ACSA

Advanced Crystal Structure AnalysisMrTailor: PDB file Preparation to generate External Restraints

Tim GrüneGeorg-August-UniversitätInstitut für Strukturchemie

http://shelx.uni-ac.gwdg.de/~tg

tg@shelx.uni-ac.gwdg.de

Tim Grüne mrtailor 1/25

ACSA

Refinement at low Resolution

Resolution[Å] refined parameters data/parameters ratio3.0 x,y,z 0.9:12.3 x,y,z; B 1.5:11.8 x,y,z; B 3.1:11.5 x,y,z; B 5.4:11.5 x,y,z; U11U12U13U23U22U33 2.4:11.1 x,y,z; U11U12U13U23U22U33 6.1:10.8 x,y,z; U11U12U13U23U22U33 16:1

G. Sheldrick

Tim Grüne mrtailor 2/25

ACSA

Restraints and Constraints

Improvement of Data:Parameters by

Restraints

• Increase data points

• e.g. Engh-Huber parameters (Engh & Huber Parameters, Acta Cryst. (1991), A47, 392–400), 1,2- and1,3-distance restraints

• shelxl: DFIX, DANG, CHIV, FLAT, . . .

Constraints

• Decrease number of parameters

• e.g. −5 < Ueq(H) < −0.5; Invariom database.

Tim Grüne mrtailor 3/25

ACSA

Refinement at d > 3 Å

• Cα trace for 2Y9Y “Chromatin Remodelling FactorISW1A”• Data resolution: 30 Å– 3.25 Å• Cell: 206.97 206.97 215.31 90 90 120 (H32)

1. How much model bias, how much data?2. Interpretation requires experience (no peak picking)3. Removing loops without density = removing phasing

power

Tim Grüne mrtailor 4/25

ACSA

Refinement at d > 3 Å

1. Data:Parameters very low

2. 1,2- and 1,3-distances (Engh-Huber-Restraints) not shape determining

3. Molecular Replacement: heavy model bias towards template

Tim Grüne mrtailor 5/25

ACSA

Improvement of Data:Parameters

• NCS

• knowledge from deposited structures

Tim Grüne mrtailor 6/25

ACSA

Multi-subunit structures

• 70S ribosome with mRNA (PDB-ID 2wdk)• 3.5 Å• PDB: 24 chains!

• Nucleosome Core Particle (PDB-ID 3mvd)• 2.9 Å• 8 Histones + double stranded DNA (190 bp)

• “hot” structures often multi-subunit structures

• subunits often known, sometimes at better resolution than complex

Tim Grüne mrtailor 7/25

ACSA

External Restraints

Idea: Additional restraints beyond 1,3-restraints from same or similar structure

Automated in most MX refinement programs:

• Buster-TNT (http://www.globalphasing.com/buster/wiki/index.cgi?AutoBusterExample2e4ytarget)

• phenix.refine (http://www.phenix-online.org/documentation/refinement.htm#anch163)

• CNS (http://cns-online.org/v1.3/)

• refmac5 (http://www2.mrc-lmb.cam.ac.uk/groups/murshudov/) via prosmart

Tim Grüne mrtailor 8/25

ACSA

ProSmart: External Restraints for Refmac5

Nicholls et al., “Low-resolution refinement tools in REFMAC5”, Acta Crystallogr. D68, 404–417

# ProSMART Restraints Fileexte dist first chain A resi 61 ins . atom N second chain A resi 61 ins . atom O value 2.79807 sigma 1.06572exte dist first chain A resi 61 ins . atom N second chain A resi 62 ins . atom N value 3.67408 sigma 1.20622exte dist first chain A resi 61 ins . atom N second chain A resi 62 ins . atom CA value 4.93533 sigma 1.38368exte dist first chain A resi 61 ins . atom N second chain A resi 62 ins . atom C value 5.8248 sigma 1.49623

Inclusion into Refmac5 script:

EXTERNAL WEIGHT SCALE 500

@../../input/restraints_A.txt

Tim Grüne mrtailor 9/25

ACSA

Model Bias

From Nicholls et al., “Low-resolution refinement tools in REFMAC5”, Acta Crystallogr. D68, 404–417:

“It is, however, necessary for local structure to be sufficiently well conserved along the chain so thatthe effect on refinement is positive.”

Tim Grüne mrtailor 10/25

ACSA

Model Bias

Original data with 1OFC, no exter-nal restraints

with 1OFC, prosmartrestraints

with 1OFC, mrtailorrestraints

2Y9Y, chain A replaced with 1OFC

Tim Grüne mrtailor 11/25

ACSA

How to avoid model bias before knowing the structure?

PDB 2Y9Y (3.25 Å); chain A replaced with 1OFC (1.9 Å)

PDB 3ee6 (orange; PhD thesis A. Pal) with 1dbi

(from the mrtailor tutorial at http://shelx.uni-ac.gwdg.de/~tg/research/programs/mrtailor/tutorial/)

Tim Grüne mrtailor 12/25

ACSA

Sequence Alignment with BLAST

• Proteins: sequence⇒ structure

• “Blast” search against various data bases http://blast.ncbi.nlm.nih.gov:

– non-redundant data base (24584348 sequences): multiple sequence alignment, conservation of residuesor outliers

– PDB (63301 sequences): search models for molecular replacement

Tim Grüne mrtailor 13/25

ACSA

Sequence Alignment with BLAST

. . . : : .. :. . * : : :. : .gi|329665881 MLLNPTKRERKENYSIDNYYKDVLNTGRSSTPSHPRMPKPHVFHSHQLQPPQLKVLYEKERMWTAKKTGYVPTMDDVKAA 80gi|329665879 MLLNPTKRERKENYSIDNYYKDVLNTGRSSTPSHPRMPKPHVFHSHQLQPPQLQVLYEKERMWTAKKTGYVPTMDDVKAA 80gi|34809857 -----GAMERKANYAVDAYFREALRVSEPKAPKAPRPPKQPIVQDFQFFPPRLFELLDQEIYYFRKTVGYKVPKN----T 71gi|158429118 ------------------------MVSEPKVPKAPRPPKQPNVQDFQFFPPRLFELLEKEILYYRKTIGYKVPRN----P 52gi|18655633 -----------------MGHLGKTRWTREEDEKLKKLVEQNGTDDWKVIANYLPNRTDVQCQHRWQKV---LNPELIKGP 60

. . : :: : .: . : : .. * *.: : . : . : * : **: :gi|329665881 YGDISDEEEKK--QKLELLKLSVNNSQPLTEEEEKMKADWESE-----GFTNWNKLEFRKFITVSGKYGRNSIQAIAREL 153gi|329665879 YGKISDQEEQK--EKLELLKLSVNNSQPLTEEEEKMKADWESE-----GFTNWNKLEFRKFITVSGKYGRNSIQAIAREL 153gi|34809857 ELGSDATKVQR--EE----QRKIDEAEPLTEEEIQEKENLLSQ-----GFTAWTKRDFNQFIKANEKYGRDDIDNIAKDV 140gi|158429118 DLPNSA-QVQK--EE----QLKIDEAEPLNDEELEEKEKLLTQ-----GFTNWNKRDFNQFIKANEKWGRDDIENIAREV 120gi|18655633 WTKEEDQRVIKLVQKYGPKRWSVIAKHLKGRIGKQCRERWHNHLNPEVKKTSWTEEEDRIIYQAHKRLG-NRWAEIAKLL 139

*:* : : : . gi|329665881 APGKTLEEVRAYAKAFWSNIERIEDYEKYLKIIENEEEKIKRVKMQQEALRRKLSEYKNPFFDLKLKHPPSSNNKRTYSE 233gi|329665879 APGKTLEEVRAYAKAFWSNIERIEDYEKYLKIIENEEEKIKRVKMQQEALRRKLSEYKNPFFDLKLKHPPSSNNKRTYSE 233gi|34809857 -EGKTPEEVIEYNAVFWERCTELQDIERIMGQIERGEGKIQRRLSIKKALDQKMSRYRAPFHQLRLQYGN--NKGKNYTE 217gi|158429118 -EGKTPEEVIEYSAVFWERCNELQDIEKTMAQIERGEARIQRRISI---LEHHHHHH----------------------- 173gi|18655633 -PGRTDNAIKNHWNSTMRRKV----------------------------------------------------------- 159

gi|329665881 EEDRFILLMLFKYGLDRDDVYELVRDEIRDCPLFELDFYFRSRTPVELARRGNTLLQCLEKEFNAGIVLDDATKDRMKKE 313gi|329665879 EEDRFILLMLFKYGLDRDDVYELVRDEIRDCPLFELDFYFRSRTPVELARRGNTLLQCLEKEFNAGIVLDDATKDRMKKE 313gi|34809857 IEDRFLVCMLHKLGFDKENVYEELRAAIRASPQFRFDWFIKSRTALELQRRCNTLITLIEREN---IELEEKERAEKKKK 294gi|158429118 -------------------------------------------------------------------------------- 173gi|18655633 -------------------------------------------------------------------------------- 159

Tim Grüne mrtailor 14/25

ACSA

MrTailor

Blast search against PDBwith target sequence

multiple alignment

prosmart

modifiedPDB

clustal

externalrestraints

refmac5

qscores

model PDB

mrtailor

pdb/mtz

Tim Grüne mrtailor 15/25

ACSA

MrTailor Applications

1. Sequence Mapping after Molecular Replacement

2. Model Preparation for Prosmart

Tim Grüne mrtailor 16/25

ACSA

Sequence Mapping

#> mrtailor -a tppI_blast.aln -t "gi|215261288" -m "gi|6573500" \

-p 1dbi.ent -o 1dbi_with_tppI-sequence.pdb

• operates on all matching chains in PDB-file

• leaves all other chains intact

• 1dbi_with_tppI-sequence.pdb: residue numbers and types match target sequence (tpp-I, "gi|215261288").

• 1dbi_with_tppI-sequence.pdb: can be used as search model for MR

• 1dbi_with_tppI-sequence.pdb: gaps allow for movement during refinement

Tim Grüne mrtailor 17/25

ACSA

Clustalx QScores: Removing Stray Residues

A Q K C H S V I T Q D2 4 43 8 34 35 28 16 11 2 2

A Q K C H S V I T Q D0 0 0 0 0 0 0 0 0 0 0

Tim Grüne mrtailor 18/25

ACSA

Clustalx QScores: Removing Stray Residues

Example TPP-I (PDB 3ee6 vs. 1dbi) Input PDB-file for ProSmart after using mrtailor with qscore cut-off:

Tim Grüne mrtailor 19/25

ACSA

Number of External Restraints

Refmac5 with unmodified 1dbi + Prosmart

Number of atoms : 4062Number of residues : 542Number of chains : 2

Standard External AllBonds: 7862 23442 31304

Refmac5 and Prosmart with mrtailored 1dbi

Number of atoms : 2334Number of residues : 464Number of chains : 2

Standard External AllBonds: 3784 14290 18074

Tim Grüne mrtailor 20/25

ACSA

Algorithm Details

1 2 3 4 4

3EE6 VTTVGGTSFQ---EPFLITNEIVDYISGG <-- Target

1DBI VIAVGAVDQYDRLASF---------SNYG <-- Reference

1. Identical residues (number & type): reference PDB unmodified

2. Different residue types: reduced to Alanine in reference PDB

3. gap in target sequence: residue removed from reference PDB

4. gap in reference sequence: renumbering and renaming (F194 S195→ F397 I407)

Tim Grüne mrtailor 21/25

ACSA

GUI

Tim Grüne mrtailor 22/25

ACSA

Tutorial

http://shelx.uni-ac.gwdg.de/~tg/research/programs/mrtailor/tutorial

• Based on TPP-I "tripeptidyl-peptidase I" (TPP), 2.35 Å

• Pal, A. et al. "Structure of Tripeptidyl-peptidase I Provides Insight into the Molecular Basis of Late InfantileNeuronal Ceroid Lipofuscinosis" J. Biol. Chem. 2009, 284: 3976-3984

Tim Grüne mrtailor 23/25

ACSA

Conclusions

• mrtailor can remove model bias from a molecular replacement solution

• mrtailor can improve the effect of external restraints

• At low resolution the answer is not unique: try several options, e.g.

1. no external restraints (freedom to move)

2. external restraints from original PDB file (more restraints)

3. external restraints after using mrtailor (with and without alignment scoring) (compromise between 1+2)

Tim Grüne mrtailor 24/25