10/28/2015 Phenix Tutorial © 2015 Thayumanasamy …

Phenix Tutorial Data analysis, molecular replacement and refinement using Phenix Suite

10/2

8/20

15

© 2

015

Thay

uman

asam

y So

mas

unda

ram

1

Overview of structure solution 10/2

8/20

15

© 2

015

Thay

uman

asam

y So

mas

unda

ram

2

Overview of the tutorial • Details of diffraction data • Overview of the hands on analysis • Introduction to Phenix Suite • Brief description of Phenix Graphical User Interface • Brief description of Phenix Programs covered in our Tutorial

• Data analysis: Xtriage | Merging Statistics • Molecular Replacement: Phaser • Data and model visualization: Coot • Refinement: Phenix.refine • Miscellaneous: PDB Tools and Density modification [time permitting]

• Tetragonal hen egg white lysozyme data will be provided • We will NOT cover indexing, processing, merging and scaling • For these topics refer to earlier presentation

10/2

8/20

15

© 2

015

Thay

uman

asam

y So

mas

unda

ram

3

Phenix Tutorial • Details of diffraction data • Overview of the hands on analysis • Introduction to Phenix Suite • Brief description of Phenix Graphical User Interface • Brief description of Phenix Programs covered in our Tutorial

• Data analysis: Xtriage | Merging Statistics • Molecular Replacement: Phaser • Data and model visualization: Coot • Refinement: Phenix.refine • Miscellaneous: PDB Tools and Density modification [time

permitting]

10/2

8/20

15

© 2

015

Thay

uman

asam

y So

mas

unda

ram

4

Details of diffraction data • Hen egg white lysozyme (HEWL) crystal • Tetragonal P43212 space group • Data collected at home at 100°K • R-Axis IV++ w/ RU-H3R copper (λ=1.541Å) rotating anode • 1°/frame; 6 min/frame; 190° data • Diffraction data processed with HKL.1.9.82 • Scaled and merged scalepack file (1.6Å) • Unmerged and original index scalepack file (1.6Å)

10/2

8/20

15

© 2

015

Thay

uman

asam

y So

mas

unda

ram

5



permitting]

10/2

8/20

15

© 2

015

Thay

uman

asam

y So

mas

unda

ram

6

Overview of hands on analysis 1 of 2

• Data quality check at 1.6Å, 2.5Å, and 3.5Å resolutions • Searching for homologous macromolecular structures • Blast-P search and results • Molecular replacement runs using 193L (tetragonal) model

• At 1.6Å, 2.5Å, and 3.5Å resolutions • At 1.6Å with 120°, 90°, and 60° of data • At 1.6Å resolution

• 1LSG orthorhombic PRS (Blast-P) search result as model • 3LZT triclinic HEWL as model • 2FBB hexagonal HEWL as model • 2ZQ3 orthorhombic HEWL as model • 135L monoclinic TEWL as model • 1LCN monoclinic HEWL (A chain) as model

10/2

8/20

15

© 2

015

Thay

uman

asam

y So

mas

unda

ram

7

Overview of hands on analysis 2 of 2

• Coot usage and its functions • Visualization of Phaser runs using Coot • Structure refinement with Phenix.refine • Visualization of refinement runs using Coot • Mutate 193L model using PDB Tools

• Single amino acid mutation • Run Phaser and Phenix.refine • Visualization of mutation in Coot • “Fixing” the mutation in Coot

• Delete five amino acids at N-terminus of 193L using PDB Tools • Run Phaser and Phenix.refine on new model • Visualization of data and model in Coot • Build the model in Coot

10/2

8/20

15

© 2

015

Thay

uman

asam

y So

mas

unda

ram

8



permitting]

10/2

8/20

15

© 2

015

Thay

uman

asam

y So

mas

unda

ram

9

Introduction to Phenix • Python-based Hierarchical ENvironment for Integrated

Xtallography (PHENIX) [versions: 1.9-1692 and 1.10.1-2155] • URL: www.phenix-online.org • Free for academic usage but registration required • Available for Linux & Windows (32- & 64-bits) and Macintosh • “Phenix is a software suite for the automated determination of

molecular structures using x-ray crystallography and other methods”

• Funded by NIH General Medical Sciences • PHENIX Reference: A comprehensive Python-based system for macromolecular

structure solution. P. D. Adams, P. V. Afonine, G. Bunkóczi, V. B. Chen, I. W. Davis, N. Echols, J. J. Headd, L.-W. Hung, G. J. Kapral, R. W. Grosse-Kunstleve, A. J. McCoy, N. W. Moriarty, R. Oeffner, R. J. Read, D. C. Richardson, J. S. Richardson, T. C. Terwilliger and P. H. Zwart. Acta Cryst. D66, 213-221 (2010).

10/2

8/20

15

© 2

015

Thay

uman

asam

y So

mas

unda

ram

10

http://www.phenix-online.org/

http://journals.iucr.org/d/issues/2010/02/00/dz5186/index.html



permitting]

10/2

8/20

15

© 2

015

Thay

uman

asam

y So

mas

unda

ram

11

Phenix Graphical User Interface

Main Menu Interface Molecular Replacement Sub-menu

10/2

8/20

15

© 2

015

Thay

uman

asam

y So

mas

unda

ram

12

Version 1.9-1692 or 1.10.1-2155 (Windows or Macintosh or Linux)

Hands on practice

10/2

8/20

15

13

© 2

015

Thay

uman

asam

y So

mas

unda

ram



permitting] • Tetragonal hen egg white lysozyme data will be provided

10/2

8/20

15

© 2

015

Thay

uman

asam

y So

mas

unda

ram

14

Xtriage and Merging Statistics • Typical first steps in Phenix Suite • Xtriage

• X-ray diffraction data collected, indexed, and processed • Merged and scaled data using many programs (HKL, Mosflm, etc) • Checks for data quality and provides lots of statistics

• Merging Statistics • X-ray diffraction data collected, indexed, and processed • Unmerged original index data using many programs • Checks for data quality and provides lots of statistics

10/2

8/20

15

© 2

015

Thay

uman

asam

y So

mas

unda

ram

15

Xtriage Graphical User Interface Xtriage Input Xtriage running 10

/28/

2015

©

201

5 Th

ayum

anas

amy

Som

asun

dara

m

16

Required: Merged reflection file Optional: Reference PDB file

Xtriage Graphical User Interface

Xtriage data quality output Xtriage summary

10/2

8/20

15

© 2

015

Thay

uman

asam

y So

mas

unda

ram

17

Merging Statistics GUI Merging statistics Input Merging statistics output 10

/28/

2015

©

201

5 Th

ayum

anas

amy

Som

asun

dara

m

18

Required: Unmerged reflection file Optional: Reference PDB file

Pitfalls of relying on R-merge alone

35-1.6A 193 deg 130 deg 65 deg 30 deg

Observations 185663 124975 62259 28319

Unique 14231 14037 12762 7582

Redundancy 13 8.9 4.9 3.7

Mean <I> 1465 1454 1377 1441

Mean I/sigma I 34 28 20.9 18.7

R-merge 0.088 0.094 0.07 0.068

R-measured 0.091 0.099 0.077 0.078

R-pim 0.024 0.032 0.032 0.037

Completeness (%) 88.18 87.51 79.56 47.2

10/2

8/20

15

© 2

015

Thay

uman

asam

y So

mas

unda

ram

19

Hands on practice

10/2

8/20

15

20

© 2

015

Thay

uman

asam

y So

mas

unda

ram




10/2

8/20

15

© 2

015

Thay

uman

asam

y So

mas

unda

ram

21

Molecular Replacement: Phaser • One of the ways (cf MIR, MAD, MIRAS) of solving the phase

problem and hence the structure • Requires homologous known protein structure • Phaser (Automated Single Ensemble)

• Software for finding the phases using a model (known structure) • Single ensemble interface • Rotation function • Translation function • Packing • Anisotropic correction

10/2

8/20

15

© 2

015

Thay

uman

asam

y So

mas

unda

ram

22

Homologous macromolecule search National Center for

Biotechnology Information Basic Local Alignment Search

Tool

10/2

8/20

15

© 2

015

Thay

uman

asam

y So

mas

unda

ram

23

Homologous protein search Protein- Basic Local Alignment

Search Tool Protein - BLAST

10/2

8/20

15

© 2

015

Thay

uman

asam

y So

mas

unda

ram

24

Homologous protein search Protein- Basic Local Alignment

Search Tool Results-1 Protein- Basic Local Alignment

Search Tool Results-2

10/2

8/20

15

© 2

015

Thay

uman

asam

y So

mas

unda

ram

25

Hands on practice

10/2

8/20

15

26

© 2

015

Thay

uman

asam

y So

mas

unda

ram

Phaser Graphical User Interface Phaser Input-01 Phaser Input-02 10

/28/

2015

©

201

5 Th

ayum

anas

amy

Som

asun

dara

m

27

Required: Merged/Unmerged reflection file Required: One search model

Required: Sequence or m. wt. Optional: Sequence identity (%)

Required: Number of copies

Phaser Graphical User Interface 10/2

8/20

15

© 2

015

Thay

uman

asam

y So

mas

unda

ram

28

• Phaser Output

LLG: “+” and high TF-Z sore: >8 or 9




10/2

8/20

15

© 2

015

Thay

uman

asam

y So

mas

unda

ram

29

Coot Graphical User Interface 10/2

8/20

15

© 2

015

Thay

uman

asam

y So

mas

unda

ram

30

• Phaser Output Opened in Coot

Coot tips • Extra useful windows to keep it open

• Draw>Sequence View • About>Hints • Display Manager • Go to Atoms

• Left-mouse button: Rotate • Ctrl+ Left-mouse button : Move • Shift + Left-mouse button : Label atom • Right-mouse button : Zoom in and out • Middle-mouse button: Center on atom • Keyboard +/- : Change contour level (e Density, etc)

10/2

8/20

15

© 2

015

Thay

uman

asam

y So

mas

unda

ram

31

Coot Graphical User Interface 10/2

8/20

15

© 2

015

Thay

uman

asam

y So

mas

unda

ram

32

• Important Coot Windows

Phaser Results @ Various Resolutions

Phaser Results 1.6Å data 2.5Å data 3.5Å data

LLG 4162.34 1509.4 643.5

TF-Z Score 52.3 33.7 22.5

10/2

8/20

15

© 2

015

Thay

uman

asam

y So

mas

unda

ram

34

Phaser Results @ Various Completions

Phaser Results

193 deg 1.6Å 130 deg 1.6Å 65 deg 1.6Å 30 deg 1.6Å

LLG 4162.0 4127.98 3753.44 2272

TF-Z Score 52.3 52.4 49.5 39.2

Lyz03b data with 193L as model

Importance of resolution

Phaser Results for 2.5Å Phaser result for 3.5Å

10/2

8/20

15

© 2

015

Thay

uman

asam

y So

mas

unda

ram

35

Importance of resolution

Phe 3 at 1.6Å Phe 3 at 3.5Å

10/2

8/20

15

© 2

015

Thay

uman

asam

y So

mas

unda

ram

36

Importance of completion

Phaser result for 30° data Phaser result for 60° data

10/2

8/20

15

© 2

015

Thay

uman

asam

y So

mas

unda

ram

37

Importance of completion

Tyr 53 with 30° data Tyr 53 with 60° data

10/2

8/20

15

© 2

015

Thay

uman

asam

y So

mas

unda

ram

38

MR: Phaser results w/ many models

35-1.6A 1LSG 3LZT 2FBB 2ZQ3 1LCN-A

Space Group Orthorhombic Triclinic Hexagonal Orthorhombic Monoclinic

SG Number P212121 P1 P6122 P212121 P21

Resolution 2.4Å 0.93Å 1.46Å 1.6Å 1.63Å

Protein HEWL+Seg Lysozyme Lysozyme Lysozyme Trky Lyso

Phaser LLG 506 1136 1478 1440 1907

Phaser TF-Z 24.4 34.9 38.1 37.8 42.0

10/2

8/20

15

© 2

015

Thay

uman

asam

y So

mas

unda

ram

39

Phenix.Refine GUI

10/2

8/20

15

© 2

015

Thay

uman

asam

y So

mas

unda

ram

45

From a different refinement Cycle 1

10/28/2015 Phenix Tutorial © 2015 Thayumanasamy …

Documents

Transcript of 10/28/2015 Phenix Tutorial © 2015 Thayumanasamy …