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Structural Biology. Semi-automatic structure solution with HKL-3000. Structural Biology. Semi -automatic structure solution with HKL-3000. Structural biology bottlneck. I have a dream: - PowerPoint PPT Presentation
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Bethesda, March 4th 2009
Semi-automatic structure solution with
HKL-3000
Structural Biology
Bethesda, March 4th 2009
Semi-automatic structure solution with
HKL-3000
Structural Biology
Bethesda, March 4th 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 4th 2009
HKL/HKL-2000 vs. HKL-3000
HKL/HKL-2000 h, k, l, F, σMethods in Enzymology (1997) 275: 307-326.
Times cited: 18018
HKL-3000 partially build model Acta Cryst. D (2006) 62: 859-66.
Times cited: 77
PDB deposits: >710
Bethesda, March 4th 2009
Integrated system – HKL-3000current 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 4th 2009
HKL-3000 six mouse click programEngage your brain !
Bethesda, March 4th 2009
or Automatic structure solution
HKL
Bethesda, March 4th 2009
Why to engage my brain ?One little detail …
O-ring
Bethesda, March 4th 2009
IntegrationHKL-3000 is a pipeline component
Other systems provide additionalcontextual up- and downstreaminformation
Pepdbexpression & purification
Xtaldbcrystallization
HKL-3000Data collection,
diffraction & structure solution
Wetlabchemicals & solutions
Bethesda, March 4th 2009
Project setup Check against PDB during data collection
Bethesda, March 4th 2009
Completeness – overlapsMulti-crystal strategy
Bethesda, March 4th 2009
Priceless crystals
Star of Africa Crystallography lab
Bethesda, March 4th 2009
Scaling
Signal from Sulfur
Bethesda, March 4th 2009
Feedback of possible hardware, sample or experiment problems
Detector Goniostat Cooling
Crystal decay Experiment Twinning …..
Bethesda, March 4th 2009
Possible Pseudo-translational Symmetry
Bethesda, March 4th 2009
SAD or MAD phasing
Bethesda, March 4th 2009
Substructure solution
Data management in HKL-3000 - substructure solution
Bethesda, March 4th 2009
Substructure enantiomorph determination
Bethesda, March 4th 2009
Double conformations
Bethesda, March 4th 2009
Optimization of solvent content worthwhile but slow
automaticoptimization
manual
Bethesda, March 4th 2009
NCS identification and use
From sites From map
Bethesda, March 4th 2009
Model building and preliminary refinement (10 minutes, 190aa)
Secondary structure after each cycle
Unidentified density -> Xtaldb
Bethesda, March 4th 2009
TM1086 (APC4579)
10 Se-Met/282 aa35.1kDa
I222a= 290Å, b= 300Å, c=316Å
~81% solvent content = 17 molecules in AU~38% solvent content = 57 molecules in AU
Bethesda, March 4th 2009
TM1086 (APC4579)
282 aa35.1kDaP3221
a= 165Å, b= 165Å, c=98Å~78% solvent content = 3 molecules in AU~40% solvent content = 8 molecules in AU
Bethesda, March 4th 2009
TM1086 (APC4579)
Bethesda, March 4th 2009
I222 crystal form
~210 Å
30 chains in AU
8460 aa
1.05MDa
Bethesda, March 4th 2009
APC81521 ( ChangSoo)
Final model with right targetAPC81521
Attempt to use automatic building with wrong targetAPC81501
To figure out the right target at the resolution of 2.5Ǻ1. 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 4th 2009
2as8 1.95Å resolution, R=18.8/17.4, Rfree=25.2/23.1
Bethesda, March 4th 2009
# of PDB deposits with given # of Mg ions
1 1273
2 943
3 210
4 358
5-10 274
11-50 80
51-100 33
>101 52
# of Mg # of deposits
Bethesda, March 4th 2009
Four deposits - 1896 Mg ions
Mg-O distance distribution
0
5
10
15
20
25
30
35
40
1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2
Mg - O distance (Å)
%
2a68,2a69,1iw7,1smy
CSD
HR PDB
Bethesda, March 4th 2009
Unknown ligands
Bethesda, March 4th 2009
Completeness of data and completeness of the model (3bqs)
Bethesda, March 4th 2009
3bqs 1ze0
Acta Cryst. D 63(2007)348-54
Bethesda, March 4th 2009
Molecular Replacement
SGCZoledronic acid
in Cryptosporidium parvum
Arun MalhotraRNase T
Bethesda, March 4th 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
70
40
50
60
10
30
20
2.0 1.52.53.03.5
Bethesda, March 4th 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 DauterTom Terwilliger Solve/ResolveGeorge Sheldrick Shelxd/ShelxePaul Emsley CootGarib Murshudov Refmac
Grants: NIH GM53163, GM62414, GM74942NIAID HHSN272200700058C DOE, NCI, HKL Research
Bethesda, March 4th 2009
Possible pseudo-translational symmetry
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