fundamental of automated structure determination · 2016. 5. 19. · sp :auto-rickshaw • what...

Macromolecular Crystallography School 2010, Madrid SP :Auto-Rickshaw

Santosh Panjikar

Fundamental ofAutomated Structure Determination

SAD

MR

MRSIRAS

SIRASMRMAD

MAD

MRRIP

RIP

S-SADMRSAD


From Crystal to Structure

Data processing+scaling

HKL suitesMosflm/Scala

Automar/marscaleXDS

D*trek

Derivatisation SeMet

Halide soakingHeavy atom soaking

Xenon/Krypton

Cryo-protection Organic solvent

Oil based Cryo-salt

Structure Solution

DiffractionData collection strategy BEST

How many degrees ?How much dose ?What resolution ?

What detector distance ?………?





Automar/marscaleXDS

D*trek

Derivatisation? SeMet


Xenon/Krypton


Oil based Cryo-salt

Structure Solution

DiffractionData collection strategyBEST







Automar/marscaleXDS

D*trek

DerivatisationSeMet


Xenon/Krypton


Oil based Cryo-salt

Structure Solution

Diffraction ?Data collection strategy BEST





Data processing+Scaling ? HKL suites

Mosflm/Scala Automar/marscale

XDSD*trek

DerivatisationSeMet


Xenon/Krypton


Oil based Cryo-salt

Structure Solution

Diffraction Data collection strategy BEST





Data processing+Scaling


Automar/marscaleXDS

D*trek

DerivatisationSeMet


Xenon/Krypton


Oil based Cryo-salt

Structure Solution ?

Diffraction Data collection strategyBEST




Phasing in Macromolecular Crystallography

How do we get from spots on a screen to a pretty picture of our protein?

BY CALCULATING AMPLITUDES AND PHASES!!


Phase Determination Methods

1. SIR, SIRAS, MIR, MIRAS(single/multiple isomorphous replacement with anomalous scattering)

2. MAD(multiple wavelength anomalous diffraction)

3. SAD (SAS)(single wavelength anomalous diffraction/scattering)

4. RIP, RIPAS(radiation damage induced phasing with anomalous scattering)

5. MR(molecular replacement)

6. Direct Methods


Data reduction

Location of Heavy atoms

Heavy atom refinementand/or

Phase calculation

Solvent flattening&

NCS averaging

Model building

Model refinement

Expe

rimen

tal P

hasi

ng

Hea

vy a

tom

der

ivat

ives

CCP4

SHELXDCRUNCH2SOLVECNSHYSSSUPERFLIP

SHARPBP3PHASERMLPHARESOLVESHELXE DM

PARROT, PIRATERESOLVE, SOLOMON

ARP/wARPRESOLVEBUCCANNERESSENSALBEMAIDTEXTAL REFMAC5

CNSSHELXL

Manual building

COOTO

Steps in structure determination (Experimental phasing)


Data reduction


Heavy atom refinementand

Phase calculation

Solvent flattening&

NCS averaging

Model building

Model refinement



Data reduction



Phase calculation

Solvent flattening&

NCS averaging

Model building

Model refinement



Primary aim

To achieve an interpretable electron density map anda partial structure in the minimal time in order toconfirm the success of the experiment at thesynchrotron while the crystal is still at or near thebeam line


Ultimate aim

To achieve a model, which is correct and ascomplete as possible from a seqence file andintensity data at low and high resolution, andachieve almost refined model (~30% Rfree) forthe data better than 3.2 Å resolution.


The Auto-Rickshaw Philosophy

Photographed by Prof. B.C. Wang


The Auto-Rickshaw Approach

Mimic What An Experienced Crystallographer would Do



Mimic What An Experienced Crystallographer would Do

Try To Be As Fast As Possible ... And (Just) As Good As necessary



Try to Mimic What An Experienced Crystallographer would Do

Try To Be As Fast As Possible ... And (Just) As Good As necessary

Minimize User Input


Softwarepipeline

Crystallographic software

Phasing methods

Decision makingand machine learning

Database ofX-ray anomalous

data

Auto-Rickshaw : An Automated Crystal Structure Determination Pipeline


Decision making process

Decision making module (DMD): Overall decisiong makingSingle choice of software at each step of structure determinationPredefined choice of parameters

SHELXD, SOLVE, CHRUNCH2

SHELXE, MLPHARE, BP3, PHASER, SHARP

DM, PIRROT, RESOLVE, SOLOMON, PIRATE

ARP/wARP, ALBE, RESOLVE, BUCANEER, SHELXE

Master

Step-representative

Program-representative

CPU usage

CCP4 (data reduction utility)


Master decision makerStep Representative decision makerProgram Representaive decision maker

Decision making


Decision making

Master decision makerStep Representative decision makerProgram Representaive decision maker


The Auto-Rickshaw System for experimental phasing

Phase improvement and model completion


Panjikar, S., Parthasarathy, V., Lamzin, V. S., Weiss, M.S. & Tucker, P. A. (2005). Auto-Rickshaw - An automatedcrystal structure determination platform as an efficienttool for the validation of an X-ray diffraction experiment.Acta Cryst. D61, 449-457.


Date: Fri, 11 Feb 2005 10:47:17 -0600From: "Dauter, Zbigniew" To: [email protected]: decision makersParts/Attachments:

Hi Santosh

We just read the proof of your Auto-Rickshaw paper very nice work!One question.

What exactly means (in Abstract) that you combine computer programs withdecision makers?

Does it mean that the intermediate results are rapidly sent to India(Bangalore or Madras) and there some twenty crystallographers makedecisions and transfer them back by telephone?

CheersZbyszek


• What should be the maximum resolution cut-off for substructure solution and phasing ?

• When to terminate the task for substructure solution ?

• How many located heavy atom sites are actually correct ?

• Which hand of the substructure is correct ?

• 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 ?

Decision making


0

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Resolution cut off

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Ano

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CC

DA

NO

/σ(I) SAD

HREM/PEAKHREM/INFLHREM/INFL

MAD

Resolution


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Resolution cut off

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Ano

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/σ(I)

Resolution

SAD

HREM/PEAKHREM/INFLHREM/INFL

MAD










Decision making


When to terminate the SHELXD trials

0

0.05

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0.15

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1 6 11 16 21 26 31 36 41 46 51 56 61 66 71 76 81 86 91 96

SHELXD TRIALS

CC

(wea

k)

CCweak : Correlation coefficient between observed and calculated E values for weak reflection as computed in SHELXD

SITCOM

Macromolecular Crystallography School 2010, Madrid SP :Auto-RickshawSP :Auto-

Substructure determination using SHELXD in Auto-Rickshaw

Initial resolution cut-off based signal/noise ratio (from the program SHELXC).

CCweak: Auto-Rickshaw usually considers better than 15%

If 15% is not achieved in the first round of SHELXD, then resolution is decreased by 0.2 and increase number of trails by 100. Checks the CCweak in the next round.

If CCweak improves, number of heavy atoms sites is increased in the next round of SHELXD trail.


Substructure determination using SHELXD in AutoRickshaw

Common site using SITCOM (Dall‘Antonia & Schneider)










Decision making


Changing hand of the substructure

System Chiral NonchiralTriclinic P1Monoclinic P2, P21, C2Orthorhombic P222, P2221, P21212, P212121, C222,

C2221, I222, I212121, F222Tetragonal P41P43

P4122P4322P41212P43212

P4, P42, I4, I41P422, P4212, P4222, P42212 I422, I4122

Trigonal P31P32P3112P3212 P3121P3221

P3, R3P312, P321, R32

Hexagonal P61P65 P62P64

P6122P6522 P6222P6422

P6, P63

P622, P6322

Cubic P4132P4332 P23, F23, I23, P213, I213P432, P4232, F432,F4132, I432, I4132

For nonchiral space groups the other hand of the heavy-atom sites is found by the operation (x, y, z) (-x, -y, -z), except for three space groups (I41, I4122 , F4132 and I4132) where there is also a change of origin.


Changing hand of the substructure

I41 (x,y,z) (x+0.5, y, z)I4122 (x,y,z) (x+0.5, y, z+0.25)F4132 (x,y,z) (x+0.75, y+0.25, z+0.75)I4132 (x,y,z) (x+0.25, y+0.25, z+0.25)

For nonchiral space groups the other hand of the heavy-atom sites is found by the operation (x, y, z) (-x, -y, -z), except for three space groups (I41, I4122 , F4132 and I4132) where there is also a change of origin.


Changing hand of the substructureFor the chiral space groups the change of hand of the heavy-atom sites with the operation (x, y, z)(-x, -y, -z) is accompanied by a change of space group to the other chiral form.

System ChiralTriclinicMonoclinic OrthorhombicTetragonal P41P43

P4122P4322P41212P43212

Trigonal P31P32P3112P3212 P3121P3221

Hexagonal P61P65 P62P64

P6122P6522 P6222P6422

Cubic P4132P4332

Chiral objectsNonsuperimposable

mirror images

Nonchiral objectssuperimposablemirror images

mirror mirror


Enantiomorph determination

Level-1: ABS SHELXE(C-value) (Contrast)

Level 2: MLPHARE/SHARP/BP3 DM(FOM) ( FOM, FreeR)

Level 3: MAPMAN CAPRA(Maximum Fragment size) (Cα-atoms)










Decision making


Example: which software to choose for Phase Calculation ?



Example: which software to choose for Phase Calculation ?

SHELXE

MLPHARE

BP3/SHARP



Determination of correct space groupSystem Space groupMonoclinic P2, P21,

Orthorhombic P222, P2221, P2212, P2122, P21212, P22121, P21221 P212121C222, C2221, I222, I212121,

Tetragonal P4, P42, (P41P43)I4, I41P422, P4212, P4222, P42212 , (P4122P4322), (P41212P43212)I422, I4122

Trigonal P3, (P31P32) R3, R32P312, (P3112P3212)P321, (P3121P3221)

Hexagonal P6, P63 ,(P61P65 ), (P62P64)P622, P6322, (P6122P6522), (P6222P6422)

Cubic P23, P213, I23, I213 P432, P4232, F432,F4132, I432, I4132


Space group determination

Vario

us jo

bs a

re la

unch

ed o

n di

ffere

ntcl

uste

r nod

es in

var

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spa

ce g

roup

s

T=0


Space group determination

T=0 + x

Vario

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unch

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ffere

ntcl

uste

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roup

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How long Auto-Rickshaw takes to complete the job

Residues/ASU(Average)

Mol./ASU Beamline version(approx. time)

Advanced version(approx. time)

500 2 15 min 180 min

2.0 GHz , 64 CPU cluster


Terminate data collection earlier

Collect more data than planned

Get direct answer to the questions:

Did my experiment work?

Do I get a structure out of it now?

Is Auto-Rickshaw printing out for me the Methods section?

Impact on Beamline Users


Example-1:Fewer Data Sufficient• A protein 593 residues long (solvent content 78%)• Diffraction to a resolution of 2.55 Å• Nine Se heavy atoms• Space group P6422and

X-ray data data collection Auto-Rickshaw(CCP4+SHELXD+SHELXE+ARP/wARP)

• peak (360°) - finished and processed • inf (180°) - initiated and halted

Energy of incoming X-rays

X-ra

y an

omal

ous

sign

alInf

Peak

Rem

solved in 8 min (369 α-helical atom)130 residues traced at cycle 0 (30 min)561 residues traced after 2.5 hours

SAD


Energy of incoming X-rays

X-ra

y an

omal

ous

sign

alInf

Peak

Rem

Example-2:More Data NeededSAD

• A protein 250 residues long (48% solvent content)• Diffraction to a resolution of 3 Å• One Pt heavy atom expectedbut…• 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 !!!


Combination of molecular replacement (MR)

and experimental (SAD)

phasing

Data reduction



Phase calculation

Solvent flattening&

NCS averaging

Model building

Model refinement

Expe

rimen

tal P

hasi

ng

Data reduction

Rotation function

Translation function

Rigid body refinement

Model building

Model refinement

MR

Pha

sing

• SAD• 2W-MAD• 3W-MAD• 4W-MAD• SIRAS• RIP

MR

Sear

ch M

odel

Hea

vy a

tom

der

ivat

ives

Steps in structure determinationAuto-Rickshaw

http://www.embl-hamburg.de/Auto-Rickshaw

A platform for automated crystal structuredetermination

MRSAD

Phased MR

MRRIP


The Search Model

Molecular replacement only works if you have a good search model.HOMOLOGY• The higher the sequence identity the easier MR.• There is a rough inverse correlation between the rms deviation of atomic

positions and the percentage sequence identity. • It is not possible to give an minimum value of homology at which MR will

work. Below 30% it is usually difficult but it can be challenging with a 100% identical molecule.

COMPLETENESS• The search model should represent a significant fraction of the unknown

structure. If it only represents 10-20% of the unknown structure MR will be difficult.

FLEXIBILITY• Some proteins (for instance antibodies) have domains that can adopt

different relative orientations with respect to each other. • MR will be difficult if the search model does not have the same relative

orientation of these domains


• AMORE (Navaza,1994)• MOLREP (Vagin & Teplyakov, 1997)• PHASER (Read., 2001)

• MRBUMP (Keegan & Winn, 2008)• BALBES (Long et al., 2008)

The MR programs and pipelines

The software pipeline makes several decisions concerning (i) truncation of the model in uncertain parts; (ii) the actual protocol for sequence alignment and homology modelling; and (iii) the choice of the MR software, the consensus approach is to derive a variety of models and try MR for all of them one by one


Molecular Replacement

• In principle, accurate phases can be obtained in a matter of hours or even minutes.

• In practice, however, sometimes structure determination by MR is not straightforward.


Why ?

• One major problem is that maps calculated from MR phases inevitably retain some memory of the search model (model bias)

• The more similar the phasing model to the unknown is, the less biased the calculated map will be.

σA -weighted


Reduction of model bias

• Repeated cycling of real-space and reciprocal-space refinement • Density modification and NCS-averaging• Free atom modelling, refinement and model building as implemented in

ARP/wARP (Perrakis et al., 1999)• SHAKE and wARP (Reddy et al., 2003)• An ML-based reciprocal-space density-modification method (Prime &

Switch ) (Terwilliger et al., 2004)

• Omission of parts of the model (Bhat et al, 1988)• Simulated-annealing OMIT maps (Bruenger 1998)• Iterative-build OMIT maps (Terwilliger et al., 2008)


The sub-structure model

Experimental phasing only works if you have a substructure model.

• A complete and accurate substructure will result in better phase estimates than partial and/or inaccurate substructure.

• High quality data is needed to calculate structure-factor amplitudes of the anomalous scatterers and to locate the anomalous scatters.

• The resolution cutoff is an important parameter to obtain the best possible substructure from a given set of diffraction data.

• Radiation damage, low redundant and large substructure can be challenging to solve a substructure.


MRSAD

• An attractive feature of MRSAD strategy is that the anomalous scatterer substructure can be quickly determined from the MR solution.

• SAD phases are virtually independent of the MR phases, MRSAD provides an experimental method to overcome the model bias inherent to MR


Basic difference in SAD phasing and MRSAD phasing

• Substructure determination

• Hand determination• Heavy atom refinement

and phase calculation• Density modification,

NCS-averaging and phase extension

• Model building

• MR + Anomalous difference Fourier

• No need of hand determination• Heavy atom refinement and

phase calculation• Density modification,

NCS-averaging and phase extension

• Model building


Validation of MR

• Collection of Anomalous data Model refinement

Calculation of Anomalous difference Fourier or LLG map

MR

Density modification

0

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ENERGY

f“

1.54 Å 1.0 – 0.8 Å

1pjk


Use of anomalous peaks in phase improvement

Phase combination of resultant phases, density

modification and/orNCS-averaging


Refinement of anomalous peaks (above 5σ) at max resolution

Dual fragment phasing usingthe partially refined/built model

and anomalous peaks

Model building




Model refinement




and anomalous peaks

Model building

MR


Combination of MR and SAD phasing

Phase improvement of poor MR or SAD phaseand model completion

Validation ofMR solution

Poor MR PhasePoor SAD Phase

Iterative model completion and

refinement


αSAD αMOD

XP,SAD

αC,SAD

αCOMB

XH

∆FO

DM

EXIT

Panjikar, S., Parthasarathy, V., Lamzin, V. S., Weiss, M. S. & Tucker, P. A. . On thecombination of molecular replacement and single anomalous diffraction phasing forautomated structure determination Acta Cryst. D 2009 D65, 1089-1097


αSAD αMOD

XP,SAD

αC,SAD

αCOMB

XH

∆FO

∆FO

DM

EXIT



αSAD αMOD

XP,SAD

αC,SAD

αCOMB

XH

∆FO

αC,MR

XP,MR

FO

∆FO

DM

EXIT

MR + refine



MRSAD: combination of MR and experimental phasing

Phase combination of resultant phases and density

modification

Model refinement

Calculation of Anomalous difference Fourier map

Refinement of anomalous peaks (above 5σ)

at maximum resolution

Dual fragment phasing usingthe partial refined model and

anomalous peaks

Model building

MRSoftware pipeline for MRSAD


2G4L hydroxynitrile lyase 257 1 18 1.84 C2221 0.022 0.067 12 0.83 -----

2G4J Glucose isomerase 386 1 11 1.85 I222 0.036 0.141 13 0.86 ----- 1.80

2G4O LeuB 355 4 32 2.00 P212121 0.019 0.06 12 0.90 -----

2G4R MogA 156 3 03 1.92 P21 0.019 0.07 06 0.91 -----

2G4W Ribonuclease A (C2) 128 2 18 1.84 C2 0.022 0.063 06 0.97 ----- 1.00

2G4I Concanavalin A 237 1 07 2.40 I222 0.037 0.104 11 1.05 ----- 1.60

2G4K hARH3 256 1 19 1.82 C2221 0.019 0.069 12 1.06 ----- 2.20

2G51 Trypsin (p1) 240 1 10 1.84 P1 0.02 0.06 03 1.09 -----

2G4X Ribonuclease A (P3221) 128 1 18 1.95 P3221 0.019 0.053 18 1.10 ----- 1.40

2G4N α-lactalbumin 122 6 54 2.30 P21212 0.03 0.071 12 1.12 -----

2G4Y Thaumatin 207 1 16 1.98 P41212 0.013 0.064 25 1.12 0.682

2G4T PPE (Na) 240 1 13 1.84 P212121 0.015 0.043 12 1.15 0.549

2G4V Proteinase K 279 1 13 2.14 P43212 0.018 0.045 26 1.18 0.559

2G4S NBR1PB1 85 1 05 2.15 P6322 0.013 0.053 34 1.18 0.558

2G52 Trypsin (P21) 240 1 12 1.84 P21 0.033 0.076 06 1.22 ----- 1.80

2G4U PPE (Ca) 240 1 13 1.84 P212121 0.033 0.123 13 1.38 0.667

2G55 Trypsin (P3121) 240 1 10 1.82 P3121 0.019 0.086 10 1.46 0.708

2G4Z Thermolysin 316 1 06 1.98 P6122 0.018 0.067 38 1.64 0.655

2G4Q Lysozyme at pH 8.0 129 1 15 1.84 P43212 0.024 0.031 23 1.73 0.675

2G4P Lysozyme at pH 4.5 129 1 17 1.84 P43212 0.022 0.04 24 2.00 0.645

2G4M Insulin 51 1 06 1.80 I213 0.019 0.057 35 2.00 0.788

Mueller-D

ieckmann et al., 2007

Long wavelength (2.0 Å) dataset Longwavelength dataset with PDB code

Total residue per molecule = 110 - 755Number of mol. in asu = 1 - 4

Total number of Se in asu = 2 - 32Resolution =3.00 - 1.80 Å

Space group

Ranom=0.013 - 0.036Rmerge=0.031 - 0.141Multiplicity= 3 - 38

Ranom/Rpim= 0.83 - 2.00

Data quality

SAD

MR

SAD


2hh6 116 1 06 2.04 P6522 2o4t 0.44 1.04/072 0.056 0.10 7.5 1.43 N Y

1vky 563 1 06 3.00 I222 1yy3 0.49 1.84/247 0.086 0.126 3.5 1.10 Y N

2gi3 452 1 13 1.80 P3221 2g5i 0.50 1.34/403 0.058 0.112 6.3 1.19 Y N

2hxv 354 1 05 2.80 I222 2d5n 0.41 2.32/316 0.057 0.078 4.0 1.27 Y N

2o08 193 2 08 2.90 C2221 2ogi 0.42 1.30/180 0.058 0.07 3.7 1.36 Y Y

1vmf 407 1 05 1.90 P212121 2cu5 0.42 1.09/124 0.107 0.134 4.1 1.41 Y N

1zbt 320 1 08 2.40 P43212 2b3t 0.49 2.43/224 0.043 0.071 6.7 1.45 Y Y

1vmi 329 1 08 2.32 P6322 1xco 0.42 1.76/311 0.046 0.062 5.0 1.48 Y Y

2f4l 276 4 28 2.50 P212121 2ii1 0.36 1.03/274 0.075 0.084 3.8 1.49 Y N

2fvg 311 1 06 2.50 P212121 1vhe 0.37 1.70/227 0.073 0.081 3.8 1.51 Y Y

1vjo 755 1 08 2.00 P212121 2ch2 0.43 1.15/371 0.061 0.064 4.1 1.68 Y Y

1vjr 269 1 06 2.40 P41212 1zjj 0.40 1.55/240 0.076 0.072 4.6 2.00 Y Y

1vkn 338 4 32 2.45 P21 1xyg 0.46 1.18/316 0.069 0.065 6.1 2.40 Y Y

MRSAD examples on JCSG SeMet data

Search model with PDB codesequence identity= 35 - 51%rmsd after optimal overlap = 1.2 - 2.4 Å

JCSG dataset with PDB code Total residue per molecule = 110 - 755

Number of mol. in asu = 1 - 4Total number of Se in asu = 2 - 32

Resolution =3.00 - 1.80 ÅSpace group

Ranom=0.032 - 0.107Rmerge=0.064 - 0.134

Multiplicity=3.4 - 7.5Ranom/Rpim=0.67 - 2.40

MR

SAD

SAD

Dataset Search model quality Data quality




Model refinement




and anomalous peaks

Model building

MR


Initial partial model built from SAD phasing protocol

Residue per monomer: 261Molecules in ASU: 4Max Resolution: 2.3 ÅSpace group: P21

Electron density after SAD phasing

Part of the complete structure, shown in experimental map


Evolution of substructure, electron density and model




Model refinement




and anomalous peaks

Model building

MR


Initial partial model built from SAD phasing protocol

Residue per monomer: 145Molecules in ASU: 4Max Resolution: 2.75 ÅSpace group: P43212

Initial partial model built from SAD phasing


Evolution of the model at low resolution in MRSAD

51.9/55.5

47.6/57.6 47.6/55.8 46.5/53.9 43.4/53.6

32.7/41.5

29.6/34.6

http://cluster.embl-hamburg.de/lresult/LV1SvIl/LOG/viewlog/result.html�


Model building module

RESOLVE

ARP/wARP

SHELXE

RESOLVE

BUCCANEER

SHELXE

Best phases after density modification

Poly-ala

Sequence docking andmodel expension

Iterative refinement, model building and sequence docking

SA

D fu

nctio

n

High resolutionbetter than 2.6 Å

Low resolution2.6 - 3.2 Å

Refinement using REFMAC5

Model Model

Refinement using REFMAC5

d50 = dmin[sc-1 - 1]1/3


Parameters to gauge the map improvement and model completion

• Standard deviation of the local r.m.s. of the electron density map after density modification

• The Rfree value from the refinement of the model • Fraction of the model built• Fraction of sequence docked• Absolute peak heights in the anomalous difference Fourier

map





Model refinement

Calculation of Anomalous/isomorphous

difference Fourier or LLG map

Refinement of anomalous/isomorphous peaks (above 5σ) at max resolution

Dual fragment phasing usingthe partialy refined/built model

and anomalous/isomorhous peaks

Model building

MR

SAD

SIRAS

2W-MAD

3W-MAD

4W-MAD

Hea

vy a

tom

refin

emen

t

Substructure determinationValidation of MR

Ligand evaluation

Phase improvementand modelcompletion

RIP/SIR


• MRSAD: (1) A means of completing your model(2) Overcome model bias from MR solution(3) Use long wavelength X-ray data for MR


Native data collection

0

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ENERGY

f“

1.54 Å 1.0 – 0.8 Å


Phasing at short wavelength

Heavy atoms/ASU

2 Zn + 12S

Wavelength (Å) 0.8

f” 1.76 (Zn) 0.15(S)

Bijvoet ratio 1.14

Space group P21Redundancy 3.7

Resolution (Å) 20-1.66

Completeness (%) 97.4 (94.3)

I/σ(I) 14.2(2.8)

Rmerge (%)b 3.7 (37.0)



Heavy atoms/ASU 6S

Wavelength (Å) 1.00

f” 0.24(S)

Bijvoet ratio 0.66

Space group I213

Redundancy 22.6


Completeness (%) 98.2(100.0)

I/σ(I) 42.0 (13.0)

Rmerge (%)b 3.2 (12.2)



Heavy atoms/ASU 17S

Wavelength (Å) 0.9

f” 0.20(S)

Bijvoet ratio (%) 0.45

Space group P41212

Redundancy 7.6


Completeness (%) 94 (68)

I/σ(I) 33.3(9.8)

Rmerge (%)b 3.9(13.7)


Exploiting Phase Information

We may also find ourselves in a situation where we have experimental phases (from a heavy atom derivative) as well as a model.

Phase information can, in fact, be exploited to help to solve the MR problem.

This is particularly useful when we have a molecular replacement model that is too poor to find a solution and phases that are too weak to provide an interpretable map.

By combining molecular replacement with experimental phases, the two together may be enough to succeed.

This technique of combining experimental phases with molecularreplacement methods is called phased molecular replacement.


Phased Molecular Replacement

Domain rotationfunction

FTFA

Orientation (αA, βA, γA)

FT

F(αA, βA, γA)Phased translationfunction

(TxA,TyA, TzA)


An Example


Another example

Phased MR model is useful for interpretability of the poor experimental mapand this can be also useful in phase enhancement

90°


New emerging phasing method: RIP X-ray Vs. UV

X-ray UV

Nano & Ravelli (2006)


Data collection for UV-RIP phasing

X-ray data collection before UV

UV exposure (10 – 30 min)

X-ray data collection after UV


CYS-158: 27.6 CYS-174: 14.9

CYS-194: 24.7CYS-127: 21.0

CYS-46:20.1CYS-30:23.8

CYS-184:14.4CYS-214: 8.8

TRP-164

TRP-12

TRP-132

TRP-39

TRP-26TRP-83

TRP-232

Elastase5 min series(2)


TRP-CYS distance

CYS-158: 27.6 CYS-174: 14.9

CYS-194: 24.7CYS-127: 21.0

CYS:46:20.1CYS:30:23.8

CYS-184:14.4CYS-214: 8.8

12-TRP 20.0 09.2 19.3 19.6

26-TRP 33.8 31.0 14.9 24.8

39-TRP 28.6 25.1 17.0 29.4

83-TRP 15.9 20.9 09.8 16.9

132-TRP 24.6 18.5 12.2 16.6

164-TRP 08.0 17.5 16.1 11.0

232-TRP 21.3 20.4 13.6 24.7

Elastase5 min series


UV radiation does not only break disulphide bridges but also damage seleno-methionine and covalently bound heavy atoms.

UV radiation induced phasing with or without

S/MAD, S/MIR(A)S and MR can provide additional or

alternative phasing information


UV-RIP phasing from Seleno-Met protein crystalFAE

H32

ChSynt


Auto-Rickshaw server

Commandline

Web-browser

BeamlineAdvanced

Version

Necessary Input required

Overview of Auto-Rickshaw

AR server is available to world wide crystallographic community

QuickLongerSAD

MR

MRSIRAS

SIRASMRMAD

MAD

MRRIP

RIP

S-SADMRSAD

No. of residues per monomer

No. of expected heavy atom sites per monomer

No. of monomers per a.u.

Space group

X-ray data

Sequence file (optional)

Model file PDB-format (optional) Email address


Input Required

integrated and scaled X-ray data files

formats Extension SCALEPACK : H K L I+ SIGI+ I- SIGI- ( .sca )

MTZ : H K L I+ SIGI+ I- SIGI- ( .mtz )

XDS : XDS-ASCII.HKL ( .HKL ) or ( .hkl )

XSCALE : ( .HKL ) or ( .hkl )

D*TREK (.REF) or (.ref)

sequence file (if available)

free format ( .pir ) or ( .seq) or ( .txt)


Beamline version(Phasing, density modification and helix/β-strand recognition)

Advanced version(Phasing, density modification and modelbuilding)

SAD, 2W-MAD, 3W-MAD, 4W-MAD, SIRAS

SAD, S-SAD,

2W-MAD, 3W-MAD, 4W-MAD,

SIRAS,

RIP

MR, MRSAD, MRSIRAS,MR-2W-MAD, MR-3W-MAD,MR-4W-MAD, MR-RIP

Web-browser Interface for job submission

Quick

longer


Strategy for data evaluation at third generation source

Data collection

Data Processing

Data evaluation

Group-1

Group-2Group-3


Text for materials and methods with acknowledgement

An automatic output for the materials and methods

It includes also the references for all the programs whichare used for the structure solution.

It guides the user how to acknowledge the pipeline along with the software used in the pipeline.


• More than 1500 novel structures solved

• Largest structure solved : asymmetric unit contains 7,500 residues

• Largest substructure solved: 140 heavy atoms sites in asymmetric unit

Status and Highlights


Auto-Rickshaw

Reduction of the number of input parameters Addition of other phasing protocols (i.e. MIR) Parallel computing at various steps of structure determination Scanning and improvement of decision making parameters• Remote connection with data collection/processing software ( i.e. XIA2 and DNA)

• Auto-Rickshaw provides results in almost real time for data collection at home sources or second generation synchrotrons. Accelerating it further for third generation sources is a great challenge.

The Future

Total registered institutes : 283 Total users from registered institutes : 754

57

7

15

45

18

39

241

24

47

13

15

38

20

1

8

5

26

7

1

3

Geographic distribution of AR users

1

3

19

7

3

1

3

310


More infos:

The Auto-Rickshaw Web Site:

http://www.embl-hamburg.de/Auto-Rickshaw

Auto-Rickshaw

Auto-Rickshaw and EMBL


Acknowledgements

Santosh PanjikarVenkataraman Parthasarathy

Manfred S. WeissVictor S. LamzinPaul A. Tucker

All the developers of the various computer programs for their kind permissionto use their software in this pipeline (Martyn Winn, Kevin Cowtan, Qang Hao,George Sheldrick, Gerard Bricogne, Clemens Vonrhein, Navraj Pannu, TomTerwilliger, Axel Brunger, Gerard Kleywegt, Alwyn Jones, G. D. Smith, VictorLamzin, Anastassis Perrakis, Randy Read, Garib Murshudov, Alexi Vagin, Hi-Fu Fan, Thomas Schneider).

Hamburg beamline/Auto-Rickshaw users and JCSG for the provision of data

Thank you for your attention

Kay Diederichs for testing DPS2AR module

Daniele De Sanctis (UV-RIP )�


Tutorial

http://www.embl-hamburg.de/~panjikar/MAD/

http://www.embl-hamburg.de/~panjikar/MAD/�http://www.embl-hamburg.de/~panjikar/MAD/�http://www.embl-hamburg.de/~panjikar/MAD/�

Fundamental of�Automated Structure Determination From Crystal to StructureFrom Crystal to StructureFrom Crystal to StructureFrom Crystal to StructureFrom Crystal to StructureFrom Crystal to StructurePhasing in Macromolecular CrystallographyPhase Determination Methods��Steps in structure determination (Experimental phasing)�Número de diapositiva 12Steps in structure determination (Experimental phasing)�Primary aimUltimate aimThe Auto-Rickshaw PhilosophyThe Auto-Rickshaw ApproachThe Auto-Rickshaw ApproachThe Auto-Rickshaw ApproachNúmero de diapositiva 20Decision making processNúmero de diapositiva 22Número de diapositiva 23The Auto-Rickshaw System for experimental phasingNúmero de diapositiva 25Número de diapositiva 26Decision makingResolution cut offResolution cut offResolution cut offDecision makingWhen to terminate the SHELXD trialsSubstructure determination using SHELXD in Auto-RickshawSubstructure determination using SHELXD in AutoRickshawDecision makingChanging hand of the substructureChanging hand of the substructureChanging hand of the substructureEnantiomorph determinationDecision makingExample: which software to choose for Phase Calculation ?Example: which software to choose for Phase Calculation ?Determination of correct space groupSpace group determinationSpace group determinationSpace group determinationSpace group determinationHow long Auto-Rickshaw takes to complete the jobNúmero de diapositiva 49Example-1:Fewer Data SufficientNúmero de diapositiva 51Combination of �molecular replacement (MR) �and �experimental (SAD)�phasing��The Search ModelThe MR programs and pipelinesMolecular ReplacementWhy ? Reduction of model biasThe sub-structure modelMRSADBasic difference in SAD phasing and MRSAD phasingValidation of MRUse of anomalous peaks in phase improvementNúmero de diapositiva 64Número de diapositiva 65Número de diapositiva 66Número de diapositiva 67MRSAD: combination of MR and experimental phasingNúmero de diapositiva 69Número de diapositiva 70Número de diapositiva 71Evolution of substructure, electron density and modelNúmero de diapositiva 73Número de diapositiva 74Model building moduleParameters to gauge the map improvement and �model completion Número de diapositiva 77Número de diapositiva 78Native data collectionPhasing at short wavelengthPhasing at short wavelengthPhasing at short wavelengthNúmero de diapositiva 83Phased Molecular ReplacementNúmero de diapositiva 85Número de diapositiva 86New emerging phasing method: RIP �X-ray Vs. UV Data collection for UV-RIP phasingNúmero de diapositiva 89TRP-CYS distanceNúmero de diapositiva 91UV-RIP phasing from Seleno-Met protein crystalNúmero de diapositiva 93Número de diapositiva 94Número de diapositiva 95Input RequiredNúmero de diapositiva 97Strategy for data evaluation at third generation sourceText for materials and methods with acknowledgementNúmero de diapositiva 100Auto-RickshawNúmero de diapositiva 102Auto-RickshawAcknowledgementsTutorial

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