Semi-automatic structure solution with HKL-3000
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Transcript of Semi-automatic structure solution with HKL-3000
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.
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HKL-3000 six mouse click programEngage your brain !
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or Automatic structure solution
HKL
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Why to engage my brain ?One little detail …
O-ring
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IntegrationHKL-3000 is a pipeline component
Other systems provide additionalcontextual up- and downstreaminformation
Pepdbexpression & purification
Xtaldbcrystallization
HKL-3000Data collection,
diffraction & structure solution
Wetlabchemicals & solutions
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Project setup Check against PDB during data collection
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Completeness – overlapsMulti-crystal strategy
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Priceless crystals
Star of Africa Crystallography lab
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Scaling
Signal from Sulfur
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Feedback of possible hardware, sample or experiment problems
Detector Goniostat Cooling
Crystal decay Experiment Twinning …..
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Possible Pseudo-translational Symmetry
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SAD or MAD phasing
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Substructure solution
Data management in HKL-3000 - substructure solution
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Substructure enantiomorph determination
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Double conformations
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Optimization of solvent content worthwhile but slow
automaticoptimization
manual
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NCS identification and use
From sites From map
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Model building and preliminary refinement (10 minutes, 190aa)
Secondary structure after each cycle
Unidentified density -> Xtaldb
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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
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TM1086 (APC4579)
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I222 crystal form
~210 Å
30 chains in AU
8460 aa
1.05MDa
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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
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2as8 1.95Å resolution, R=18.8/17.4, Rfree=25.2/23.1
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# 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
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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
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Unknown ligands
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Completeness of data and completeness of the model (3bqs)
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3bqs 1ze0
Acta Cryst. D 63(2007)348-54
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Molecular Replacement
SGCZoledronic acid
in Cryptosporidium parvum
Arun MalhotraRNase T
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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
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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