
- Relatively low cost
- Slow readout (gt 1 minute)
- CCD detector
- Fast readout
- Initial CCD detectors were small
- Modular to increase area (200mm2)
- Latest technology high cost
- Limited dynamic range
19Sample environment
Goniometer and heat shield
Sample xyz androtation stage
Beamstop
Shutter box
Cold stream
Guard shield
Sample viewing camera
20BLU-ICE beam line control software
tim_at_slac.stanford.edu
- Easy to use beam lines
- Intuitive and flexible interface
- Portable to all PX beam lines
- Support all detector types
- Provide automation features
- Easy to maintain beam lines
- Powerful features for staff
- Full control of beam line hardware
- Remote operation for trouble-shooting
Periodic table to select heavy atom
Automated wavelengths for MAD
21Flexible interface for staff and users
Easy to maintain and optimize the beam line for
staff
Easy to collect and visualise datafor the users
22Step-by-step data collection 1
- Mount crystal on diffractometer
- Mount empty loop on goniometer
- Check cold-stream is aligned
- Match loop size to the crystal
- Match beam size to the crystal
- Center crystal in beam
- Collect test images
- Process images
- Data collection strategy
- Collect data
23Step-by-step data collection 2
- Mount crystal on diffractometer
- Collect test images
- Set wavelength and detector distance
- 0.5-2.0 degree oscillation
- 30-120 second exposure at synchrotron
- Check for overloads, adjust exposure time
- Check spot shape and separation
- Collect an image 90 degrees away
- Check for anisotropic diffraction and mosaicity
- Process images
- Data collection strategy
- Collect data
X-ray beam smaller than crystal
X-ray beam bigger than crystal
24Step-by-step data collection 3
- Mount crystal on diffractometer
- Collect test images
- Process images
- Autoindex
- Calculate cell parameters
- Calculate crystal orientation
- Determine crystal symmetry
- Estimate mosaicity
- Use free software (CCP4 - Mosflm)
- Data collection strategy
- Collect data
25A note on overlapping reflections
- Overlapping reflections from large cell dimension
- Reflections within a zone are too close
- Move detector backwards
- Overlapping reflections from too large rocking
width - Combination of mosaicity and oscillation angle
- Reflections from adjacent zones run into each
other - Decrease oscillation angle and move detector back
26Step-by-step data collection 4
- Mount crystal on diffractometer
- Collect test images
- Process images
- Data collection strategy
- Dont use the American way shoot first ask
questions later - Calculate the best starting orientation
- Calculate the angular range needed for complete
data - Calculate the maximum range per image
- Use free software (CCP4 Mosflm)
- Collect data
27Step-by-step data collection 5
- Mount crystal on diffractometer
- Collect test images
- Process images
- Data collection strategy
- Collect data
- Process data as it is collected
- Check data quality is good
- Check space-group / symmetry as data is collected
- Watch out for signs of radiation damage, icing,
misalignment - Adjust data collection strategy to cope with
unforeseen problems
28Collaboratory for protein crystallography
chiu_at_slac.stanford.edu
Remote access toexperimental facilities
Continued access todata and computing
High-speed Video
29Automated sample mounting
4-axis Seikorobot
96 crystal storagecassette
30Ultra-high resolution crystallography
Diffraction beyond 1.0Å resolution
Enhanced atomic detailvisualise hydrogen atoms
andprotonated side-chains
Small CCD detector (80mm2)
31MAD phasing of large structures
70 selenium atoms in the asymmetric unit
32MAD phasing from Krypton
SSRL Xe/Kr pressurisation cell
Myoglobin test experiments show feasibility
33Summary
- Conducting structural biology research
- Collaborate and gain experience with labs abroad
- Learn from the WWW free tools / software
- Gain experience conducting synchrotron
experiments - SSRL collaboratory provides remote access
- Developing a crystallography beam line
- Develop a productive environment for structural
biology research - One good beam line is better than many average
ones - Collaborate and gain experience from synchrotron
labs abroad - SSRL software and hardware is freely available
- Visitors are welcome to learn about our facilities
34Acknowledgements
- The Structural Molecular Biology Group at SSRL
- Peter Kuhn and Mike Soltis (PX Group leaders)
- Aina Cohen and Paul Ellis (Beam line engineers)
- Hsiu-Ju Chiu (Collaboratory Scientist), Ana
Gonzalez (Staff scientist) - Scott McPhillips and Tim McPhllips (Software
developers) - Paul Phizackerley (SSRL faculty)
- See http//smb.slac.stanford.edu/ for more
details - Contact us (e-mail) for advice and to access
collaboratory tools