Web-based Auto-Rickshaw for validation of the X-ray experiment at the synchrotron beamline Auto-Rickshaw http://www.embl-hamburg.de/auto-rickshaw A platform for automated crystal structure determination Santosh Panjikar EMBL Hamburg Outstation X-ray anomalous signal lrem Peak Inf hrem 1 wavelength SAD 2 wavelengths 2W-MAD 3 wavelengths 3W-MAD 4 wavelengths 4W-MAD 5 Native + Der. SIRAS Energy of incoming X-rays 6. Native/Der. MR Panjikar, S., Parthasarathy, V., Lamzin, V. S., Weiss, M. S. & Tucker, P. A. (2005). Auto-Rickshaw - An automated crystal structure determination platform as an efficient tool for the validation of an X-ray diffraction experiment. Acta Cryst. D61, 449-457.
Steps in PX Structure Solution? Derivatisation SeMet Halide soaking Heavy atom soaking Xenon/Krypton Data processing+ Scaling HKL suites Mosflm/Scala Automar/marscale XDS D*trek Cryo-protection Organic solvent Oil based Cryo-salt Data collection strategy How many degrees? How much dose? What resolution? What detector distance?? Diffraction
Steps in PX Structure Solution? Derivatisation SeMet Halide soaking Heavy atom soaking Xenon/Krypton Data processing+ Scaling HKL suites Mosflm/Scala Automar/marscale XDS D*trek Cryo-protection Organic solvent Oil based Cryo-salt Data collection strategy How many degrees? How much dose? What resolution? What detector distance?? Diffraction
Why? Primary aim: To achieve an interpretable electron density map and a partial structure in the minimal time in order to confirm the success of the experiment at the synchrotron while the crystal is still at or near the beam line Ultimate aim: To achieve a model, which is correct and as complete as possible, both at low and high resolution
Inter-component communications-i Decision i Layer i Decision i+1 Layer i+1 Decision i+2 Layer i+2 Decision i+2
Inter-component communications-ii D D D D D D D D D
Decision making and PX software What should be the maximum resolution cut-off for substructure solution and phasing? 34 defined paths through the pipeline for each phasing protocol When to terminate the task for substructure solution? How many located heavy atom sites are actually correct? Which enantiomorph to choose? Which is the best way to calculate phases? How to exploit multiple copies (dimers, etc.) if present How to get the model? How to improve the model?
Heavy atom refinement and/or phase calculation Which software to choose for phase calculation? CC weak : Correlation coefficient between observed and calculated E values for weak reflection as computed in SHELXD
Two different flavors of Auto-Rickshaw Beamline Version (Quick, no restriction on resolution) Advanced Version (Takes time, accept better than 2.7 Å data )
User interface for input parameters
User- interface for X-ray data input Input X-ray data format SCALEPACK : H K L I+ SIGI+ I- SIGI- MTZ : H K L I+ SIGI+ I- SIGI- XDS Scaled data
Example-1 A protein 250 residues long (48% solvent content) Diffraction to a resolution of 3 Å One Pt heavy atom expected but SAD at peak - no solution 2W-MAD at peak and inflection - no solution 3W-MAD at peak, inflection and remote - no solution SAD on 16-fold redundancy (peak+remote) YES!!! X-ray anomalous signal Inf Peak Rem Energy of incoming X-rays
Example-2 A protein 593 residues long (solvent content 78%) Diffraction to a resolution of 2.55 Å Nine Se heavy atoms Space group P6 4 22 and X-ray data data collection Auto-Rickshaw (CCP4+SHELXD+SHELXE+ARP/wARP) peak (360 ) - finished and processed solved in 8 min (369 α-helical atom) inf (180 ) - initiated and halted 561 residues traced after 2.5 hours 130 residues traced at cycle 0 (30 min) X-ray anomalous signal Inf Peak Rem Energy of incoming X-rays
Example-3 In an asymmetric unit 680 residues, 4 molecules (57% solvent content) 3 Se expected Diffraction to a resolution of 2.2 Å but data was good to 2.7 Å MAD experiment at Se edge Space group R3 Data processed with XDS Quick check with Beamline Version of Auto-Rickshaw to resolution 3.0 Å 3W-MAD (peak, inflection and remote): No solution 2W-MAD (remote and inflection) : No solution SAD (peak ) : Success!! (8 heavy atoms in asymmetric unit and 2-fold NCS operator found, After NCSaveraging, co-relation b/w density of two dimers 85%, 188 α -helical atoms placed) Courtesy of Koh Cha San
Example-3 Phased molecular replacement Experimental map contoured at 1.3 σ Courtesy of Koh Cha San
Range of real case runs with Auto-Rickshaw Residues in ASU Sites in ASU Mol in ASU Max. Resolution Solvent Content 85-1500 2-40 1-10 3.50-1.50 Å 0.35-0.78
How long Auto-Rickshaw takes to complete the job 2.4 GHz, 16 CPU cluster Residues/ASU Mol./ASU Beamline version Advanced version (Average) (approx. time) (approx. time) 500 <3 6 min 180 min 1000 >2 30 min 300 min
Text for materials and methods with acknowledgement An automatic output for materials and methods It includes also the references for all the programs which are used for the structure solution. It guides the user how to acknowledge the pipeline along with the software used in the pipeline.
Current status Auto-Rickshaw http://www.embl-hamburg.de/auto-rickshaw A platform for automated crystal structure determination Auto-Rickshaw is running on a 16 CPU Linux cluster since January 2005 (Four updates until now) Available to Hamburg beamline users We are planning to open the Auto-Rickshaw server to whole world Online Job submission and data retrieval Stats over 13 months: 50 distinct users 68 novel structures solved Future perspective: Parallelization (space group) (Under testing ) Ligand evaluation and identification (Under testing ) Addition other model building programs Addition of other phasing protocol Selection criteria of search models for MR Scanning of decision making parameters
Acknowledgement: Auto-Rickshaw Team V. Parthasarathy M. S. Weiss V. S. Lamzin P. A. Tucker All the developers of the various computer programs for their kind permission to use their software in this pipeline. (Martyn Winn, Kevin Cowtan, Qang Hao, George Sheldrick, Gerard Bricogne, Clemens Vonrhein, Navraj Pannu, Tom Terwilliger, Gerard Kleywegt, Alwyn Jones,G.D. Smith, Victor Lamzin, Anastassis Perrakis) Hamburg beamline/auto-rickshaw users for provision of data. Thank you for your attention