Bethesda, March 4 th 2009 Semi-automatic structure solution with HKL-3000 Structural Biology

Bethesda, March 4 th 2009 Semi-automatic structure solution with HKL-3000 Structural Biology

Bethesda, March 4 th 2009 Structural biology bottlneck I have a dream: To convert high throughput, high output SG center into, not necessary high throughput, but very high output center. Can we use sequence to predict: solubility, crystallization, ….. structure, … function, ….

Bethesda, March 4 th 2009 HKL/HKL-2000 vs. HKL-3000 HKL/HKL-2000 h, k, l, F, σ Methods in Enzymology (1997) 275: Times cited: HKL-3000partially build model Acta Cryst. D (2006) 62: Times cited: 77 PDB deposits: >710

Bethesda, March 4 th 2009 Integrated system – HKL-3000 current status HKL-2000, DENZO, SCALEPACK SHELXD, SHELXE MLPHARE, CYCLMLPHARE DM, REFMAC SOLVE, RESOLVE ARP/WARP STATBUILD O, COOT,CCP4 Interactive development: Integrated approach shows how the choice in one step influences subsequent steps.

Bethesda, March 4 th 2009 HKL-3000 six mouse click program Engage your brain !

Bethesda, March 4 th 2009 or Automatic structure solution HKL

Bethesda, March 4 th 2009 Why to engage my brain ? One little detail … O-ring

Bethesda, March 4 th 2009 Integration HKL-3000 is a pipeline component Other systems provide additional contextual up- and downstream information Pepdb expression & purification Xtaldb crystallization HKL-3000 Data collection, diffraction & structure solution Wetlab chemicals & solutions

Bethesda, March 4 th 2009 Project setup Check against PDB during data collection

Bethesda, March 4 th 2009 Completeness – overlaps Multi-crystal strategy

Bethesda, March 4 th 2009 Priceless crystals Star of Africa Crystallography lab

Bethesda, March 4 th 2009 Scaling Signal from Sulfur

Bethesda, March 4 th 2009 Feedback of possible hardware, sample or experiment problems Detector Goniostat Cooling Crystal decay Experiment Twinning …..

Bethesda, March 4 th 2009 Possible Pseudo-translational Symmetry

Bethesda, March 4 th 2009 SAD or MAD phasing

Bethesda, March 4 th 2009 Substructure solution Data management in HKL substructure solution

Bethesda, March 4 th 2009 Substructure enantiomorph determination

Bethesda, March 4 th 2009 Double conformations

Bethesda, March 4 th 2009 Optimization of solvent content worthwhile but slow automatic optimization manual

Bethesda, March 4 th 2009 NCS identification and use From sites From map

Bethesda, March 4 th 2009 Model building and preliminary refinement (10 minutes, 190aa) Secondary structure after each cycle Unidentified density -> Xtaldb

Bethesda, March 4 th 2009 TM1086 (APC4579) 10 Se-Met/282 aa 35.1kDa I222 a= 290Å, b= 300Å, c=316Å ~81% solvent content = 17 molecules in AU ~38% solvent content = 57 molecules in AU

Bethesda, March 4 th 2009 TM1086 (APC4579) 282 aa 35.1kDa P3221 a= 165Å, b= 165Å, c=98Å ~78% solvent content = 3 molecules in AU ~40% solvent content = 8 molecules in AU

Bethesda, March 4 th 2009 TM1086 (APC4579)

Bethesda, March 4 th 2009 I222 crystal form ~210 Å 30 chains in AU 8460 aa 1.05MDa

Bethesda, March 4 th 2009 APC81521 ( ChangSoo) Final model with right target APC81521 Attempt to use automatic building with wrong target APC81501 To figure out the right target at the resolution of 2.5Ǻ 1. Manually build helixes and stands; 2. Submit this model to dali to find out several similar structures in PDB; 3. Search targetDB(MCSG) using the sequence of these structures to find out the right target. MCSG sequence from electron density map

Bethesda, March 4 th as8 1.95Å resolution, R=18.8/17.4, R free =25.2/23.1

Bethesda, March 4 th 2009 # of PDB deposits with given # of Mg ions >10152 # of Mg # of deposits

Bethesda, March 4 th 2009 Four deposits Mg ions

Bethesda, March 4 th 2009 Unknown ligands

Bethesda, March 4 th 2009 Completeness of data and completeness of the model (3bqs)

Bethesda, March 4 th bqs 1ze0 Acta Cryst. D 63(2007)348-54

Bethesda, March 4 th 2009 Molecular Replacement SGC Zoledronic acid in Cryptosporidium parvum Arun Malhotra RNase T

Bethesda, March 4 th 2009 Directions for a future Using NCS in structure solution Using high-resolution native data with low- resolution phasing data The boundaries can be moved by improving diffraction data quality or by using more elaborate methods Resolution [Å] 4.0 Unique content in ASU [%] = = (1 – solvent content)/NCS factor No methods to interpret such maps Arp/wARP builds most of the model Atomicity based phasing e.g. ME Challenging projects

Bethesda, March 4 th 2009 People involved Wladek Minor Marcin Cymborowski Maks Chruszcz Matt Zimmerman Heping Zheng Marek Grabowski Zbyszek Otwinowski  Dominika Borek  Andrzej Kudlicki Andrzej Joachimiak Rongguang Zhan Youngchang Kim Marianne Cuff Chris Lazarski and SBC/MCSG staff Zbyszek Dauter Tom Terwilliger Solve/Resolve George SheldrickShelxd/Shelxe Paul EmsleyCoot Garib MurshudovRefmac Grants: NIH GM53163, GM62414, GM74942 NIAID HHSN C DOE, NCI, HKL Research

Bethesda, March 4 th 2009 Possible pseudo-translational symmetry